ES2561499T3 - Methods of treatment of diseases through the transplantation of allogeneic or xenogeneic organs or tissues - Google Patents

Abstract

A graft of a splenic organ or of porcine splenic tissue in the gestation phase for use in the treatment of a disease or disorder selected from the group consisting of a coagulation disorder, a deficiency in a coagulation factor , a defect in the lymphoid stroma, a disorder related to immunity and a lysosomal storage disease / enzyme deficiency, said graft being selected from a splenic organ or porcine splenic tissue so that it does not express or present at least one molecule capable of stimulating or of potentiating an immune response in a xenogenic subject, wherein said at least one molecule is a lymphocyte correceptor or a ligand of a lymphocyte correceptor, and wherein said phase of differentiation of said graft of a splenic organ or of a Porcine splenic tissue corresponds to a pig between 20 and 63 days of gestation.

Description

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DESCRIPTION

Methods of treatment of diseases through the transplantation of allogeneic or xenogeneic organs or tissues

The present invention relates to a graft of an organ or a porcine splenic tissue in gestation phase for use in the treatment of a hematological and / or genetic disorder and / or a lysosomal storage disease / an enzyme deficiency, associated with a pathological physiology or morphology of an organ or tissue according to claim 1. More particularly, the present disclosure relates to methods of treatment of diseases through the transplantation of human or porcine allogeneic organs / tissues in one phase. pregnancy between 7 and 9 weeks or between 20 and 28 days, respectively.

The transplantation of completely differentiated organs / tissues is a widely used and life-saving medical procedure of choice for the treatment of many very debilitating or fatal diseases, including kidney, heart, pancreatic, pulmonary, hematological, genetic and liver diseases. For example, the number of human kidney transplants has increased rapidly in recent years, but demand greatly exceeds the availability of organs. In the case of kidney failure, permanent hemodialysis can be used to prolong life, however, this is a very debilitating, uncomfortable and expensive procedure with limited efficacy, which implies a significant risk of opportunistic infections. In the case of diabetes, a disease with a tremendous medical and economic impact, daily insulin injection, the usual treatment of the prior art, does not satisfactorily prevent the limiting or fatal consequences of this disease. Worldwide, diabetes appears in virtually 5 percent of the population between the ages of 20 and 79, and therefore affects 150 million people. In the United States alone, an estimated 17 million people - more than 6 percent of the population - have saccharine diabetes, and approximately 1 million Americans with 20 years or more are diagnosed with the disease each year. In 1999 there were approximately 450,000 deaths in diabetic adults in the United States. Heart disease is the predominant cause of disability and death in all industrialized nations, and in addition, the incidence of heart failure is increasing in the United States, as more than half a million Americans die each year from this disease (Braunwald E. , 1997. N Eng J Med. 337: 1360; Eriksson H., 1995. J Inter Med. 237: 135). In addition, in the United States, heart disease accounts for approximately 335 deaths per 100,000 individuals (approximately 40% of total mortality) eclipsing cancer, which follows with 183 deaths per 100,000 individuals. In many acute and chronic clinical conditions, liver damage occurs, including drug-induced hepatotoxicity, viral infections, vascular lesions, autoimmune diseases and blunt trauma. In addition, patients with congenital metabolopathy may be at risk of developing liver damage. Symptoms of liver damage that occur as a result of these clinical conditions include, for example, fulminant liver failure with cholestasis, liver lesions and liver tissue necrosis, and in many cases, restoring normal liver function is vital for survival. from the patients.

Therapeutic transplantation in humans is normally carried out by the transplantation of completely differentiated allogeneic organs / tissues between suitably haplocompatible donors and recipients. However, said treatment modality suffers from considerable drawbacks. Allogeneic transplantation of differentiated organs / tissues is impossible to implement in a large number of cases due to the lack of availability of adequately immunologically and morphologically compatible transplant donors. Additionally, the use of human donors to provide organs / tissues for transplantation requires subjecting living donors to major surgery, for example, in the case of a kidney transplant. Alternatively, the use of cadaveric organs / tissues often also presents ethical dilemmas. In the case of diabetes, it has been demonstrated that the transplantation of pancreatic islets of an adult cadaveric donor is technically feasible, however, this methodology cannot be routinely implemented due to an insufficient number of pancreas from immunologically allogeneic donor pancreas. compatible of which isolate the sufficient amount of islets necessary.

Therefore, a large number of patients who could otherwise benefit from a therapeutic transplant succumb to diseases associated with renal, heart, liver, pancreatic, pulmonary or hematological insufficiency, while waiting for compatible transplant donors. In addition, even when suitably compatible haplocompatible transplant donors are found, permanent and harmful immunosuppressive treatments, such as daily administration of toxic drugs such as cyclosporin A, are generally necessary to prevent graft rejection. The use of drugs such as cyclosporine A is very undesirable since they cause serious side effects such as carcinogenicity, nephrotoxicity and an increased susceptibility to opportunistic infections. Such immunosuppressive treatments contribute to the disadvantages of allogeneic transplantation, since these are often unsuccessful in preventing short-term rejection, and are generally unable to prevent rejection indefinitely in the long term. Acute rejection of heart or liver grafts is often fatal. In the case of kidney transplantation, the inability of current immunosuppressive regimens to prevent acute graft rejection often requires emergency surgery to remove the graft, followed by the need to be placed on renal dialysis pending availability. of another compatible organ for transplantation.

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An alternative that has been proposed to the transplantation of an allograft involves the transplantation of a xenograft, that is, the transplantation of grafts derived from animals, in particular of porcine grafts, which are well established as a potential alternative animal of choice for human grafts. The great advantages of using xenografts for transplants would be their availability on demand for all patients in need of a transplant, as well as the avoidance of the medical and ethical burden of collecting grafts from living human donors or cadavers. However, to date, organ / tissue xenografts have been ruled out for transplantation in humans due to their so far Insurmountable immune incompatibility with human receptors.

Therefore, the ability to generate organs / tissues, such as pancreatic, renal, hepatic, cardiac or lymphoid organs / tissues, suitable for therapeutic transplantation in sufficient amounts and optimally tolerated in immunocompetent human beings without immunosuppression or With minimal immunosuppression, it is a very desired goal. A strategy that has been proposed to fulfill this aspiration involves the use of organs / tissues in early stages of development for transplantation. This methodology is promising since it has been shown that immunological tolerance to grafts derived from a developing tissue is better than grafts derived from adult tissues (Dekel B. ef al., 1997. Transplantation 64, 1550; Dekel B. et al., 1997. Transplantation 64, 1541; Dekel B. et al., 1999. Int Immunol. 11, 1673; Hammerman MR, 2000. Pediatr Nephrol. 14, 513). Additionally, the greater potential for growth and differentiation of developing organs / tissues is very desirable for the generation of optimally functional grafts integrated into the host. For example, it has been shown that grafts derived from developing human renal tissue show a reduction in apoptosis and tlsular destruction, as well as a sustained growth phase (Dekel B. et al., 1997. Transplantation 64, 550; Dekel B. ef al., 2000. Transplantation 69, 1470). In the developing human kidney, recent stem cells are induced in the nephrogenic pathway to form nephrons until 34 weeks gestation. Said nephrogenic differentiation route involves the invasion of a specialized region of the Intermediate mesoderm by an epithelial source (urethral primordium), which grows and branches to form a system of collecting ducts and induces the clustering of disorganized metarrenal mesenchymal stem cells and its differentiation in nephrons [Woolf, AS in: Pediatric Nephrology, 4th ed. Barratt, T. M., Avner, A. and Harmon, W. (eds.), Williams & Wilkins, Baltimore, Maryland. pages 1-19 (1999)]. Therefore, gestational phase kidney tissue transplants can be a potential source of renal regenerative cells and a promising solution for the current organ shortage for kidney transplantation. In the case of pancreatic tissues, pancreatic islet cells, such as insulin-producing beta cells, show an improvement in cell growth and differentiation with respect to the beta cells of already differentiated islets. For example, it has been shown that human fetal islets, which include the earliest insulin secretory cells, transplanted into mice and athymic rats, which are immunodeficient hosts, show continued growth and development, including the production of the other hormones pancreatic; glucagon, somatostatin and pancreatic polypeptide (Usadel et al., 1980. Diabetes 29 Suppl. 1: 74-9). Similarly, it has been shown that grafts derived from human embryonic pancreas in NOD / SCID mice, which are also immunodeficient hosts, generated human beta-producing insulin-derived graft cells (Castaing M. et al., 2001. Dlabetologla 44 : 2066). It has also been shown that transplants of porcine Islets in the gestation phase in mice can show a similar differentiation program, with a chronology similar to that of normal non-transplanted tissues.

Several mechanisms have been suggested to explain the reduced immunogenicity of grafts of developing tissues. It has been suggested that such grafts derived from developing tissues induce attenuated immune responses of the host against the Graft compared to grafts derived from adult tissues, because the former are predominantly vascularized by the vasculature derived from the host, as opposed to vascularization of the Graft derived predominantly from the Graft itself observed in the latter (Hyink DP et al., 1996. Am J Physlol. 270, F886). Additionally, it has been suggested that low levels of expression of the major hlstocompatibility complex (MHC) and adhesion molecules, and of the antigen presenting cells in the gestational phase tissue grafts, decrease the ability of said grafts to activate Host immune responses.

However, the methodologies that involve the use of developing human organs / tissues are impeded by the practical and ethical obstacles involved in obtaining a sufficient number of human embryos / fetuses, as well as the ethical problems involved in the use of tissue. human embryonic In order to avoid such obstacles, the use of developing organs / tissues derived from animals has been suggested, in particular porcine developing organs / tissues (Auchincloss, H. and Sachs, DH, 1998. Annu. Rev. Immunol. 16, 433 - 470; Hammerman, MR, 2002. Curr. Opln. Nephrol. Hypertens. 11.11-16).

In the interior technique, various methodologies for the use of non-syngeneic developing organ / tissue transplants for the treatment of diseases have been attempted.

One methodology involves the transplantation of gestational kidneys in non-immunosuppressed allogeneic receptors, in an attempt to generate functional renal organs derived from graft immunologically tolerated in said receptors, as shown by the use of graft embryo graft transplantation. 15 days under the renal capsule or in the eplplon of non-immunosuppressed adult host rats, without (Rogers, SA et al., 1998. Kldney Int. 54, 27-37; Rogers, SA et al., 2001. Am. J. Physiol. Regul. Integr. Comp. Physlol. 280,

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R132-136) or with (Rogers, S. A. and Hammerman, M. R., 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R661-665) prior in vitro preservation of the grafts.

Another methodology involves the transplantation of gestational kidneys in xenogeneic receptors treated with a blockade of costimulation with CTLA4-lg, in an attempt to generate functional renal organs derived from the graft immunologically tolerated in said receptors, as shown by the use of 15 day rat embryo graft transplantation in host mice (Rogers, SA and Hammerman, MR, 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1865-1869).

Another methodology involves the transplantation of renal tissue in the gestation phase in immunodeficient xenogeneic receptors reconstituted with human PBMC, in an attempt to generate functional renal organs derived from the graft immunologically tolerated in said receptors, as shown by the transplantation of human tissue into a 12 to 22 week gestation phase in chimeric rats SCID / Lewls and SCID / athymic (Dekel B. et al., 1997. Transplantation 64, 1550), and organ / tissue transplantation in a 70 day gestation phase in NOD / SCID mice (Dekel B. et al., 2001. J Am Soc Nephrol. 13, 977-90; Dekel B. et al., 2000. Transplantation 69, 1470).

Still another methodology involves the transplantation of pancreatic grafts in gestation phase grown in immunodeficient xenogeneic receptors, in an attempt to generate functional pancreatic cells and tissues derived from the graft immunologically tolerated in said receptors, as attempted by transplanting cells of the porcine islets in gestation phase in athymic mice (Otonkoski T. et al., 1999. Transplantation 68, 1674), of human fetal islets in mice and athymic rats (Usadel et al., 1980. Diabetes 29 Suppl 1: 74-9 ) and human embryonic pancreas in NOD / SCID mice (Castaing M. et al., 2001. Diabetologia 44: 2066).

An additional methodology involves the transplantation of porcine fetal islet cell groups intra-temporally or under the renal capsule into diabetic human receptors, in an attempt to treat diabetes in said recipients (Groth CG. Etal., 1999. J Mol Med. 77, 153).

A further methodology involves the transplantation of ventral mesocephalic allogeneic striatum tissue in human recipients with Parkinson's disease, in an attempt to treat this disease (Subramanian, T., 2001. Semin Neurol. 21, 103; Schumacher JM. Etal., 2000. Neurology54, 1042).

However, all prior art methodologies that involve transplantation of non-syngeneic developing tissues suffer from some or all of the following disadvantages: (I) a suboptimal tolerance by allogeneic / xenogenic human lymphocytes; (ii) a suboptimal structural and functional differentiation, for example, with respect to the production of urine by renal grafts, or with respect to the production of insulin by pancreatic grafts; (iii) a vascularization derived predominantly from the Graft, as opposed to that derived from the host; (iv) suboptimal growth; (v) inadequate availability of transplanted organs / tissues; and / or (vi) a sub-optimal security for administration to humans, especially with regard to preventing the generation of graft-derived teratomas.

Therefore, all prior art methodologies have failed to provide a suitable solution for the use of non-syngeneic developing organ / tissue transplants for the treatment of human diseases susceptible to a therapeutic transplant.

There is therefore a widely recognized need for, and it would be very advantageous to have, a method for the treatment of human diseases susceptible to a therapeutic transplant through the transplantation of developing and / or non-syngeneic organs / tissues devoid of the above limitations.

Summary of the invention

The present invention relates to a graft of a splenic tissue or a porcine splenic organ during pregnancy for use in the treatment of a disease or a disorder selected from the group consisting of a coagulation disorder, a deficiency in a coagulation factor, a defect in the lymphoid stroma, a disorder related to immunity and a lysosomal storage disease / an enzymatic deficiency, said graft being selected or from a splenic organ or from a tissue that does not express or present the at least one molecule capable of stimulating the potentiation of an immune response in a xenogeneic subject, wherein said at least one molecule is a lymphocyte correceptor or a ligand of a lymphocyte correceptor, and in which said phase of differentiation of said graft of Splenic organ or porcine splenic tissue corresponds to a pig between 20 and 63 days of gestation.

Preferably in the present invention the subject is a human being.

Preferably in the present invention the differentiation phase corresponds to between 20 and 56 days of gestation.

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Preferably in the present invention the differentiation phase corresponds to between 20 and 42 days gestation.

Preferably in the present invention the differentiation phase corresponds to 42 days gestation. Preferably in the present invention the graft of a splenic organ or of splenic tissue for its use further comprises the use of an immunosuppressive drug, thus favoring the incorporation of said graft of a splenic organ or of a splenic tissue in said subject. Preferably in the present invention the immunosuppressive drug is capable of blocking the binding of a lymphocyte correceptor with a ligand of said lymphocyte correceptor.

Preferably in the present invention the immunosuppressive drug is CTLA4-lg. Preferably in the present invention the lymphocyte correceptor or the lymphocyte correceptor ligand is chosen from the group consisting of B7-1, CD40 and CD40L. Preferably in the present invention the selection of the graft of a splenic organ or of splenic tissue is carried out through an analysis by an RT-PCR of said graft of a splenic organ.

or of splenic tissue.

Preferably in the present invention the deficiency in a coagulation factor comprises hemophilia. Preferably in the present invention the lysosomal storage disease / enzyme deficiency comprises Gaucher's disease. Preferably in the present invention the defect in the lymphoid stroma comprises a defect in the lymphoid stroma that results in a deterioration in the growth and / or differentiation of hematological cells.

The present invention successfully addresses the drawbacks of the currently known configurations by providing an optimal and generally applicable method for the treatment of essentially any disease susceptible to a therapeutic transplant through the use of organ graft or allogeneic / xenogeneic tissue transplantation. , by virtue of the ability of said grafts to generate graft-derived organs / tissues that show a specific differentiation of the optimal structural and functional lineage, and are optimally tolerated by human alloreactive / xenoreactive lymphocytes in a recipient without immunosuppression or with minimal immunosuppression . Unless otherwise defined, all technical and scientific terms used herein have the same meaning as usually understood by the person skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in practice or in the assay of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification will predominate, including the definitions. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

Brief description of the drawings

The invention is described herein by way of example only, with reference to the accompanying drawings. By now making a specific reference to the drawings in detail, it is emphasized that the details shown are by way of example and only for the purpose of illustrating the analysis of the preferred embodiments of the present invention, and are presented for the purpose of providing what is believed to be the most useful and easily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, allowing the description, taken together with the drawings, for those skilled in the art to appreciate how they can be carried out. practice the various forms of the invention.

In the drawings:

FIGs. 1 a - j are photographs that represent the growth and differentiation of early human and porcine renal precursors after transplantation. Figures 1 a-b, respectively, represent a macroscopic image and histology (Figure 1 b; H&E; magnification x 10 of the original) of a graft of human renal tissue in a gestation phase of 8 weeks, 8 weeks after transplantation . Note the massive growth and formation of the shape of a kidney (arrow) and the appearance of the layers of the glomeruli and tubules. Figures 1 c-d are a macroscopic image and histological analysis (H&E; magnification x 10 of the original), respectively, of a porcine renal tissue graft in a 4-week gestation phase, 8 weeks after transplantation. Note the massive growth (arrow) and the external vascular beds and the numerous glomeruli and tubules. Transplanted early embryonic kidney cells differentiate into other cell types after transplantation of porcine renal grafts in a gestation phase between 20 and 21 days (Figures 1 e-g) and porcine renal grafts in a gestation phase between 24 and 25 days (Figures 1 h - j). Figure 1 e is an x 4 magnification of the original of a photomicrograph of the H&E histology showing blood vessels (arrowheads), cartilage (large arrow) and bone (small arrows). Figures 1 f-g are magnifications x 40 of the original photomicrograph of the H&E histology depicting bone and cartilage, respectively. Figure 1 h is an x 10 magnification of the original of a photomicrograph of H&E histology showing myofibroblasts (arrowheads) and cartilage (large arrow). Figures 1 i-j are x 40 enlargements of the original H&E histology photomicrographs depicting myofibroblasts and a representative glandular tissue structure, respectively.

FIGs. 2 a-j are photomicrographs that represent the vascularization of grafts derived from renal tissue at an early gestation stage by the blood vessels of the recipient mouse. Immunostaining of human renal tissue grafts is shown in an 8-week gestation phase (Figures 2 a, 2 c and 2 e) and pig in one phase

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4 weeks gestation (Figures 2 b, 2 d and 2 f), 4 weeks after transplantation, with the mouse anti-CD31 antibody (PECAM) (magnification x 40 of the original). Figures 2 c-d represent positive staining (arrowheads) in the larger vessels, Figures 2 e-f represent the capillaries of medium and small size, and Figures 2 g-h represent the developing glomeruli. Figures 2 g - h represent the absence of staining of mature glomeruli and small capillary vessels of human fetal renal tissue in a 16-week gestation phase and of pigs in an 8-week, 4-week gestation phase. after transplant Figures 2 I-j show the absence of vessels derived from the host in fetal kidneys of human and pig vascular vascular control, respectively (magnification x 20 of the original).

FIGs. 3 a-b are photographs of the complete graft representing cysts filled with a urine-like fluid generated by transplants of early human and porcine embryonic renal precursors. Figures 3 a-b represent, respectively, macroscopic images of intra-abdominal grafts derived from human tissue in a gestation phase of 8 weeks and pigs in a gestation phase of 4 weeks containing large cysts (Indicated by arrows), 8 weeks later of the transplant Cyst fluid analysis identified it as diluted urine.

FIGs. 4 a-d are graphical representations of data that represent the growth curves of human renal tissue grafts in a gestation phase of 14, 10, 8 and 7 weeks, respectively, in the presence (A, black triangles) or in the absence ( □, white squares) of human reactive PBMC. In grafts of renal tissue in a gestation phase of 14 or 10 weeks, 8 weeks after transplantation, P <0.01 and P <0.05 compared to controls, respectively.

FIGs. 4 e-f are photomicrographs that represent the transplantation of a graft of human renal tissue in a 14-week gestation phase immunostained with antibodies against human CD3 (magnification x 40 of the original) showing the destruction of the glomerulus (Figure 4 e) and of the tubule (Figure 4 f) by human T lymphocytes. FIGs. 4 g-h are photomicrographs that represent a transplant derived from a renal tissue in an 8 week gestation phase immunostained with specific antibodies against human CD3 (magnification x 40 of the original). Note the absence of infiltration of T lymphocytes and the presence of intact glomeruli and tubules (Figures 4 g-h, respectively).

FIGs. 5 a-b are graphical representations of data representing the graft growth curves derived from human renal tissue in an 8-week gestation phase (Figure 5 a) in recipients receiving two Alloreactive Human PBMC Independent Infusions at the time of transplant and 6 weeks after transplant (▲, black triangles), or in recipients that have not received PBMC (□, white squares). The growth curve of transplants that originate from human fetuses 14 weeks of age show growth arrest (Figure 5b; ▲, black triangles) when the latter are transplanted into recipients along with the second dose of PBMC human, compared to those who do not receive the infusion of PBMC (□, white squares; P <0.05, 8 weeks after transplantation).

FIGs. 6 a-c are photomicrographs that represent the rejection of grafts derived from adult porcine renal tissue by human leukocytes. Figures 6 a-b are enlarged images at x 4 and at x 20, respectively, representing the histological staining with hematoxylin and eosin (H&E) of grafts derived from adult subcapsular porcine renal tissue 4 weeks after intraperitoneal infusion of human PBMC. Figure 6c represents the infiltration of T lymphocytes into the transplanted tissue, as determined by the use of an immunohistochemical detection of human CD3.

FIGs. 7 a-d are graphical representations of data representing the growth curves of porcine renal tissue grafts in a gestation phase of 8, 6, 4 and 3 weeks (Figures 7 a-d, respectively) in presence (A, triangles black) or in the absence (□, white squares) of xenorreactive human PBMCs. In transplants from porcine fetuses of 8 or 6 weeks of age, 8 weeks after transplantation, P <0.01 and P <0.05 compared to controls, respectively.

FIGs. 8 a-c are photomicrographs that represent the destruction of the transplanted tissue by invading human T lymphocytes in a transplant derived from porcine renal tissue in an 8-week gestation phase, 4 weeks after the transplant. Figures 8 a-b (magnification x 40 of the original) represent immunostaining with antibodies against human CD3, and FIG. 8 c represents histological staining with H&E (magnification x 10 of

original).

FIGs. 9 a - b are photomicrographs of a transplant derived from porcine renal tissue in a 4-week gestation phase that shows the glomeruli and tubules preserved without any positive CD3 infiltrate (magnification x 40 of the original), 4 weeks after the transplant.

FIGs. 10 a-b are graphical representations of data that represent similar growth curves of grafts derived from porcine renal tissue in a 4-week gestation phase (Figure 10 a) in recipients receiving 2 independent infusions of human x-reactive PBMC at the time of the transplant and 4 weeks after the transplant (A, black triangles), or in recipients that have not received the PBMC (□, white squares). The growth curve (Figure 10 b) of transplants from porcine fetuses of 8 weeks of age show growth arrest (A, black triangles) when the latter are transplanted into recipients along with the second dose of human PBMC and in comparison with those who do not receive the infusion of PBMC (□, white squares) (P <0.05, 8 weeks after transplantation).

FIGs. 11 a - c are UV photographs of an agarose gel electrophoresis that represent the analysis by RT-PCR of the mRNA expression of the costimulant molecule in normal developing human renal tissue (prior to transplantation), in human renal tissue undergoing transplantation immediately after transplantation, but before administration of all-reactive human PBMCs (after transplantation) and 2, 4 and 6

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weeks after the recipient mice were reconstituted with human PBMCs. The transplants analyzed were derived from human renal tissues in gestation phases of 8, 14 and 22 weeks (Figures 11a-

c, respectively).

FIGs. 12 a-c represent the patterns of differential gene expression of genes related to the immunity of normal adult human renal tissues against those in the gestation phase. Figure 12 a is a hierarchical grouping dendrogram (Zuo, F. et al., 2002. Proc. Nati. Acad. Sci. USA 99, 6292-6297) of the experimental groups generated on the basis of similarity of their expression profiles, which represent that adult and fetal expression patterns are grouped independently. Figure 12b is a diagram of the result of an analysis in mlchromatrlz representing the gene expression patterns of the 231 Immunity-related genes analyzed, showing that 122 of these genes had a TNoM score = 0 or 1 (Kaminski, N and Friedman, N., 2002. Am. J. Respir. Cell Mol. Blol. 27, 125-132). Gene expression values were divided by the geometric mean of all samples, transformed logarithmically and then plotted using the PLOTTOPGENE software (Kaminski, N. and Friedman, N., 2002. Am. J. Respir Cell Mol. Blol. 27, 125-132). The color yellow and purple represent the maximum and minimum expression, respectively. Note that most of the genes related to humanity were expressed at lower levels in the gestation phase compared to adult renal tissue. Figure 12 c is a representation of data representing the gene expression of 68 genes with a TNoM = 0 (P <0.05). The graphical representations are the values of the average expression of all the genes of the group. To eliminate the effect of the atypical values, the genes were normalized to an Interval of [0, 1], which means that the maximum value of each gene was set to 1, the minimum value was zero and the rest of the values they adjusted linearly in this Interval. Note again that the majority of the statistically significant genes (57/68) were smaller in the gestation phase compared to the renal tissue in the adult phase.

FIG. 13 is a photograph of the complete Graft depicting a Graft derived from human pancreatic tissue in a 12-week gestation phase, 8 weeks after transplantation, transplanted into a mouse mouse NOD / SCID carrier carrying human PBMC alorreactlvos. Note the pronounced graft growth and the absence of any sign of graft rejection.

FIGs. 14 a - b are mlchrographs representing a porcine liver derived graft in a 21-day gestation phase stained with H&E transplanted in a NOD / SCID recipient mouse, 7 weeks after transplantation at an x 4 and x 20 magnification of the original, respectively. Figure 14 a shows the clear development of a teratoma with broad cartilage differentiation (Figure 14 b).

FIGs. 15 a - d are mlchrographs that represent liver differentiation in histological sections of porcine liver derived grafts in a 28-day gestation phase transplanted in the spleen of a NOD / SCID mouse, 6 weeks after transplantation, stained with H&E (Figure 15 a), periodic acid-Schiff (PAS; Figure 15 b), porcine anti albumin antibody (Figure 15 c) and anti Ki67 antibody (Figure 15 d). Note the lobular patterns of hepatocyte configuration in Figures 15 a-c. The functionality of the growing liver is suggested by staining for PAS and albumin (Figures 15 b and 15 c, respectively). Original enlargement of the photophotographs of Figures 15 a - c: x 10. In Figure 15 d, positive staining of the hepatocyte nuclei (arrows) with anti-K67 antibody demonstrates the proliferation of graft-derived hepatocytes.

FIG. 16 a-b are mlchrographs that represent liver differentiation in histological sections of a graft derived from porcine liver in a 28-day gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. Sections were stained with PAS and with porcine anti albumin antibody (Figures 16 a and 16 b, respectively). Original enlargement: x 4. The functional activity of the transplanted liver is demonstrated by the synthesis of glycogen (PAS positivity) and albumin.

FIGS. 17 a-b are photomicrographs that represent liver differentiation in a histological section of a graft derived from human liver in a 7-week gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. Figure 17 a depicts staining with H&E, at a magnification x 4 of the original, note the differentiation of the bile duct (arrows). Figure 17 b represents staining with PAS, at a magnification x 40 of the original, note the presence of hepatocytes differentiated with stored glycogen.

FIG. 18 a is a stereo photomicrograph that represents the pancreatic growth of a complete porcine pancreas graft in a 28-day gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 5 weeks after transplantation.

FIGs. 18 b-c are photomicrographs that represent the differentiation of pancreatic tissue in a histological section of a graft derived from porcine pancreas in a 27-day gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. Figures 18b and 18c represent photomicrographs of the section with little and much increase, respectively. The section was stained with H&E, note the differentiation of the pancreatic lobe, with ductal structures and pancreatic acinars.

FIGs. 19 a-c are photomicrographs that represent the functional pancreatic differentiation in histological sections of grafts derived from porcine pancreas in the gestation phase transplanted under the renal capsule of NOD / SCID mice, 6 weeks after transplantation. Sections stained with anti insulin antibody (Figure 19 a) and anti pancreatic polypeptide antibody (PP) (Figure 19 b) demonstrate the synthesis of insulin and PP, respectively, in a tissue derived graft in a gestation phase of 27 days. Figure 19 c represents a histological section of a porcine pancreas derived graft in a 28-day gestation phase immunostained with anti cytokeratin antibody (the antibody is not reactive with the mouse epithelium). Note the differentiation of the graft derived from the pancreatic ductal epithelium.

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FIGs. 20 a-b are microfotograffas that represent functional pancreatic differentiation in a section of a graft derived from human pancreas in an 8-week gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. The section was immunostained with anti insulin antibody, note the foci of insulin-positive beta cells. Figure 20 a is a photomicrograph taken with little magnification, and Figure 20 b is a photomicrograph taken with much magnification that highlights the expression of insulin on a islet of Langerhans.

FIGs. 20 c-d are microfotograffas that represent pancreatic differentiation in a histological section of a graft derived from human pancreas in an 8-week gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. The section was immunostained with anti vimentin antibody (not reactive with mouse tissues), note the differentiation of mesenchymal cells of human origin in the graft (Figure 20 d). Figures 20 c and 20 d are micrographs of the stained section taken with little and much increase of the original, respectively.

FIGs. 21 a-c are microfotograffas that represent cardiac differentiation in histological sections of a graft derived from a human heart in a 9-week gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. Figure 21 a represents a section stained with H&E, note that the transplant contains two different types of cardiac-specific cellular components: cardiomyocytic structures ("CM") and basal ganglionics ("BG"). Figures 21b and 21c represent sections immunostained with anti-alpha-sarcomeric anti-actin antibody and with anti-neurofilament protein, respectively. Note the groups of cardiomyocytic cells identified by the positivity for alpha-sarcomeric actin, and the basal ganglion cells by the positivity of the neurofilament protein.

FIG. 22 is a photomicrograph depicting splenic differentiation in an H&E stained histological section of the porcine spleen-derived graft in a 28-day gestation phase transplanted under the renal capsule of a NOD / SCID mouse, 6 weeks after transplantation. Note the differentiation of the vascularized mesenchymal tissue. The photomicrograph was taken with an x 4 magnification of the original.

Description of the preferred embodiments

The present disclosure is about methods for the treatment of diseases by transplantation of developing non-syngeneic organs / tissues and about methods for assessing the suitability of graft transplantation. Specifically, the present disclosure refers to the transplantation of grafts of organs or human tissues in the gestation phase between 7 and 9 weeks, or pigs in a gestation phase of between 27 and 28 weeks, for the treatment of diseases in humans , such as kidney, pancreatic, liver, heart, genetic and / or hematological diseases. Human and porcine grafts in said gestation phase have the capacity to generate, in the absence of graft-derived teratomas, structurally and functionally differentiated organs / tissues, vascularized by the host, optimally tolerated by human alloreactive / xenoreactive lymphocytes, without immunosuppression from the host or with minimal immunosuppression. In particular, said grafts, when derived from renal organs / tissues, have the ability to generate urine producing renal organs vascularized by the host; when derived from pancreatic organs / tissues, it has the ability to generate pancreatic islets that comprise insulin-producing beta cells; when derived from liver organs / tissues, they have the ability to generate structurally and functionally differentiated liver cells and tissues. Such human grafts, when derived from cardiac organs / tissues, have the ability to generate proliferating cells and cardiac tissues. These porcine grafts, when derived from lymphoid organs / tissues, have the ability to differentiate into well-differentiated and vascularized mesenchymal / stromal lymphoid tissues. As such, the transplantation of said human or porcine grafts during pregnancy can be used for the treatment of human subjects suffering from a disease that is susceptible to a therapeutic transplant of cells, tissues and / or organs, such as a renal, pancreatic disease. , hepatic, cardiac, genetic and / or hematological, without immunosuppression of graft recipients or with minimal immunosuppression.

Before explaining in detail at least one embodiment of the invention, it should be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is susceptible to other embodiments or to be carried out or carried out in various ways. It should also be understood that the wording and terminology used in this document are descriptive and should not be considered as limiting.

Organ or tissue transplantation is the optimal or unique therapy for many devastating and deadly diseases, such as kidney, pancreatic, liver, heart, hematological and / or genetic diseases. However, current transplant methods are severely impeded by inadequate sources of adequate organ / tissue donors and by the requirement of permanent and harmful immunosuppressive treatment of graft recipients to prevent graft rejection. Suggested strategies to overcome these obstacles involve the use of xenogeneic organ or tissue grafts, which are available in sufficient quantities, and / or the development of organ or tissue grafts that have been shown to be better tolerated by non-compatible receptors than completely differentiated organ or tissue grafts.

In the prior art, several methodologies have been tried for the use of organ and tissue transplants in development and / or non-syngeneic for the treatment of diseases in humans.

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For example, transplantation of renal organs / tissues in the gestation phase has been attempted in non-immunosuppressed allogeneic receptors, in xenogeneic receptors treated by a blockade of the costimulaclone with CTLA4-lg, or in reconstituted immunodeficiency xenogeneic receptors with human PBMC. Transplantation of pancreatic organs / tissues in the gestation phase has been attempted through the transplantation of islet cells, pancreases or pancreatic islets in the gestation phase, in immunodeficiency xenogeneic receptors, or the transplantation of clusters of porcine fetal islets in diabetic human receptors. Another methodology has attempted the transplantation of allogeneic fetal mesencephalic ventral striated tissue in human recipients with Parkinson's disease.

However, all prior art methodologies that employ the transplantation of grafts of organs or non-mutagenic tissues in development, such as human or porcine grafts, in a recipient, have failed to provide a method for generating organs / graft-derived tissues that: (I) are optimally structurally / funconally differentiated; (I) are fully / optimally tolerated by human alloreactive / xenoreactive lymphocytes in a recipient without immunosuppression of the graft recipient or with minimal Immunosuppression (i¡¡) are optimally vascularized by the host; and (iv) can be generated in the absence of graft derived teratomas.

In particular, prior art methodologies have failed to provide a method for generating human or porcine graft derived organs / tissues in a recipient that show optimal structural / functional differentiation, including urine production, and that can be generated. in the absence of graft-derived teratomas that are optimally tolerated by human alloreactive / xenoreactive lymphocytes in the recipient without immunosuppression of the recipient or with minimal immunosuppression.

In addition, the previous methodologies have failed to provide a method to generate human or porcine graft-derived organs / tissues in a recipient that include pancreatic islets and insulin-producing beta cells, which can be generated in the absence of teratomas and that are optimally tolerated by human alloreactive / xenoreactive lymphocytes in the recipient.

Additionally, prior art methodologies have failed to provide a method for generating in a recipient human or porcine liver cells / tissues structurally and functionally differentiated from the graft, which can be generated in the absence of graft derived teratomas and that are optimally tolerated by human alloreactive / xenoreactive lymphocytes in the recipient.

Additionally, prior art methodologies have failed to provide a method for generating proliferative human graft-derived cells / tissues in a recipient that can be generated in the absence of graft-derived teratomas and that are optimally tolerated by alloreactive human lymphocytes in the receptor.

Likewise, prior art methodologies have failed to provide a method to generate well-differentiated and vascularized porcine lymphoid tissues derived from the graft in a recipient that can be generated in the absence of graft-derived teratomas and that are optimally tolerated by human x-reactive lymphocytes .

In carrying out the present invention, the existence of specific gestation phases was unexpectedly discovered for which the organs / tissues do not substantially show / express specific receptors of the lymphocytes or ligands thereof, and for which they can organs / tissues are transplanted into a recipient so that, in the absence of graft-derived teratomas, cells, organs and tissues that show optimal structural and functional differentiation, such as, in the case of kidney grafts, the production of urine are generated; and that they are optically tolerated by human alloreactive / xenoreactive lymphocytes in the recipient, without immunosuppression of the recipient or with minimal immunosuppression.

In particular, by practicing the present invention, specific gestation phases were discovered and defined during which human or porcine renal grafts can be transplanted into a recipient so that they are generated, in the absence of a formation of derived teratomas. of the urine-producing graft, organs and renal tissues that are optimally structurally and functionally differentiated that are optimally tolerated by human alloreactive / xenoreactive lymphocytes in the recipient in the absence of immunosuppression of the recipient or with minimal immunosuppression.

Additionally, by carrying out the present invention, specific gestation phases were discovered and defined during which human or porcine liver grafts can be transplanted into a recipient so that they are generated, in the absence of a formation of teratomas derived from the graft, liver organs / tissues optimally structurally and functionally differentiated that are optimally tolerated by human alloreactive / xenoreactive lymphocytes.

In addition, by practicing the present invention, specific gestation phases were discovered and defined during which human or porcine pancreatic grafts can be transplanted into a

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receptor so that, in the absence of a graft-derived teratoma formation, structurally and functionally differentiated pancreatic organs / tissues that include pancreatic islets and insulin-producing beta cells that are optimally tolerated by human alloreactive / xenoreactive lymphocytes are generated.

Additionally, by carrying out the present invention, specific gestation phases were discovered and defined during which human cardiac grafts can be transplanted into a recipient so that they are generated, in the absence of a formation of graft-derived teratomas, Differentiated and proliferative cardiac cells / tissues that are optimally tolerated by human alloreactive lymphocytes.

Also, by carrying out the present invention, specific gestation phases were discovered and defined during which porcine lymphoid grafts can be transplanted into a recipient so that they are generated, in the absence of a teratoma formation, cells / tissues Well-differentiated and vascularized mesenchymal / stromal lymphoids that will be well tolerated by human xeno-reactive lymphocytes.

Therefore, transplantation of grafts derived from human or porcine organs / tissues in the gestation phases mentioned above can be used for the structural / functional replacement / repair of organs / tissues that show pathological physiology / morphology in the recipients of said grafts. , and that therefore can be used for the treatment of diseases related to said organs / tissues showing said pathological physiology / morphology, without any immunosuppression of the recipient or with minimal immunosuppression.

Therefore, according to one aspect of the present disclosure, a method is provided for the treatment of a disorder related to the pathological physiology or morphology of an organ or tissue.

The method is carried out by transplanting in a subject in need thereof a graft of a selected therapeutically effective organ or mammalian tissue: (i) so that it does not express or substantially present any molecule capable of stimulating or potentiating an immune response in the subject; (ii) in a predetermined phase of differentiation advanced enough to be able to generate structurally and functionally differentiated organs / tissues of essentially a single type in the subject, preferably in the absence of a formation of graft-derived teratomas, but sufficiently early to allow the graft to be optimally tolerated by non-syngeneic lymphocytes; or (iii) preferably both.

Depending on the context of the transplant, in order to facilitate the incorporation of the graft, the method may additionally advantageously comprise the treatment of the subject with an immunosuppressive regimen before, together with or after the graft transplant.

As used herein, "treating" the disorder includes curing, alleviating or stabilizing the disorder, or inhibiting the future onset or development of the disorder.

As used herein, the term "disorder" refers to any disease, or to any pathological or unwanted condition, condition or syndrome, or to any physical, morphological or physiological abnormality.

As used herein, the phrase "therapeutically effective graft" refers to a graft that has structural and / or functional characteristics such that transplantation of the same in the subject serves for the treatment of the disorder.

Methods for the selection of a graft that does not express or substantially present the molecule, or in a predetermined differentiation phase, are described below.

Graft transplantation can be carried out in several ways, depending on various parameters, such as, for example, the type, phase or severity of the disorder, the physical or physiological parameters specific to the individual subject and / or the desired therapeutic result. . For example, depending on the application and the purpose, graft transplantation can be carried out by using a graft from any of the various species of mammals and / or types of organs or tissues, by implanting the graft. in various anatomical locations of the subject, by the use of a graft consisting of a total or partial organ or tissue, and / or by the use of a graft consisting of several individual organs or tissues and / or portions thereof.

The person skilled in the art, such as a doctor, in particular a transplant surgeon specialized in the disorder, will possess the necessary expertise for the selection and transplantation of the appropriate graft, so that the disorder is treated according to the teachings of The present invention.

Depending on the application and the purpose, the method can be carried out by using a syngeneic, allogeneic or xenogenic graft derived essentially from any mammalian species.

As used herein, a "syngeneic" graft refers to a graft that is essentially genetically identical to the subject or essentially all lymphocytes of the subject.

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Some examples of syngeneic grafts include a graft derived from the subject (also referred to as "autologous graft"), a clone of the subject or an identical twin of the subject.

As used herein, a "non-syngeneic" graft refers to an allogeneic graft or a xenogenic graft.

As used herein, an "allogeneic graft" refers to a graft derived from a non-syngeneic donor with the subject or non-syngeneic donor with a substantial proportion of the lymphocytes present in the subject, in which the donor is of the same species as the subject or the same species as substantially all of the subject's lymphocytes.

Normally, two mammals of the same species that are not clones or twins are allogeneic to each other.

As used herein, a "xenogeneic graft" refers to a graft derived from a non-syngeneic donor with the subject or non-syngeneic donor with a substantial proportion of the lymphocytes present in a subject, in which the donor is from a a different species from that of the subject or from a different species from a substantial proportion of the lymphocytes present in the subject

Normally, mammals of different species are xenogeneic with each other.

As described and illustrated in the following Examples section, transplantation of a human or animal graft of the present disclosure into a recipient can be used to generate organs and tissues optimally structurally and functionally differentiated, in the absence of a teratoma formation, which are optionally tolerated by human alloreactive / xenoreactive receptor lymphocytes without immunosuppression of the recipient or with minimal immunosuppression.

As used herein, an "optimally tolerated" graft is a graft that is not rejected or that is not substantially infiltrated by the non-syngeneic T lymphocytes of the subject in the graft.

As used herein, a graft that is "rejected" is a graft that causes what is commonly known in the art as hyperacute rejection, acute rejection or chronic rejection. Extensive guidance is provided in the literature for the determination of graft rejection (for example, see: Kirkpatrick CH. And Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE et al., 1997. Mayo Clin Proc. 72, 175; Morrison VA et al., 1994. Am J Med. 97, 14; Hanto DW., 1995. Annu Rev Med. 46, 381; Senderowicz AM et al., 1997. Ann Intern Med. 126, 882; Vincenti F. et al., 1998. New Engl. J Med. 338, 161; Dantal J. et al. 1998. Lancet 351, 623). The infiltration of an object by the T lymphocytes of the graft receptor is usually correlated with graft rejection.

As used herein, a "minimum immunosuppressive treatment" of the subject refers to an immunosuppressive treatment of the subject restricted to the administration of a drug capable of blocking an interaction between a lymphocyte correceptor and an analogous ligand thereof, or an immunosuppressive treatment of the subject applied for a single period of 20 days or less.

As described hereinbefore, the graft can be derived from several mammalian species.

Depending on the application and the purpose, the graft is preferably a human graft or a pig graft.

In carrying out the present invention, the gestation phases of human organ / tissue grafts were identified for which they are in a predetermined differentiation phase suitable for carrying out the method, as described in the following section of Examples

Preferably, the human graft is selected in a differentiation phase corresponding to between 5 and 16 weeks of gestation, more preferably between 6 and 15 weeks of gestation, more preferably between 7 and 14 weeks of gestation, more preferably between 7 and 9 weeks gestation and most preferably between 7 and 8 weeks gestation.

As described and shown in the Examples section that follows, the human grafts of the present disclosure selected in a gestation phase of between 7 and 8 weeks gestation can be used to generate organs and tissues optimally functionally and structurally differentiated, in the absence of the formation of a teratoma derived from the graft, which are optimally tolerated by human alloreactive lymphocytes of the recipient without immunosuppression of the subject or with minimal immunosuppression.

As used herein, the phrase "alloreactive lymphocytes" refers to lymphocytes substantially capable of rejecting an essentially completely differentiated allogeneic graft.

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By practicing the method of the present invention, the universal applicability of the method with respect to different types of cells / organs / tissues was demonstrated by the use of allogeneic renal, hepatic, pancreatic and cardiac grafts of human origin.

As described and shown in Example 1 of the Examples section that follows, transplantation into a recipient of a human renal graft of the present disclosure selected in a differentiation phase corresponding to between 7 and 8 weeks gestation can be used for generate, in the absence of the formation of a graft-derived teratoma, kidney organs and tissues that are optimally funconally and structurally differentiated that are capable of producing urine and that are optimally tolerated by the human alloreactive lymphocytes of the recipient, without immunosuppression of the recipient or with a minimal immunosuppression

As described and shown in Example 6 of the Examples section that follows, a human liver graft of the present disclosure selected in a differentiation phase corresponding to 7 weeks gestation can be used to generate, in the absence of the formation of a graft-derived teratoma, funclonally and structurally differentiated hepatic organs / tissues that will be optimally tolerated by the human alloreactive lymphocytes of the recipient, without immunosuppression of the recipient or with minimal immunosuppression.

As described and shown in Example 7 of the Examples section that follows, transplantation into a recipient of a human pancreatic graft of the present disclosure selected in a differentiation phase corresponding to 8 weeks gestation can be used to generate, in absence of the formation of a graft-derived teratoma, funconally and structurally differentiated pancreatic organs / tissues derived from the Graft that include pancreatic islets and beta-producing insulin cells that will be optimally tolerated by human alloreactive lymphocytes of the recipient, without immunosuppression of the recipient or with minimal immunosuppression.

As described and shown in Example 8 of the Examples section that follows, transplantation into a recipient of a human cardiac graft of the present disclosure selected in a differentiation phase corresponding to 9 weeks gestation can be used to generate, in absence of the formation of a graft-derived teratoma, graft-derived cells / tissues that show a significantly proliferative cardiac phenotype that will be well tolerated by the human alloreactive lymphocytes of the recipient.

In carrying out the present invention, the gestation phases of the porcine organs / tissues were identified during which they are in a suitable predetermined differentiation phase to implement the method, as described in the following section of Examples .

Preferably, the porcine graft is selected in a differentiation phase corresponding to between 20 and 63 days of gestation, more preferably between 20 and 56 days of gestation, more preferably between 20 and 42 days of gestation, more preferably between 20 and 35 days of gestation, more preferably between 20 and 28 days of gestation, more preferably between 24 and 28 days of gestation and most preferably between 27 and 28 days of gestation.

Alternatively, the pig graft can be advantageously selected in a differentiation phase corresponding to between 22 and 33 days of gestation, between 23 and 32 days of gestation, between 24 and 31 days of gestation, between 25 and 30 days of gestation, between 26 and 29 days of gestation, between 22 and 63 days of gestation, between 22 and 56 days of gestation, between 22 and 42 days of gestation, between 22 and 35 days of gestation, between 22 and 34 days of gestation, between 22 and 32 days of gestation, between 22 and 31 days of gestation, between 22 and 30 days of gestation, between 22 and 29 days of gestation, between 22 and 28 days of gestation or between 22 and 27 days of gestation.

In order to avoid the formation of teratomas, the porcine graft is preferably selected in a differentiation phase corresponding to at least 22 days of gestation since, as shown in Example 6 of the Examples section that follows, a porcine graft in a differentiation phase corresponding to a gestation phase as early as 21 days presents the risk of causing teratomas when it is transplanted into a host.

As described and shown in the Examples section that follows, transplantation into a porcine graft recipient between 27 and 28 days gestation can be used to generate graft-derived organs and tissues optimally functionally and structurally differentiated, in the absence of formation of a teratoma derived from the graft, which are optimally tolerated by human xeno-reactive lymphocytes of the recipient, without immunosuppression of the recipient or with minimal immunosuppression.

As used herein, the phrase "xenoreactive lymphocytes" refers to lymphocytes substantially capable of rejecting an adult xenogeneic graft.

By practicing the method of the present invention, the universal applicability of the method with respect to different types of cells / organs / tissues was demonstrated by the use of renal, hepatic, pancreatic and lymphoid grafts of porcine origin.

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As described and shown in Reference Example 1 of the following Examples section, transplantation into a recipient of a porcine renal graft of the present disclosure selected in a differentiation phase corresponding to between 27 and 28 days gestation can be used. to generate, in the absence of graft-derived teratomas, graft-derived organs and renal tissues that are optimally functionally and structurally differentiated that are capable of producing urine and that are optimally tolerated by the xenorreactive human lymphocytes of the recipient, without immunosuppression of the recipient or with a minimal immunosuppression

As described and shown in Reference Example 6 of the Examples section that follows, transplantation into a recipient of a porcine liver graft of the present disclosure selected in a differentiation phase corresponding to 28 days gestation can be used to generate Functionally and structurally differentiated hepatic organs / tissues of the graft that, in the absence of the formation of a graft-derived teratoma, will be optimally tolerated by the xenorreactive human lymphocytes of the recipient, without immunosuppression of the recipient or with minimal immunosuppression.

As described and shown in Reference Example 7 of the Examples section that follows, transplantation into a recipient of a porcine pancreatic graft of the present disclosure selected in a differentiation phase corresponding to 27-28 days gestation can be used. to generate functionally and structurally differentiated pancreatic organs / tissues from the graft, including pancreatic islets and insulin-producing beta cells that, in the absence of the formation of a graft-derived teratoma, will be optimally tolerated by the recipient's xeno-reactive human lymphocytes, without immunosuppression of the recipient or with minimal immunosuppression.

As described and shown in Example 9 of the Examples section that follows, transplantation into a recipient of a graft of a porcine lymphoid organ / tissue of the present disclosure selected in a differentiation phase corresponding to 28 days of gestation to generate, in the absence of the formation of a graft-derived teratoma, mesenchymal / stromal lymphoid cells / tissues derived from the host well differentiated and vascularized that will be well tolerated by the xenorreactive human lymphocytes of the recipient.

In carrying out the present invention, it was unexpectedly discovered that the structurally and functionally differentiated organs / tissues generated by the grafts show a vasculature predominantly derived from the subject (for further details please refer to the following Examples section). Without being bound by a paradigm, the present inventors are of the opinion that pig grafts of between 27 and 28 days of gestation, respectively, or grafts derived from other species in equivalent differentiation phases, are optimally incorporated into the subject due to its ability to generate organs / tissues that have said vasculature predominantly derived from the subject. Based on this point of view, it has been suggested in the art that the degree of vasculature derived from the host in a transplanted graft correlates with the tolerance of said graft.

The discovery that the transplantation of human or porcine organs / tissues at differentiation stages corresponding to said early gestation phases can be used to generate in a recipient some graft-derived organs and tissues optimally structurally and funconally differentiated from the Graft line that they are optimally tolerated by human alloreactive / xenorreactive lymphocytes in the recipient, without immunosuppression of the recipient or with minimal immunosuppression, in the absence of the formation of a graft-derived teratoma, it was unexpected given that: (i) the prior art teaches that organ / tissue transplantation at more advanced stages of differentiation is optimal for such application (for example, see Otonkoskl T. et al., 1999. Transplantation 68, 1674); and (ii) since the earliest stages of pregnancy - and therefore the least immunogenic ones - of the grafts during which they would be able to generate graft-derived organs / tissues in the absence of graft-derived teratomas were unknown.

Grafts of non-human origin originating from numerous species, other than pig, can also be used in optimal differentiation stages corresponding to the aforementioned optimal human or porcine gestation phases, in order to implement the method of the present disclosure. Some animals, such as the main domesticated or farm animals and primates, which have been widely characterized with respect to the correlation between the differentiation phase and the gestation phase, may be suitable for carrying out the method. Such animals include bovids (e.g., cow), equidae (e.g., horse), ovids (e.g., goat, sheep), felids (e.g., Felis domestica), canids (e.g., Canis domestica), rodents (per eg, mouse, rat, rabbit, guinea pig, gerbil, hamster) and primates (for example, chimpanzee, rhesus monkey, macaque, marmoset).

Various methods can be used to obtain a graft in a differentiation phase corresponding to a specific gestation period.

Obtaining said graft is carried out optimally by removing the graft from an embryo or a donor from the developing graft in said gestation stage.

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Someone usually versed in the subject will understand that the gestation phase of an organism is the period of time that has elapsed since the fertilization of the oocyte generated by the organism.

Alternatively, a graft in a differentiation phase corresponding to a specific gestation phase can be obtained by in vitro culture of cells, organs / tissues that are in a differentiation phase earlier than that of the graft, such as specific precursor cells of the organ, so that a graft is generated in a desired phase of differentiation. Said in vitro controlled differentiation of cells, tissues or organs is carried out routinely, for example, by using the culture of embryonic stem cell lines, to generate cultures containing cells / tissues / organs of the desired strains. For example, for the generation of several strains, including endodermal strains such as the liver; ectodermal lines such as the brain, skin and adrenal; and mesodermal lines such as muscle, cartilage, Müllerian duct and heart, see, for example: Schuldiner M. et al., 2000. Proc Nati Acad Sel USA 97: 11307-1112 and Itskovitz-Eldor J. et al., 2000 Mol Med 6: 88; for pancreatic differentiation of embryonic stem cells, see, for example: Lee S. H., et al., 2000. Nature Biotechnol. 18: 675; Lumelsky et al., 2001. Science 292: 1389-1394; Soria et al., 2000. Diabetes 49: 1-6; Schuldiner M. et al., 2000. Proc Nati Acad Sci USA 97: 11307-11121). For the differentiation of lung lines, see, for example, Otto WR, 1997. Int J Exp Pathol. 78: 291-310.

In order to optimally treat the disorder, graft transplantation is preferably carried out in such a way that therapeutically replaces or replaces the organ or tissue that shows the physiology or pathological morphology related to the disorder.

Therefore, the graft is preferably selected from a type of organ or tissue corresponding to that of the organ or tissue with pathological physiology or morphology. For example, for the treatment of a renal, pancreatic, hepatic or cardiac graft, respectively. For example, for the treatment of a hematological and / or genetic disorder, the graft is preferably a lymphoid graft. Alternatively, for the treatment of a hematological and / or genetic disorder, the lymphoid graft can be derived from any other suitable lymphoid tissue, depending on the application and purpose, such as the lymph nodes, Peyer's plaques, the thymus or of the bone marrow.

As described in the following Examples section, the transplant of a graft selected from a type of organ or tissue corresponding to that of the organ or tissue showing a physiology or pathological morphology related to the disorder according to the protocol established in This document can be used for the treatment of the disorder.

As described hereinbefore, graft transplantation can be performed by transplanting it in various anatomical locations. Preferably, the graft is transplanted in an anatomical location where it will have an optimal therapeutic effect.

Depending on the application and purpose, the graft can be transplanted in a homotopic anatomical location (the normal anatomical location for the type of organ or tissue of the graft), or in an ectopic anatomical location (an abnormal anatomical location for the type of organ or tissue of the graft). Optionally, when the graft is transplanted, the organ or tissue that shows a pathological physiology or morphology can be removed, for example, so as to allow growth and incorporation of the graft, for example, in the context of substitution. of organs by graft transplantation in a homotopic anatomical location.

As used herein, a "homotopic anatomical location" of a graft whose type of organ or tissue exists in the form of multiple individual homologues (eg, right and left kidney, different fingers of the same hand, etc.) includes the anatomical location of any such homologues.

Depending on the application and the purpose, the graft can be advantageously implanted under the renal capsule or in the kidney, testicular fat, hypodermis, omentum, portal vein, liver, spleen, cardiac cavity, heart, heart pancreas and / or intra-abdominal space.

Preferably, the transplantation of a renal graft of the present disclosure is carried out by transplantation of the graft into the intra-abdominal space, or more preferably, in the renal capsule. Preferably, the kidney capsule transplant is carried out by a subcapsular transplant. The subcapsular transplant advantageously allows the insertion of a catheter that only requires a short distance to the skin, where urine from the renal graft can be extracted. Intra-abdominal transplantation advantageously allows the host's excretory system to create anastomosis with the ureter or renal pelvis of the developing transplanted renal tissue. As shown in Example 1 of the following Examples section, the method can be practiced by transplanting a renal graft of the present disclosure under the renal capsule of a recipient. Alternatively, renal graft transplantation can be performed by transplanting the graft into the portal vein, liver, spleen, testicular fat, hypodermis or omentum.

Transplantation of a liver graft of the present disclosure can be advantageously carried out by transplanting the graft into the portal vein, liver, renal capsule, testicular fat, hypodermis, epiploon, spleen and / or intra-abdominal space. . Preferably, the transplantation of a liver graft of the present disclosure

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It is carried out by transplanting the graft under the renal capsule or in the spleen. As shown in Example 6 of the following Examples section, the method can be practiced by transplanting a liver graft of the present disclosure under the renal capsule or in the spleen.

The transplantation of a pancreatic graft of the present disclosure can be carried out advantageously by transplanting the graft into the portal vein, the liver, the pancreas, the testlcular fat, the hypodermis, the omentum and / or the intra-abdominal space. Preferably, the transplantation of a pancreatic graft of the present disclosure is carried out by transplanting the graft under the renal capsule. Preferably, for the transplantation of a pancreatic graft into the portal vein, the pancreatic graft is a graft of a pancreatic Islet. As shown in Example 7 of the following Examples section, the method can be practiced by transplanting a pancreatic graft of the present disclosure under the renal capsule. A guide to the practice of therapeutic transplantation of pancreatic grafts in accordance with the teachings of the present disclosure is provided in Example 5 of the following Examples section.

The transplantation of a cardiac graft of the present disclosure can be carried out advantageously, depending on the application and the purpose, by transplanting the graft into the cardiac cavity, the heart, the myocardium and the intra-abdominal space. As shown in Example 8 of the following Examples section, the method can be practiced by transplanting a cardiac graft of the present disclosure under the renal capsule.

Preferably, for the treatment of a cardiac disorder related to myocardial ischemia due, for example, to a myocardial infarction, the cardiac graft is administered in the area of the Infarction and / or in the perimeter area of the infarction. As the person skilled in the art will be aware, the infarcted area is easily visible, allowing the specific location of the application of therapeutic grafts to be possible. The precise determination and chronology of an effective dose in this particular case may depend, for example, on the size of the infarction and on the time elapsed after the onset of myocardial ischemia. In the subject a broad guide is provided for the therapeutic implantation of cardiac tissues, such as a cardiac graft of the present disclosure, in the injured myocardium, according to the teachings of the present invention (see, for example: Strauer et al. ., 2001. Dtsch Med Wochenschr. 126: 932; Strauer et al., 2002. Circular 106: 1913; Stamm et al., 2003. Lancet 361: 45; Perin et al., 2003. Circulation 107: 2294; Assmus et al., 2002. Circulation 106: 3009; Britten et al., 2003. Circulation 108: 2212; and US Pat. Nos. 5,733,727, 6,395,016 and 6,592,623).

Transplantation of a lymphoid graft of the present disclosure, such as a splenic graft of the present disclosure, can be advantageously carried out, depending on the application and the purpose, by transplanting the graft into the portal vein, the liver, the renal capsule, hypodermis, omentum, spleen and intra-abdominal space. As shown in Example 9 of the following Examples section, the method can be practiced by transplanting a lymphoid graft of the present disclosure under the renal capsule.

As described hereinbefore, depending on the application and the purpose, graft transplantation can be carried out by transplanting a graft consisting of a complete or partial organ. The method can be advantageously carried out by transplanting a graft consisting of a partial organ for organ grafting in a differentiation phase corresponding to that of a human organ in a gestation phase of 9 weeks or greater, or to a porcine organ in a gestation phase of 5 weeks or more.

As shown in Reference Example 1 of the following Examples section, the transplantation of said partial organ grafts in differentiation phases corresponding to said gestation phases results in a significantly improved incorporation and / or functional and structural differentiation. of the graft with respect to the transplantation of a graft of a complete organ.

As described hereinbefore, depending on the application and the purpose, graft transplantation can be carried out by transplanting a graft consisting of several organs, individual tissues and / or portions thereof.

For example, the transplantation of an increasing number of grafts of individual organs or tissues can be advantageously employed to increase the physiological or physical therapeutic effect of the graft to the desired levels. For example, increasing the number of individual organs / tissues of an endocrine tissue graft (for example, from a Graft of a pancreatic islet) can be used to obtain sufficiently high levels of secretion of the hormone derived from the Graft (for example , of Insulin), so that a desired effect is achieved (for example, an increase in insulin secretion capacity). In the case of renal grafts, the increase in the number of grafts derived from individual organs or renal tissues can be used to obtain a sufficient number of renal organs to achieve, for example, a urine production capacity sufficiently high as to relieve or cure a kidney disorder in the subject.

As described hereinbefore, the method for the treatment of the disorder may advantageously comprise the treatment of the subject with an immunosuppressive regimen before, during or after graft transplantation.

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Various types of immunosuppressive regimens can be used for immunosuppression of the subject.

Some examples of suitable types of immunosuppressive regimens include the administration of immunosuppressive drugs, tolerance-inducing cell populations and / or immunosuppressive radiation.

Extensive guidance for the selection and administration of immunosuppressive regimens suitable for transplantation is provided in the literature of the technique (for example, see: Kirkpatrick CH and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE et al., 1997. Mayo Clin Proc. 72, 175; Morrison VA et al., 1994. Am J Med. 97, 14; Hanto DW., 1995. Annu Rev Med. 46, 381; Senderowlcz AM et al., 1997. Ann Intern Med. 126, 882; Vincenti F. et al., 1998. New Engl. J. Med. 338, 161; Dantal J. etal. 1998. Lancet 351, 623).

Preferably, the immunosuppressive regimen consists in the administration of an immunosuppressive drug to the subject.

Some examples of suitable immunosuppressive drugs include, but are not limited to, CTLA4-lg, anti CD40 antibodies, anti CD40 ligand antibodies, anti B7 antibodies, anti CD3 antibodies (eg, human OKT3 anti CD3 antibody), methotrexate (MTX), rapamlclna, prednlsona, metll prednlsolone, azathioprene, cyclosporine A (CsA), tacrolimus, clclofosfamide and fludarablna, mlofophenolate mofetllo, dacllzumab [a humanized antibody (lgG1 Fe) anti-chain T alpha IL2R antibodies (lymphocytes with T25 alpha antibodies) (for example, cholera A chain or Pseudomonas toxin).

Preferably, the immunosuppressive drug is capable of blocking the binding of a lymphocyte correceptor to a ligand of the analogous lymphocyte correceptor thereof.

Some examples of suitable drugs capable of blocking the binding of a lymphocyte correceptor to an analogous lymphocyte correceptor ligand include, but are not limited to, CTLA4-lg, anti CD40 antibodies, anti CD40 ligand antibodies, anti B7-1 antibodies or -2 and anti CD28 antibodies.

Such polypeptide drugs are particularly advantageous since these are, unlike the commonly used immunosuppressive drugs such as cyclosporin A, essentially non-toxic and / or non-carcinogenic, and by virtue of passive blockade of Interactions with the cell surface receptor , provide a reversible and temporary immunosuppression in the subject.

Preferably, the drug that is capable of blocking the binding of a lymphocyte correceptor to the ligand of the analogous lymphocyte correceptor thereof is CTLA4-lg. CTLA4-lg is a genetically engineered fusion protein of human CTLA4 and the Fe domain of lgG1. It prevents the activation of T lymphocytes by binding to human B7, which jointly stimulates T lymphocytes through CD28.

An extensive guide for the administration of immunosuppressive drugs such as CTLA4-lg, so that the immunosuppression of a transplant recipient is facilitated, is provided in the literature of the technique (for example, see: Benhamou PY., 2002. Transplantation 73, S40; Najafian N, and Sayegh MH., 2000. Expert Opin Investlg Drugs 9, 2147-57).

Preferably, administration of the immunosuppressive drug to the subject is carried out for a single period of time between 1 and 20 days, more preferably between 1 and 18 days, more preferably between 1 and 16 days and most preferably between 1 and 14 days, as described in Reference Example 1 of the Examples section that follows.

As described and shown in Reference Example 1 of the following section of Examples, the transplantation of a non-syngeneic graft, such as a xenogeneic graft, together with the administration of CTLA4-lg to a normal immunocompetent subject In accordance with the protocol established herein, it can be used to generate structurally and funconally differentiated organs / tissues optimally tolerated by the lymphocytes of the subject.

As such, the method of the present disclosure is superior to all prior art methods for the treatment of disorders by transplantation of non-syngeneic or developing organs / tissues, since it can be carried out satisfactorily by the temporary administration of a blocker of the interaction of the lymphocyte-ligand corrector of the lymphocyte correceptor, instead of the permanent administration of harmful immunosuppressive agents, such as cyclosporin A, the usual method employed in the prior art.

Although the method can be practiced for the treatment of the disorder in a subject of essentially any mammalian species, the method is preferably used for the treatment of the disorder in a human subject.

The method can be used to treat essentially any disorder related to the physiology or pathological morphology of an organ or tissue that is susceptible to treatment through a transplant.

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Such disorders include renal, pancreatic, cardiac, hepatic, hematological, genetic, pulmonary, cerebral, gastrointestinal, muscular, endocrine, bone, neurological, metabolic, dermal, cosmetic, ophthalmological and vascular disorders.

Preferably, the method is used for the treatment of a renal, hepatic, pancreatic, cardiac, genetic and / or hematological disorder.

The method can be used for the treatment of any of the various disorders of such types.

Some examples of renal disorders that can be treated by the use of a renal graft of the present disclosure include acute renal failure, acute nephritic syndrome, analgesic nephropathy, atheroembolic renal disease, chronic renal failure, chronic nephritis, congenital nephrotic syndrome, renal disease in terminal phase, Goodpasture's syndrome, mesangial proliferative glomerulonephritis due to IgM, interstitial nephritis, kidney cancer, kidney damage, kidney infections, kidney lesions, kidney stones, lupus nephritis, membranoproliferative glomerulonephritis, membranoproliferative II glomerulonephritis, nephritis necrotizing, nephroblastoma, nephrocalcinosis, nephrogenic diabetes insipidus, IgA-mediated nephropathy, nephrosis, nephrotic syndrome, polchondrial kidney disease, post-streptococcal glomerulonephritis, reflux nephropathy, renal artery embolism, stenosis of renal artery, renal papillary necrosis, renal tubular acldosls type I, renal tubular acidosis type II, renal infraperfusion and renal venous thrombosis.

Some examples of pancreatic disorders that can be treated by using a pancreatic graft of the present disclosure include type I or type II diabetes.

Preferably the method is used for the treatment of type I diabetes ("diabetes").

Some examples of liver disorders that can be treated by using a liver graft of the present disclosure include a Hepatitis C infection (Rosen HR, 2003, liver cirrhosis, primary sclerosing colangltls (Crlppln JS, 2002. Can J Gastroenterol. 16: 700), hepatocellular carcinoma (Molmentl EP, Klintmalm GB., 2001. J Hepatobillary Pancreat Surg. 8: 427-34), alcoholic liver disease (Podevln P. et al., 2001. J Chir (Paris). 138: 147) and hepatitis B (Samuel D., 2000. Acta Gastroenterol Belg. 63: 197-9).

In the case of cardiac disorders, disorders that can be treated by the use of a cardiac graft of the present disclosure include ischemic heart failure (Pouzet B. et al., 2001. J Soc Biol. 195: 47-9), arrhythmia ventrlcular (Olivari MT, Wlndle JR, 2000. J Heart Lung Transplant. 19: S38-42), heart failure (Koerner MM., 2000. CurrOpin Cardiol. 15: 178-82), congenital heart defects (Spezlall G. et al. ., 1998. Mayo Clin Proc. 73: 923) and cardiac tumors (Michler RE, Goldstein DJ., 1997. Semln Oncol. 24: 534-9).

It will be appreciated that, since a lymphoid graft of the present disclosure can be used to generate well-differentiated and vascularized mesenchymal / stromal lymphoid cells / tissues, transplantation of said grafts can be used for the treatment of any of the various hematological diseases and / or genetic, such as § disorders / deficiencies in coagulation factors such as hemophilia (Liu et al., 1994. Transpl Int. 7: 201) and lysosomal storage diseases / enzymatic deficiencies such as Gaucher's disease (Groth CG et al., Birth Defects Orig Artic Ser. 9: 102-5).

Additionally it will be appreciated that, by allowing the generation of well-differentiated and vascularized lymphoid stromal tissues derived from the graft, transplantation of a lymphoid graft of the present disclosure can be used to allow differentiation of the host-derived lymphoid graft, and therefore can be used for the treatment of a host disease related to a defect in the lymphoid stroma, such as a defect in the lymphoid stroma that results in an alteration in growth and / or in hematological cell differentiation.

By allowing the generation of well-differentiated and vascularized lymphoid grafts, transplantation of a lymphoid graft of the present disclosure can be used for the treatment of any of the various immunity-related disorders.

As used herein, the phrase "immunity related disorder" refers to any disease related to an immunodeficiency, a pathogenic immune response and / or a potentially therapeutic immune response.

The person skilled in the art will appreciate that, by virtue of the ability of lymphoid grafts to support the growth and differentiation of immune effector cells, such as B lymphocytes, T lymphocytes, cytotoxic T lymphocytes (NK), granulocytes, macrophages, as well as hematopoietic stem cells (HSC), a lymphoid graft of the present disclosure, such as a splenic graft of the present disclosure, can provide immune effector functionality, a corrective immunoregulation and support differentiation of various hematopoietic strains, and therefore, transplantation of said graft can be used for the treatment of essentially any disorder related to immunity.

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Some examples of immunodeficiency diseases that can be treated by transplanting a lymphoid graft of the present disclosure include the acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV), severe combined immunodeficiencies ( SCID), such as adenosine deaminase deficiency (ADA) and immunodeficiencies resulting from therapeutic myeloreduction / myeloablation, such as in the context of cancer treatment, such as hematologic malignancies. A lymphoid graft of the present disclosure will allow a therapeutic immune reconstitution of an immunodeficient subject in such contexts. The usual skilled artisan will possess the expertise necessary for the treatment of an immunodeficiency disease by transplanting a graft of a lymphoid organ / tissue of the present disclosure in accordance with the teachings of the present invention and will have a broad guide available for carrying to practice this aspect of the method of the present disclosure in the literature of the technique (see, for example: FischerA. et al., 1998. Springer Semin Immunopathol. 19: 479-92; Kane L. et al., 2001. Arch Dis Child Fetal Neonatal Ed. 85: F110; Horwitz ME., 2000. PediatrClin North Am. 47: 1371; Friedrich W., 1996. Ann Med. 28: 115-9; Parkman R., 1993. Leukemia. 7: 1100-2). The usual skilled artisan will appreciate that this aspect of the method of the present invention will be used for the treatment of any of the various infectious diseases in a subject whose own immune system does not create adequate protective immune responses. Such infectious diseases include those caused by microbial pathogens, such as viruses, bacteria, mycoplasmas, protozoa, fungi, and the like.

Some examples of diseases related to a potentially therapeutic immune response also include neoplasms. The usual skilled artisan will possess the expertise necessary for the treatment of a hematological and / or genetic disease by transplanting a graft of a lymphoid organ / tissue of the present disclosure in accordance with the teachings of the present invention and will have a broad guide available. To implement this aspect of the method of the present invention in the literature of the technique (see, for example: Toungouz M, Goldman M. et al., 2001. Adv Nephrol Necker Hosp. 31: 257-72; Parkman R ., 1993. Leukemia. 7: 1100-2; Porter DL., 2001. J Hematother Stem Cell Res. 10: 465-80).

Some examples of diseases related to pathogenic immune responses include autoimmune diseases. By providing the transplantation of immunoregulatory immune effector cells of a suitable lymphoid graft of the present disclosure, such as those that include suitable suppressor T lymphocytes, it can be used for suppression of pathogenic immune responses. The usual skilled artisan will possess the expertise necessary for the treatment of a disease related to a pathological immune response by transplanting a graft of a lymphoid organ / tissue of the present disclosure in accordance with the teachings of the present invention and will have extensive guidance. available to implement this aspect of the method of the present disclosure in the literature of the technique (see, for example: Toungouz M, Goldman M. et al., 2001. Adv Nephrol Necker Hosp. 31: 257-72; Moore J, Brooks P., 2001. 23: 193-213).

After transplantation, growth and / or differentiation of the graft and the effect can be advantageously controlled.

Therapeutic graft.

For example, as described in Reference Example 1 of the following Examples section, the functionality of a renal graft of the present disclosure can be advantageously controlled after transplantation by an analysis of fluid production by the graft, in particular by analyzing the specific metabolites of urine or by-product content in said fluid. Supraplasmic concentrations of urine specific by-products, such as, for example, urea nitrogen and creatinine, are indicative of graft functionality.

As described in Reference Example 5 of the following Examples section, the functionality of a pancreatic islet graft can be advantageously controlled by analyzing serum glucose levels. The normalization of serum glucose levels in the serum of a diabetic subject after transplantation of a graft from a pancreatic islet is indicative of the functionality of the graft (i.e., of physiologically regulated insulin secretion by the graft).

The functionality of a splenic graft of the present disclosure can be easily controlled after transplantation through numerous tests routinely performed by the skilled artisan, including through the analysis of appropriate liver metabolism specific proteins in the subject's serum.

The functionality of a cardiac graft of the present disclosure can also be conveniently controlled after transplantation through numerous methods carried out by the usual skilled artisan, including an echocardiogram, an electrocardiogram and the analysis of specific cardiac function proteins. in the subject's serum, depending on the application and the purpose.

Likewise, the functionality of a lymphoid graft of the present disclosure can be controlled in a similar manner after transplantation, depending on the application and the purpose, through numerous methods practiced by the usual skilled artisan routinely, for example, by collecting peripheral blood cells of the subject and analyzing them with respect to the appropriate cell types by means of a fluorescence activated cell classification (FACS), through the appropriate lymphocyte stimulation assays with specific antigens and through the assay of the appropriate cytokines, chemokines, antibodies,

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coagulation factors, lysosomal storage enzymes, and the like, in the subject's serum through an ELISA, or through the testing of said molecules in the subject's blood cells through an FACS.

After transplantation, the immune tolerance of the subject to the graft, and / or the growth and differentiation of the graft can be advantageously controlled.

Various methods can be used for the evaluation of the immune tolerance of the graft subject.

For example, tolerance can be assessed by controlling the infiltration of specific leukocytes of the subject or of specific graft lymphocytes, by controlling the origin of the graft vasculature, and / or by controlling the histological appearance of specific structures. of the organ or tissue. Said control can be carried out advantageously as described in Reference Example 1 of the following Examples section. Infiltration into the graft of leukocytes, neutrophils, cytotoxic T lymphocytes or T lymphocytes of the subject, or the absence thereof, are usually indicative of a suboptimal or optimal graft incorporation into the subject, respectively.

In cases where the tolerance of the subject to the graft is suboptimal, a therapeutic adjuvant therapeutic immunosuppressive treatment of the subject can be advantageously carried out, as described hereinbefore.

In carrying out the present invention, it was unexpectedly found that the organs / tissues in defined differentiation phases corresponding to a specific gestation phase that did not express or present a molecule capable of stimulating or potentiating an immune response before and / or after transplanting them into a receptor, they were able to generate structurally and functionally differentiated organs / tissues tolerated optimally by non-syngeneic lymphocytes when they are transplanted into a subject.

Therefore, in accordance with a further aspect of the present disclosure, a method is provided for the evaluation of the suitability of a mammalian organ or tissue in a differentiation phase corresponding to a specific gestation stage for the transplantation of a grafting of the organ or tissue in a mammalian subject.

The method is preferably carried out by evaluating a test transplant taken from the organ or tissue to check the expression or presentation of the molecule capable of stimulating or enhancing an immune response (hereinafter "the molecule" ) in the subject before and / or after the transplant of the test transplant in a mammalian test recipient.

In accordance with the teachings of the present disclosure, a test transplant that does not express or substantially present the molecule before and / or after the transplant of the test transplant in the test recipient will be optimal for transplantation. In general, the lower the level of expression or presentation of the molecule in the test transplant, the more suitable the organ or tissue graft will be for transplantation. In particular, the lower the level of expression or presentation of the molecule in the test transplant, the more optimally the graft will be structurally differentiated, functionally differentiated and well tolerated by non-syngeneic lymphocytes after transplantation in the subject.

It will be appreciated that since test transplants in the differentiation phases corresponding to the various gestation phases can be tested to check the expression of the molecule, the method according to this aspect of the present disclosure allows the identification of a phase of optimal differentiation of the organ or tissue for transplantation of a graft thereof in the subject.

In accordance with the teachings of the present disclosure, the test transplant test to check the presence of the molecule is preferably carried out before the transplant of the test transplant in the test recipient, and / or after a period after the Transplant selected from an interval of 1 second to 45 days, depending on the type of molecule tested, as described in more detail below.

The method according to this aspect of the present disclosure can be implemented by using a test receiver of any of the various mammalian species, and / or showing any of the various characteristics, depending on the application and the finish.

In accordance with the teachings of the present disclosure, the test receiver is preferably a rodent, and / or the subject.

Preferably, the rodent is a mouse.

The use of a mouse as a test receiver is very advantageous because this species, for many reasons, is by far the most convenient, economical and effective experimental mammal available.

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In accordance with the additional teachings of the present disclosure, the test recipient is a carrier of functional human T lymphocytes.

Preferably, human T lymphocytes are non-syngeneic with the organ or tissue.

As described and illustrated with emphasis on Example 1 of the following Examples section, the method can be effectively implemented by transplanting a human test transplant into a test recipient that carries non-slnggenic human T lymphocytes with The organ or tissue.

Therefore, the method can be used for the determination of the phase of differentiation or gestation of an organ or of a human tissue optimal for the transplantation of said organ or tissue in an allogeneic human subject.

Therefore, the method of evaluating the phase of differentiation of a graft more suitable for the transplantation of the present disclosure is unique and optimal with respect to all prior art methods, and can be conveniently used for the identification of the phase. of differentiation or gestation of essentially any type of organ or tissue optimally suitable for therapeutic transplantation of a graft thereof in a human being.

Although the method according to this aspect of the present disclosure can be practiced by using a graft derived essentially from any mammalian species, the organ or tissue is preferably a porcine organ or tissue, more preferably an organ or a human tissue

According to the teachings of the present disclosure, the method according to this aspect of the present disclosure can be advantageously carried out by using an organ or a human tissue in a specific phase of differentiation selected to correspond with 5 and 16 weeks of gestation, more preferably with between 6 and 15 weeks of gestation, more preferably with between 7 and 14 weeks of gestation, more preferably with between 7 and 9 weeks of gestation and most preferably with 8 weeks of gestation.

As described and illustrated in Reference Example 1 of the following Examples section, the method can be effectively implemented by using an organ or human tissue in a differentiation phase.

corresponding to 8 weeks gestation.

Alternatively, the method can be advantageously carried out by using an organ or a pig tissue in a specific phase of differentiation selected to correspond between 20 and 63 days of gestation, more preferably with between 20 and 56 days of gestation. , more preferably with between 20 and 42 days of gestation, more preferably with between 20 and 35 days of gestation, more preferably with between 20 and 28 days of gestation, more preferably with between 24 and 28 days of gestation and most preferably with between 27 and 28 days gestation.

The method of evaluating the phase of differentiation of a graft suitable for transplantation of the present disclosure can be carried out by testing the test graft to check the various types of molecules capable of stimulating or enhancing an immune response in the subject.

Some examples of such types of molecules include cytokines, chemokines, inflammatory mediators, immune cell receptors, immune cell co-receptors, MHC molecules, antigen presenting molecules, adhesion molecules, innate immunity mediators, apoptosis mediators, metalloproteinases , immunomodulators, lymphocyte co-receptors and lymphocyte co-receptor ligands.

Preferably, a graft suitable for transplantation does not express or substantially exhibit a lymphocyte correceptor or a ligand of a lymphocyte correceptor.

Examples of such lymphocyte co-receptors and lymphocyte co-receptor ligands include CD28, B7-1 (CD80), B7-2 (CD86), CD40, CD40L (CD40 ligand, CD154), CD2, CD58 [lymphocyte function associated antigen 3 (LFA-3)], intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function associated antigen 1 (LFA-1).

Preferably, a graft suitable for transplantation does not express or substantially present B7-1, more preferably CD40 or CD40L, more preferably CD40 and CD40L and most preferably B7-1, CD40 and CD40L.

As mentioned hereinbefore, the test transplant test to check the presence of the molecule is preferably carried out before the transplant of the test transplant in the test recipient, and / or after a period after the Transplant selected from an interval of 1 second to 45 days, depending on the type of molecule tested.

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Preferably, the test transplant test to verify the presence of CD40 or CD40L is carried out both before and after the transplant of the test transplant in the test receiver.

Preferably, the test transplant test to verify the presence of CD40 after the transplant of the test transplant in the test receiver is carried out after a period after the selected test transplant in a range of between 1 second and 45 days. , more preferably between 11 days and 45 days, more preferably between 11 days and 42 days, more preferably between 11 days and 31 days and most preferably between 14 days and 28 days.

Preferably, the test transplant test to verify the presence of CD40L after the transplant of the test transplant in the test recipient is carried out after a period after the selected test transplant in a range of: between 1 second and 45 days ; more preferably between 11 days and 45 days; more preferably between 14 days and 45 days; more preferably between 14 days and 31 days; more preferably between 17 days and 31 days or between 14 days and 28 days; more preferably between 25 days and 31 days; more preferably between 27 days and 29 days; more preferably between 27.5 days and 28.5 days; and most preferably 28 days.

Preferably, the test transplant test to check the presence of B7-1 is carried out before the transplant of the test transplant.

As described and shown in Reference Example 1 of the following Examples section, grafts that do not express substantially B7-1, CD40 and CD40L in the respective optimal test transplant periods prior to transplantation / after transplantation set forth above In this document, they are suitable for transplantation. In particular, said grafts can generate structurally and functionally differentiated organs / tissues optimally tolerated by non-syngeneic / alloreactive human lymphocytes.

Without being bound by any paradigm, the present inventors are of the opinion that a graft that does not express or substantially present said molecules, which are the main cell-specific antigen presenting molecules, is optimally tolerated by an allogeneic or xenogeneic subject at least partially as a result that said grafts substantially lack the antigen presenting cells that have been proposed in the art as critical for the activation of graft rejection.

Numerous methods, well known to the usual skilled artisan, can be used to analyze an organ, tissue or cells, such as the test graft or a portion thereof, to check the expression or presentation of a specific molecule.

In cases where the molecule is a protein or an RNA molecule, the expression or presentation of the molecule is preferably evaluated by analyzing the cells or tissues to check for the presence of the mRNA encoding the protein molecule. or to check the presence of the RNA molecule, respectively.

The analysis of an mRNA or RNA molecule in cells or tissues is preferably carried out through an analysis by RT-PCR. The analysis by RT-PCR can be carried out advantageously as described and illustrated in Example 1 of the following Examples section. Alternatively, analysis of the presence of an mRNA or RNA molecule in cells can be carried out by modifications of the RT-PCR protocol described in Reference Example 1 of the following Examples section (for example, by the use of a PCR with internal primers, of a competitive RT-PCR, etc.), by a Northern blot or by a microarray analysis.

Alternatively, the expression or presentation of the protein molecule can be detected directly by direct detection of the protein molecule by the use of various biochemical techniques.

The various methods for detecting the expression or presentation of a specific protein in an organ, in a tissue or in cells are well known to those of ordinary skill in the art. Such methods include immunofluorescence flow cytometry, Western blot analysis, in situ fluorescence hybridization (FISH), enzyme immunoadsorption assay (ELISA), microarray hybridization, immunofluorescence confocal microscopy, and the like.

In cases where the molecule is a protein, the expression or presentation of both the molecule and the mRNA encoding the molecule is tested.

In cases where the molecule is a protein, an optimal graft is one that does not substantially express the molecule, more preferably the mRNA encoding the molecule, or most preferably neither the molecule nor the mRNA encoding the molecule.

According to the teachings of the present invention, the evaluation of the suitability of the graft for transplantation can advantageously comprise graft analysis to check the expression or presentation of some

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substantially lower levels than in the adult organ / tissue grafts of the immunity-related molecules listed in Table 3 of the following Examples section and in the World Web / Internet Wlde (
http://www.weizmann.ac.il/immunology/reisner/immunogenlclty.xls).

As described and illustrated in Reference Example 1 of the following Examples section, Grafts expressing substantially lower levels of said immunity-related molecules than Grafts / tissues grafts in adult phase are more suitable for their transplant than the latter. An Graft that expresses or shows substantially lower levels than the Grafts of organs / tissues in adult phase of Grafts of type organ or tissue with the largest possible number of such molecules related to immunity, may be optimally suitable for transplantation.

Preferably, the analysis of the expression or presentation of said graft-related immunity molecules is carried out by microarray hybridization analysis of the graft derived mRNA, preferably as described and illustrated in Example 1. from the following section of Examples.

Alternatively, the analysis of the expression or presentation of said graft related Immunity molecules can be carried out by using any of the analytical techniques described hereinbefore for Graft analysis to check the expression or the presentation of the molecule capable of stimulating or potentiating an immune response in the subject.

Therefore, the present disclosure can be used to optimally treat disorders by the use of transplantation of grafts derived from an organ or non-syngeneic or developing tissue, and for the identification of grafts of optimally suitable organs or tissues to lead to Practice a therapeutic transplant.

The therapeutic transplant method of the present disclosure is unique and drastically superior with respect to all other prior art methods, since it allows for the first time a generalized optimal treatment of a wide range of disorders susceptible to therapeutic transplantation in humans by the transplantation of an allograft and / or a xenograft without immunosuppression or with minimal suppression of the graft recipient, through the use of developing xenogeneic or allogeneic grafts that have, respectively, essentially a limited supply or are of essentially any allogeneic origin.

It is expected that during the life of this patient many relevant medical diagnostic techniques will be developed, and that the scope of the phrase "method of evaluation of the phase of differentiation of an organ of a mammal more suitable for transplantation of the same in a mammalian subject "pretend a priori to include all such new technologies.

The objects, advantages and new additional features of the present invention will be apparent to the person skilled in the art after the analysis of the following examples, which are not intended to be limiting. Adlclonally, each of the various embodiments and aspects of the present invention as described above herein and are claimed in the section of claims that follow, find experimental support in the following examples.

Reference Examples

Reference is now made to the following examples, which, together with the above descriptions, illustrate the invention in a non-limiting manner.

Generally, the nomenclature used in the present document and in the laboratory procedures used in the present invention includes molecular, biochemical, mlcroblologlic and recombinant DNA techniques. These techniques are explained in depth in the bibliography. See, for example, "Molecular Clonlng: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Blology" Volumes I - III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wlley and Sons, Baltimore, Maryland (1989); Perbal, "A Practlcal Gulde to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recomblnant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Coid Sprlng Harbor Laboratory Press, New York (1998); the methodologies as set forth in US Pat. No. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Blology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols ¡n Immunology" Volumes I - III Coligan J. E., ed. (1994); Stltes et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mlshell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); The available immunoassays are widely described in the patent and scientific literature, see, for example, US Pat. No. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Ollgonucleotide Synthesls" Galt, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D. and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D. and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immoblllzed Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Clonlng" Perbal, B., (1984) and "Methods ¡n Enzymology" Vol. 1 - 317, Academlc Press; "PCR Protocols: A Gulde To Methods And

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Applications ", Academic Press, San Diego, CA (1990); Marshak et al.," Strategies for Protein Purification and Characterization - A Laboratory Course Manual "CSHL Press (1996). Other general references are provided throughout this document. It is believed that the procedures thereof are well known in the art, and are provided for convenience to the reader.

REFERENCE EXAMPLE 1

Transplantation of human or porcine renal organs / tissues at an early stage of pregnancy generates structurally and functionally differentiated renal organs / tissues tolerated by human alloreactive / xenoreactive lymphocytes

Organ / tissue diseases for which the transplantation of an organ / tissue from an allogeneic donor remains the optimal therapeutic option, such as kidney disease, is very debilitating and is related to significant mortality rates. However, transplantation of an allogeneic donor organ / tissue is often impossible to implement due to the difficulty in finding a donor of a haplocompatible organ / tissue. In addition, even when a compatible donor is found, in order to prevent graft rejection, such a transplant requires permanent immunosuppression of the graft recipient, usually through the administration of toxic immunosuppressive drugs, such as cyclosporine A. Such immunosuppressive treatments contribute to the disadvantages of allogeneic transplantation, since these are often unsuccessful in preventing graft rejection in the short term, and are usually unable to prevent graft rejection indefinitely. An alternative to allograft transplantation involves the transplantation of xenografts, particularly porcine grafts, which are considered the optimal animal alternative to human grafts. However, xenografts generally cannot be used for transplantation due to the very high suboptimal tolerance of such grafts by human lymphocytes. Therefore, suitable organs / tissues for a non-syngeneic therapeutic transplant in humans and tolerated by human lymphocytes, and suitable sources of such organs / tissues are highly desired. A proposed strategy to provide such organs / tissues involves the use of grafts in early stages of development, since it has been shown that the earlier the stage of development of an organ / tissue is, the better tolerated it will be when transplanted into a host not syngeneic. However, to date, satisfactory growth and differentiation of grafts of a developing or non-syngeneic organ / tissue has not been achieved nor a satisfactory immunological tolerance of said grafts by human lymphocytes in the absence of graft-derived teratomas.

In the conception of the present invention, it was hypothesized that there is a developmental phase during which the organs / tissues are sufficiently differentiated to be involved in the specific development of an organ / tissue in the absence of graft derived teratomas, still undifferentiated enough to be optimally tolerated when they are transplanted into a non-syngeneic host. In carrying out the present invention, it was unexpectedly discovered the existence of specific gestation phases during which human or porcine organs / tissues can be transplanted into a host so that they generate, in the absence of graft-derived teratomas, organs / structurally and functionally differentiated graft derived tissues that are optimally tolerated by human alloreactive / xenoreactive host lymphocytes. In particular, the existence of specific gestation phases was unexpectedly discovered during which human or porcine renal organs / tissues can be transplanted into a recipient so as to generate, in the absence of graft-derived teratomas, optimally structurally differentiated renal organs and functionally that they are optimally tolerated by human alloreactive / xenoreactive receptor lymphocytes, as described below and / or as previously described (Dekel B. et al., 2002. Nature Medicine).

Materials and methods:

Preparation of murine hosts for transplantation: three-month-old Balb / c mice (Harían Olac, Shaw’s Farm, Blackthorn, Bicester, Oxon., United Kingdom) were used as hosts for transplant studies. For the generation of immunodeficient receptors, Balb / c mice were mortally irradiated by divided doses of a total body irradiation (TBI; 3.5 Gy followed 3 days later by 9.5 Gy) with a 150-A gamma ray source (60) Co (produced by the Atomic Energy Commission of Canada, Kanata, Ontario) with a focal length of the skin of 75 centimeters and a dose rate of 0.7 Gy / minute, as previously described (Lubin 1. et al., 1994. Blood 83, 2368; Reisner, Y. and Dagan, S., 1998. Trends Biotechnol. 16, 242-246; Segall, H. et al., 1996. Blood 88, 721-730). Bone marrow cells of the NOD / SCID mice (Weizmann Institute Animal Breeding Center, Rehovot, Israel) were extracted from the diaphysis of the femur and tibia of mice 4-8 weeks old, as previously described (Levite M et al., 1995. Cell Immunol. 162, 138). Balb / c receptor mice were immunologically reconstituted with 3 X 10 6 bone marrow cells from NOD / SCID mice administered intravenously in 1 milliliter of phosphate buffer saline one day after the second fraction of total body irradiation ( TBI), as previously described (Reisner, Y. and Dagan, S., 1998. Trends Biotechnol. 16, 242-246; Segall, H. et al., 1996. Blood 88, 721-730). The resulting SCID mouse (severe combined immunodeficiency) animals have been shown to allow excellent incorporation of hematopoietic cells or viable human solid tissues (Marcus H. et al., 1995. Blood 86, 398; Segall H. et

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al., 1996. Blood 88, 88; Reisner Y. and Dagan S., 1998. Trends Biotechnol. 16, 242; Bocher WO. et al., 2001. Eur J Immunol. 31,2071). NOD / SCID donor mice were obtained from the Weizmann Institute Animal Breeding Center, Rehovot, Israel and animal experiments were carried out according to the National Instltutes of Health Gulde for Care and Use of Experimental Animáis and were approved by the Weizmann Institute of Science Animal Care Commlttee.

Removal of developing renal tissue: developing human renal tissues were obtained by scraping with the approval of a Helsinki committee and developing renal tissues were dissected surgically from embryos under a dissecting stereoscope as previously described ( Rogers S. et al., 1998. Kidney Int. 54, 27). Samples of adult kidneys were obtained from normal kidneys extracted from a clear cell carcinoma in phase I. Porcine renal tissues in the gestation phase and adults were obtained with the help of the Lahav Institute for Animal Research, Kibbutz Lahav. The porcine renal tissues in the gestation phase were isolated from animals by using the techniques previously described (Rogers S, et al., 1998. Kidney Int. 54, 27). All samples intended for an analysis of gene expression were deep frozen in liquid nitrogen. Transplant tissues were maintained under sterile conditions at 4 ° C (for approximately 2 hours) either in RPMI 1640 or in Dulbecco-modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Biological Industries, Beit HaEmek, Israel).

Transplantation of developing renal tissue: Transplantation of renal tissue under the renal capsule of recipient mice was performed as previously described (Dekel, B. et al., 1997. Transplantation 64, 1541-1550). In the transplants, complete human renal precursors (up to 8 weeks gestation) or pigs (up to 4 weeks gestation), or complete renal tissues or fragments of 1-2 mm in diameter were used in the late gestation phases. The transplant was carried out 7-10 days after reconstitution of the irradiated hosts with the bone marrow of the NOD / SCID. For growth assays, renal tissues were transplanted into SCID receptor mice. For transplantation, the renal tissues were maintained under sterile conditions at 4 ° C for approximately two hours either in RPMI 1640 or in medium of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FCS; Biological Industries, Beit Haemek, Israel). Kidney tissue transplantation was performed under general anesthesia induced by intraperitoneal injection of 2.5% of Avertin in phosphate buffer saline (10 milliliters per kilogram of body weight). Both host kidneys were exposed through a bilateral incision, a 1.5 mm incision was made at the caudal end of the renal capsule and a fragment of approximately one cubic millimeter of renal tissue was implanted under each renal capsule. Kidney tissues were also transplanted intra-abdominally to control the specific immune support of the renal subcapsular space. In some experiments the renal tissues were implanted and sutured (5-0 suture) in the testicular fat pad along with a left nephrectomy. The transplanted mice were treated postoperatively with ciprofloxacin in their drinking water for 7 days.

Administration to human PBMC mice: between one and three days after kidney tissue transplantation, as described above, 108 human PBMCs were injected intraperitoneally into host mice. Control mice did not receive human BMCs. The generation of human PBMCs, their infusion into recipient mice and the analysis of the incorporation of the infused cells were carried out as previously described (Segall, H. ef al., 1996. Blood 88, 721-730) . Human PBMCs were generated from lymphocyte layers obtained from normal volunteers, as follows. Blood samples were placed in a pad of Lymphoprep solution (Nycomed, Oslo, Norway) and centrifuged at 2,000 rpm for 20 minutes. The interfacial layer was collected and washed twice and the cells were counted and resuspended in phosphate buffer saline (at pH 7.4) at the desired concentration. For the analysis of human lymphocyte incorporation, cells were recovered from the peritoneum 10 to 14 days after PBMC infusion. Individual cell suspensions were incubated for 30 minutes on ice with labeled human anti-CD3-PE and CD45-PerCP antibodies (panhuman leukocyte antigen) (Becton-Dickinson, Mountain View, CA). After washing, two or three color fluorescent analyzes of these human antigens were carried out using a FACScan analyzer (Becton-Dickinson). Data from lymphocytes selectively classified by their standard forward and lateral scattering characteristics were collected.

In some experiments, double infusions of PBMC from different human donors were administered to the graft recipient mice.

Analysis of infiltration, growth and differentiation of the graft: the infiltration of human immune cells, as well as the growth and development of grafts derived from renal tissue in the glomeruli and mature tubules, were monitored after transplantation as follows . Graft recipients were sacrificed 4-10 weeks after transplantation, as indicated. The recipient's kidneys and capsules were then removed and fixed in 10% paraffin. The transplants were sectioned and mounted on slides coated with poly-L-lysine and the sections were stained with hematoxylin and eosin (H&E). For the determination of graft growth from the moment of transplantation until the time of extraction, the graft size was measured before the transplant and at the time of the extraction (after the transplant) and the graft size ratios were calculated between before the transplant and after the transplant. The graft size was determined according to the graft size formula = L x W, in which L and W represent the major and

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minor graft, respectively. The graft development evaluation was carried out by counting the number of glomeruli and mature tubules in 10 consecutive microscopic fields (magnification x 40) per transplant in 3 transplants per group. The determination of infiltration of human T lymphocytes in the Graft sections was determined as previously described (Naveh MT. Et al., 1992. J Clin Invest. 90, 2434). In summary, the graft sections were immunostained with rabbit anti human CD3 antibody (Zymed, San Francisco, CA; T-cell pan), as previously described (Dekel, B. et al., 1999. Int. Immunol. 11, 1673-1683) and the number of human CD3 positive cells in 10 consecutive microscopic fields (magnification of x 100) per transplant in 3 transplants per group was counted. The paraffin tissue blocks of the transplants were cut at 4-6 pm thick, deparaffinized with xylene, rehydrated and placed for 15 min in ethanol containing 3% H2O2 to block endogenous peroxidase. The slides were washed thoroughly with tap water and transferred to PBS. The sections were then treated with 1% bovine serum albumin to prevent background staining and incubated for 1 hour with anti-CD3 antibody at room temperature in a humidifying chamber. The slides were rinsed with phosphate buffer saline for 3 minutes and incubated with a biotinylated anti-mouse antibody for 30 minutes, and then incubated with peroxidase-conjugated streptavidin (StrAvigen; Biogenex, San Ramón, CA) for 30 minutes. After rinsing, the peroxidase marker was visualized by incubation for 15 minutes and stained with Mayer's hematoxylin using an immunohistochemical staining kit (Biomeda, Foster City, CA), according to the manufacturer's instructions. The 3-amino-9-ethylcarbazole reagent produced a red product that is soluble in alcohol and can be used with an aqueous mounting medium (Kaiser glycerol gelatin). A negative control for the staining of T lymphocytes was performed following all the steps mentioned above but omitting the addition of the primary antibody. Staining was found to be uniformly negative in the transplants of control mice not infused with human PBMC.

Vascularization analysis of the host vessels: five micrometer thick paraffin sections were immunostained with a rat antibody specific for mouse PECAM-1 (Pharmingen, San Diego, California) by using a Histostain Plus kit (Zymed, San Francisco, California), according to the manufacturer's instructions. Vessels were counted in similar regions of the renal grafts according to the high power fields (5 consecutive fields per transplant in 5 transplants per group).

Urine collection and analysis: the fluid produced by the cysts derived from the graft of renal tissue in early gestation phase was collected and analyzed to check the content in urinary markers, as follows. Human and porcine developing transplants were surgically excised from anesthetized mice through a middle incision or on the left flank and a plastic microcatheter was inserted into an identifiable area of fluid concentration. At the insertion site, the graft walls were sutured around the catheter by using a 5-0 nylon suture and the graft fluid was collected through the microcatheter in small volume PCR tubes sutured in the skin of the mice. The drained fluid was subsequently analyzed to check the concentrations of urea nitrogen and creatinine.

Analysis by RT-PCR of the expression of the costimulant molecule in the grafts: among the multiple costimulatory routes identified, more and more evidence suggests that the interaction of the costimulant receptor ligands of the CD28 and CD40 T lymphocytes (CD40L, CD154) with their respective B7-1 / -2 and CD40 ligands expressed in antigen presenting cells are critical for T cell responses to alloantigens (Sayegh MH. And Turka LA., 1998. N Engl J Med. 338 , 1813). Therefore, experiments were carried out to test whether the alloreactive human immune cells did not reject allograft derived human allograft tissue grafts in a gestation phase of between 7 and 8 weeks as a result of the expression regulated by decrease by said tissues of costimulant molecules B7-1, B7-2, CD40, CD40L and HLA-DR, as follows. Messenger RNA from grafts derived from renal tissue was analyzed in a gestation phase of 8, 14 and 22 weeks through RT-PCR at the following time points: (i) before transplantation; (ii) immediately after the transplant but before the infusion of the alloreactive human PBMCs; and (iii) 2, 4 and 6 weeks after reconstitution of the mice with human PBMCs, as follows. The grafts were carefully dissected from the subcapsular implantation site and total RNA was isolated from the dissected grafts, as previously described (Dekel, B. et al., 1999. Int. Immunol. 11, 1673-1683). In summary, the renal graft tissues were homogenized with a glass-Teflon tissue homogenizer in Tri- reagent (Molecular Research Center Inc., Cincinnati, OH) for total RNA isolation, according to the manufacturer's instructions. The isolated RNA was air dried, resuspended in nuclease-free water and quantified by spectrophotometry. 1 microgram aliquots of the total RNA preparation in cDNA with AMV reverse transcriptase were re-transcribed using an Access RT-PCR kit (Promega Corp., Madison, Wisconsin), according to the manufacturer's instructions. The specific sequences of the costimulatory molecules and the constitutive gene of the (3-actin control (Pratt, JR et al., 2002. Nature Med. 8, 582-587) were subsequently amplified by PCR from the synthesized cDNA, as follows: In summary, the reverse transcription cDNA product was diluted to 1:50, 1: 100 and 1: 500 in sterile water and amplification was carried out by PCR using Tfl DNA polymerase thermostable in a 50 microliter reaction mixture containing 40 micromolar of each dNTP, 0.4 micromolar of each primer (Table 1), 10 millimolar of Tris HCI (at pH 8.3) and 1.5 millimolar of MgCh. The sample was tested at least three times, and the compared tissue samples were amplified by a parallel PCR using a single master reagent mixture In order to minimize non-specific amplification of non-target sequences,

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established a high temperature of PCR hybridization (64 ° C), and, in order to detect the PCR signals in the linear phase of product amplification, the PCR reaction was carried out with 20-35 thermal cycles . In all experiments, the possibility of amplification of contaminating DNA through control reactions was ruled out through the use of reverse transcription reactions in which reverse transcriptase or template cDNA was omitted. The products of the PCR reaction were electrophoretically separated into 1.5% agarose gels, the gels were stained with ethidium bromide and photographed using a UV dlafanoscope, as previously described (Sharma VK. Et al. ., 1996. Transplantation 62, 1860).

Transplantation of grafts of xenogenic renal tissue in early gestation phase in normal immunocompetent mice together with a mild blockage of short-term costimulation: to test the immunogenicity of grafts of xenogeneic renal tissue at an early gestation stage in an immunocompetent normal mammal , immunocompetent Balb / c mice were transplanted with grafts of porcine renal tissue in a gestation phase between 27 and 28 days, as described above for graft transplantation in immune reconstituted mice, and underwent a mild blockage of the costimulation by an intraperitoneal injection of 250 micrograms of CTLA4-lg (kindly provided by Steffen Jung, Hadassa Medical School, Jerusalem, Israel) every 48 hours for 2 weeks. CTLA4-lg is a fusion protein that comprises the extracellular portion of mouse CTLA-4 fused with the constant region of human IgG, which blocks the costimulant interaction of the CD28 costimulant receptor of T lymphocytes with their costimulant cell ligands presenters of the B7-1 and -2 antigen. Control mice were injected with phosphate buffered saline or a control immunoglobulin.

Table 1. Oligonucleotide primers used for amplHicadon by a cDNA PCR prepared from ____________________________ of developing human renal tissues .____________________________

 Amplified sequences

 Oligonucleotide primers * (sense / antisense) PCR product length (bp)

 B7-1

 5-GACCAAGGAAGTGAAGTGGC-3 ’(SEQ ID N °: 1) / 5-AGGAGAGGTGAGGCTCTGGAAAAC-3’ (SEQ ID N °: 2) 410

 B7-2

 5-CACTATGGGACTGAGTAACATTC-3 ’(SEQ ID NO: 3) / 5-GCACTGACAGTTCAGAATTCATC-3’ (SEQ ID NO: 4) 383

 CD40

 5’-CTCTGCAGTGCGTCCTCTGGGG-3 ’(SEQ ID NO: 5) / 5-GATGGTATCAGAAACCCCTGTAGC-3’ (SEQ ID NO: 6) 410

 CD40L

 5-TATCACCCAGATGATTGGGTCAGC-3 ’(SEQ ID N °: 7) 1 5-CCAGGGTTACCAAGTTGTTGCTCA-3’ (SEQ ID N °: 8) 349

 HLA-DR

 5’-ATGAAGGTCTCCGCGGCAGCCC-3 ’(SEQ ID NO: 9) 1 5’-CTAGCT CAT CT CCAAAGAGTT G-3’ (SEQ ID NO: 10) 215

 (3-actin

 5’-ACCATCAAGCTCTGCGTGACTG-3 ’(SEQ ID N °: 11) / 5-GCAGGTCAGTTCAGTTCCAGGTC-3’ (SEQ ID N °: 12) 310

 Homology searches for all primer sequences were performed by using the NCBI GenBank database to ensure non-specificity of primers for mouse genes.

Statistical analysis: the comparisons between the groups were evaluated by the Student t test. Data were expressed as the mean ± e.t.m. and were considered statistically significant if P values were 0.05 or less.

Microarray analysis: preparation of the labeled cRNA and hybridization was carried out in a Genechip Human Genome HU95A (Affymetrix) matrix as recommended by the microarray manufacturer. The analysis of Genechip data was carried out as previously described (Zuo, F. et al., 2002. Proc. Nati. Acad. Sci. USA 99, 6292-6297; Kaminski, N. et. al., 2000. Proc. Nati. Acad. Sci. USA 97, 1778-1783). For the clustered analysis, the CLUSTER, GENE CLUSTER and TREEVIEW programs (Eisen, MB et al., 1998. Proc. Nati. Acad. Sci. USA 95, 14863-14868) and the SCOREGENE software package (
http://FGUSheba.cs.hujl.ac.il/). Increment factors for each sample were calculated against the geometric mean of all samples. A detailed description of the scoring methods and the methodology used for the analysis of the microarray data has been published (Kaminski N. and Friedman N., 2002. Am. J. Respir. Cell Mol. Biol. 27, 125 -132).

Experimental results:

Gestational age determines growth and differentiation: an initial experiment was carried out to determine the viability of adult human kidney tissue transplants in immunodeficiency mice. At 8 weeks after the transplant it was found that all adult transplants (5/5) were sclerotic and not viable. To assess the influence of the development phase on the potential for growth and differentiation, human renal tissues were transplanted in a gestation phase between 7 and 14 weeks in immunodeficiency mice (Table 2). Globally, more than 80% of human kidney grafts from donors from

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all ages, and all grafts recovered had increased in size, with no sign of neoplasia in any of the grafts recovered.

Table 2. Transplantation of human or porcine renal organs / tissues in the gestation phase in non-syngeneic human leukocyte receptors ______________________________ ______________________________

 Graft origin

 Graft gestation phase No. of grafts Type of graft Graft growth * Renal differentiation of the graft ** Non-renal differentiation of the graft Necrosis

 Human

 14 weeks 3 full 3/3 none none 3/3

 14 weeks 8 fragments 7/8 7/7 none none

 10 weeks 2 full 2/2 none none 2/2

 10 weeks 6 fragments 6/6 6/6 none none

 8 weeks 5 full 5/5 5/5 none none

 7 weeks 3 full 3/3 3/3 none none

 Porcine

 8 weeks 7 full 5/7 none none 5/5

 8 weeks 6 fragments 6/6 6/6 none none

 6 weeks 5 full 4/5 none none 4/4

 6 weeks 6 fragments 6/6 6/6 none none

 between 27 and 28 days 12 full 12/12 12/12 none none

 between 24 and 25 days 9 full 8/9 5/8 3/8 none

 between 20 and 21 days 9 full 6/9 3/6 3/6 none

 * The growth and differentiation of the transplant were evaluated at 8 weeks after the transplant. ** Differentiation was classified as renal (nephrons only), non-renal (differentiated derivatives other than renal) and necrosis (in addition to nephrons, the appearance of necrotic areas mainly in the center of the transplant).

The results were remarkably different when human kidney grafts were compared in a gestation phase between 7 and 8 weeks with human kidney grafts in a late gestation phase. At 8 weeks after transplantation, grafts derived from renal tissue in a gestation phase between 7 and 8 weeks (n = 8) had experienced remarkable growth (the proportion of transplant size was 20.1 ± 2, 7). A complete nephrogenesis [5.5 ± 0.8 glomeruli and 19.3 ± 2.7 tubules per field (magnification of x 40)] was observed in transplants derived from these grafts, which originally contained primarily mesenchymal stem cells and primordiae urethral, but no glomerulus. The macroscopic morphology and histological appearance of said graft derived from human renal tissue in a gestation phase of 8 weeks, 8 weeks after transplantation, are shown in Figures 1 a-b, respectively. Beyond this time point of gestation, the transplantation of complete fetal kidneys in development resulted in a central necrosis of the Graft and the viability was reduced. Therefore, small fragments of human fetal renal tissue were inserted into immunodeficient hosts, as previously described (Dekel, B. et al., 1997. Transplantation 64, 1550-1558; Dekel, B. et al., 2000 Transplantation 69, 1470-1478). Under identical conditions, the sections of the tissue-derived transplants in a gestation phase between 10 and 14 weeks (n = 14) had significantly smaller transplant size ratios (14.8 + 2.2 and 12.3 ± 1.8, respectively, P <0.01) as well as glomerular and tubular counts (4.2 ± 0.8 and 15.3 ± 2.7; 3.5 ± 0.8 and 11.2 ± 2.2 by HPF, respectively; P <0.05). Therefore, in contrast to the transplantation of more mature human fetal renal fragments, Graft of renal tissues at an earlier stage of pregnancy resulted in optimal growth and nephrogenesis.

The same methodology described above was used to evaluate the growth and differentiation potential of porcine renal tissues during pregnancy (Table 2). In this case, renal transplants of porcine tissue in a gestation phase of between 6 and 8 weeks showed fibrosis and central necrosis and deterioration of the Graft, while the sectioned grafts had a proportion of the transplant size of 10.5 + 2.2 and 8.2 ± 1.2, respectively, at 8 weeks after transplantation. For the characterization of the porcine renal tissues in an early gestation phase, transplants were analyzed in a gestation phase of between 20-21, between 24-25 and between 27-28 days (combined data of 30 transplants). All swine transplants in a gestation phase of 27-28 days showed significant growth, with a proportion of the transplant size at 8 weeks after the transplant of 28.3 ± 4.1 and complete differentiation in mature glomeruli and tubules (7.0 ± 1.0 glomeruli and 35.5 ± 5.1 tubules per high resolution microscopic field). The macroscopic morphology and histological aspects of said graft derived from porcine renal tissue in a 4-week gestation phase are shown in Figures 1 c-d, respectively. On the contrary, six of the nine pig transplants in a gestation phase between 20-21 days failed to develop or had evolved in growths that contain few glomeruli and tubules, but that contain other derivatives

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differentiated, such as blood vessels (Figure 1 e) and cartilage and bone (Figures 1 e-f). Additionally, porcine renal tissues in a gestation phase between 24-25 days were inferior to transplants in a gestation phase between 27-28 days for nephrogenesis, since non-renal cell types and disorganized cell clusters were found in three of the nine transplants (Figures 1 h - j). These findings complement the recent in vitro data (Oliver, JA et al., 2002. Am. J. Physiol. Renal Physiol. 283, F799-809), indicating, both, that early in gestation, the developing kidney contains cells pluripotent progenitors, or renal embryonic stem cells, with the ability to generate many cell types.

Vascularization derived from the specific host of grafts derived from renal tissue at an early gestation stage is associated with an immune tolerance to the graft: the ability of transplants to grow as tissues in non-syngeneic hosts depends primarily on their ability to sustain angiogenesis in said hosts (Gritsch, FI.A. et al., 1994. Transplantation 57, 906-917). In the case of a xenotransplant, the immune barrier is largely conditioned by the way in which the transplant derives its blood supply (Cascalho, M. and Platt, J. L., 2001. Immunity 14, 437-446). To determine the ability of recipient mice to favor the angiogenesis of avascular grafts derived from human or porcine renal tissue at an early gestation stage by interstitial growth of the recipient's vessels, the expression of mouse PECAM (CD31) was analyzed immunohistochemically , a marker of proliferating endothelial cells, of developing transplants. Immunoreactive vessels counts reflecting the combined total number of capillaries and large vessels originating from the host per high resolution microscopic field were performed (Vermeulen, PB et al., 1996. Eur. J. Cancer 32A, 2474-2484 ). At 4 weeks after transplantation, 23.5 + 4.0 and 21.3 ± 3.2 vessels originating from the host were found by high-resolution microscopic field supplying developing human and porcine transplants, respectively. Among these grafts of human and porcine tissue, all external vessels stained positively for mouse CD31 (Figures 2 a and b, respectively). In addition, medium and small size capillaries originating from the host were detected in both glomeruli (Figures 2 c and 2 d) and in the parenchyma (Figures 2 e and f) of human and porcine transplants in the gestation phase. In transplants from mature and vascularized porcine renal tissues in a 16-week gestation phase and 8-week porcine renal tissues, there was a significant reduction in the positive vessel count for mouse CD31 (10.2 ± 1.8 and 11.5 ± 2.2, respectively, P <0.001) formed mainly by larger external vessels, but not by endothelial cells in the glomeruli and small capillaries (Figures 2 gyh). The control sections of vascularized human and porcine renal tissues did not show host-derived vessels positive for CD31 (Figures 2 i-j, respectively). Therefore, recipient mice have a significantly greater contribution to the vasculogenesis of human and porcine renal tissue derivatives at an early gestation stage, including their microcirculation formation.

Human and porcine renal grafts in an early gestation phase differ in functional renal organs that produce diluted urine: grafts derived from renal tissue in an early gestation phase were found to form large fluid-filled cysts. Figures 3 a and b, respectively, represent the appearance of said cysts in grafts derived from human renal tissue in a gestation phase of 8 weeks, and pigs in a gestation phase of 4 weeks, respectively. It is observed that these cysts are formed in particular in abdominal grafts, where their growth was not limited by the renal subcapsular space. To assess whether the fluid of these cysts represented the by-products of renal function, the fluid was collected from grafts cysts derived from human renal tissue in a gestation phase of 8 weeks (n = 2) and swine in a gestation phase of 4 weeks (n = 4) and analyzed for urea nitrogen and creatinine content, 6-8 weeks after transplantation. Since the transplants cannot use the host's excretory system for urine drainage, the fluid was drained by inserting a permanent microcatheter into developing renal grafts. It was found that the average levels (mg / dl) of urea nitrogen and creatinine were higher in the cyst fluid compared to those found in the serum of the transplanted mice (518 ± 169 versus 45 ± 8 and 7 , 2 ± 1.9 versus 0.46 ± 0.048, respectively; P <0.001), indicating that human and pig transplants had produced urine. These results are in agreement with the demonstration that murine renal precursors can develop in functional nephrons (Rogers, SA et al., 1998. Kidney Int. 54, 27-37; Rogers, SA et al., 2001. Am. J Physiol. Regul. Integr. Comp. Physiol. 280, R132-136; Rogers, SA and Hammerman, MR, 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1865 - 1869; Rogers, SA and Hammerman, MR, 2001. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R661-665). The levels of urea nitrogen and creatinine in the cyst fluid were significantly lower compared to natural bladder urine (518 + 169 versus 4,279 ± 402 and 7.2 ± 1.9 versus 54 ± 6 , respectively; P <0.001). The diluted urine of the cyst fluid is compatible with the reduced ability to concentrate the urine of the kidneys at an early gestation stage.

Grafts derived from human or porcine renal tissue at an early gestation stage are less susceptible to human alloreactive / xenoreactive lymphocytes than grafts at a late development stage: the question of whether grafts derived from renal tissue at a gestation stage Early they have an immunological advantage over tissue derived grafts in a late gestation phase addressed as follows. Preliminary experiments to establish the experimental conditions at the initial time demonstrated that the minimum number of infused PBMCs capable of inducing complete rejection of grafts derived from adult human renal tissue grafted into recipient mice was 108 cells (data not shown). Four weeks after the transplant of

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adult human kidney fragments in immunodeficient receptors together with 100 x 106 human-reactive PBMCs, massive lymphocytic infiltration, tissue destruction and rejection were observed, as previously described (Dekel, B. et al., 1997. Transplantation 64, 1541 - 1550; Dekel, B. et al., 1999. Int. Immunol. 11, 1673-1683). At 4 weeks after transplantation, grafts from renal tissue grafts in a 14-week gestation phase showed an average of 39.8 ± 7.8 human donor T lymphocytes per high resolution microscopic field. Despite the presence of T lymphocytes in these grafts, there was no early rejection similar to that of adult transplants and transplant growth occurred in a gestation phase of 14, 10, 8 and 7 weeks during the first month (Dekel, B. etal., 1997. Transplantation 64, 1550-1558; Dekel, B. et al., 2000. Transplantation 69, 1470-1478), as shown in Figures 4 a-d, respectively. However, the analysis of infiltration of T lymphocytes in grafts derived from renal tissue in a 14-week gestation phase at later time points (6-8 weeks after transplantation) revealed the damaging effects of infiltrating cells according to Graft deterioration was noticeable, in the form of the destruction of tubules and glomeruli (Figures 4 e-f, respectively), and the growth of the transplant stopped significantly compared to transplants from animals that had not undergone infusion of human PBMC (Figure 4 a). Similar results were obtained for human renal grafts in a 10-week gestation phase (Figure 4b). On the contrary, after the infusion of 100 x 106 human cells in the peritoneum of the host, grafts derived from renal tissue in a gestation phase of 8 or 7 weeks showed glomeruli and tubules preserved without any infiltration of human T lymphocytes from the donor, (Figures 4 g-h, respectively) and grew in a manner similar to the transplants of control mice (Figures 4 c-d, respectively), and therefore showed no obvious signs of destruction or rejection. In addition, when the experimental protocol was altered so that two inoculums of 100 x 106 alloreactive human PBMCs were infused from different donors 6 weeks apart, the immune cells did not reject the human renal graft in an 8-week gestation phase, but human renal grafts in a 14-week gestation phase transplanted together with the PBMC of the second donor were rejected (Figures 5 a-b, respectively). Therefore, the immunogenicity of the differentiated tissue, which developed over 6 weeks after implantation of the renal tissues in a gestation phase of 8 weeks phase, was still greatly reduced compared to the renal tissues in a gestation phase of 14 weeks

An analysis of the infiltration of T lymphocytes in pig kidney tissue transplants in hosts undergoing an intraperitoneal infusion of 100 x 106 human PBMCs was also carried out. Preliminary experiments for the establishment of the experimental conditions of the initial moment demonstrated that the minimum number of infused PBMCs capable of inducing a complete rejection of grafts derived from porcine adult renal tissue grafted in recipient mice that was 108 (data not shown) . In 3 weeks, five of the six grafts derived from adult porcine renal tissue were infiltrated and histological damage and destruction were appreciable (data not shown). Lymphocyte infiltration, analyzed by staining with FI&E, and destruction of renal parenchymal tissue in the presence of human T lymphocyte infiltrates in the graft derived from renal tissue in adult phase at 4 weeks after transplantation, are shown in the Figures 6 a - b and in Figure 6 c, respectively.

In grafts derived from porcine renal tissue in an 8-week gestation phase, the infiltration of human T lymphocytes into the six grafts was easily detectable, with an average of 40.5 ± 6.75 lymphocytes per high resolution microscopic field, 4 weeks after transplant. Kidney tissue transplants in a gestation phase of between 8 and 6 weeks showed signs of rejection, evidenced by their small size after transplantation with respect to before transplantation (Figures 7 a-b, respectively) compared to derived grafts of renal tissue in a gestation phase between 3 and 4 weeks at 6 or 8 weeks after transplantation (Figures 7 c-d, respectively). The analysis at later time points indicated that five of the six grafts derived from renal tissue in an 8-week gestation phase showed signs of rejection in the form of tissue damage concomitant with infiltration of T lymphocytes, as demonstrated by immunohistochemical staining. with human anti-CD3 antibody and by histochemical staining with H&E (Figures 8 a-b and 8 c, respectively) of graft sections. These results, and similar findings from other experiments with grafts derived from porcine renal tissue in a 6-week gestation phase, demonstrated that xenogeneic human PBMCs induce rejection of porcine renal transplants developing at these stages of organogenesis. In sharp contrast, the grafts derived from porcine renal tissue in a gestation phase of between 28 and 21 days were not rejected (Figures 7 c-d, respectively), nor did these grafts show infiltration of T lymphocytes, as demonstrated for graft derived from porcine renal tissue in a 28-day gestation phase of Figures 9 a-b. Additionally, transplants of renal tissue grafts in a 28-day gestation phase in hosts undergoing a second infusion of 100 x 10® human xenogeneic PBMCs, 4 weeks after transplantation, were not rejected, while renal tissue grafts in an 8-week gestation phase transplanted simultaneously with these cells they were finally rejected (Figures 10 a-b, respectively).

The costimulant molecules of the donor antigen presenting cells are crucial in the alloimmune response (Boussiotis, VA et al., 1996. J. Exp. Med. 184, 365-376; Schwartz, RH, 1996. J. Exp. Med 184, 1-8; Sayegh, MH and Turka, LA, 1998. N. Engl. J. Med. 338, 1813-1821; Li, Y. etal., 1999. Nature Med. 5, 1298-1302). Therefore, mRNA expression of costimulant molecules B7-1, B7-2, CD40, CD40L and HLA-DR was analyzed before and after transplantation of developing human renal tissue in the absence and in the presence of

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Alloreactive human lymphocytes. The results obtained show the differential expression of the co-formulating molecules in both human renal tissues both normal and transplanted adults in different stages of pregnancy, with a clear deficiency (especially of CD40 and B7-1) in tissue derived transplants

Renal in a phase of gestation of 8 weeks with respect to those of 14 and those of 22 weeks (Figures 11 a - c,

respectively). CD40 or CD40L mRNA expression was not detected in tissue derived grafts

Renal in an 8-week gestation phase by PCR analysis for up to 6 weeks

after transplantation (Figure 11 a). On the contrary, this expression was detected in grafts derived from renal tissue in a gestation phase between 14 and 22 weeks at 4 weeks after transplantation (Figures 11 a-c, respectively). In addition, the expression of B7-1 after transplantation and infusion of PBMC was found to be significantly lower in grafts derived from renal tissue at an 8-week gestation phase (Figure 11 a) compared to derived grafts. of renal tissue in a gestation phase between 14 and 22 weeks (Figures 11 a - c, respectively). This expression pattern of the costimulatory molecule gene is consistent with in vivo data that show the total absence of immune responses by human allogeneic receptors against human kidney tissue transplants from human fetuses of 7 or 8 weeks, and therefore provides an underlying mechanism to the allogeneic immunological tolerance achieved against said tissues.

Advantages of grafts derived from renal tissue at an early gestation stage in immunocompetent mice: the optimally low immunogenicity of porcine renal tissues at an early gestation stage was further demonstrated by transplantation of adult porcine renal tissues or at a gestation stage from 27 to 28 days in immunocompetent Balb / c mice. Evaluation of adult renal tissue grafts (n = 10) and in a gestation phase of 27 to 28 days (n = 10) after 2 weeks showed rejection of both tissues. After a short treatment with CTLA4-lg, an immunoglobulin fusion protein that directly affects recognition by B7 T cells in antigen presenting cells (Llnsley, PS et al., 1991. J. Exp. Med 174, 561-569), between 2 and 4 weeks after transplantation, all adult grafts (8/8) had an altered morphology, necrotic tissue and a high degree of lymphocyte infiltration. On the contrary, at the same time point, the infusion of CTLA4-lg resulted in the growth and differentiation of 6 of the 10 transplants in the early gestation phase, which was not observed in the untreated animals, indicating the advantage Immune of transplants derived from renal tissue in an early gestation phase on transplants derived from adult renal tissue developed in fully immunocompetent hosts.

Decrease in the expression of multiple genes related to immunity in renal tissue in an early gestation phase: to investigate the inherent immunogenic properties of renal tissue in an early gestation phase that could justify its reduced immunogenicity with respect to more mature tissue, He analyzed the global gene expression in human renal tissues in an early gestation phase and in adults by means of an analysis in mlcromatrlz. In addition, 231 genes were analyzed that have roles directly related to Immunity (the complete list of genes can be found on the World Wide Web / Internet at
http://www.weizmann.ac.il/lmmunology/relsner/lmmunogenicity.xls. These would include the genes that code for HLA molecules, cltoclnas, qulmloclnas, chemokine receptors, apoptosis mediators, adhesion molecules, metalloprotelnases, Innate Immunity molecules and other immunomodulators. Hierarchical clustering (Eisen, MB et al., 1998. Proc. Nati. Acad. Sel. USA 95, 14863-14868) of all genes based on similarity in gene expression between experimental groups revealed two main groups, which separated the adult tissues from the fetal ones. In addition, immunity-related genes were grouped according to the gestation phase with a group of renal tissue genes in the earliest gestation phase and a group of adult renal tissue genes on opposite sides of a hierarchical clustering dendrogram (Figure 12 a). Patterns of "Immune" gene expression are presented through the use of the PLOTTOPGENE program (Zuo, F. et al., 2002. Proc. Nati. Acad. Sel. USA 99, 6292-6297) (Figure 12 b ). Said analysis unexpectedly found that 68 genes were significantly modified between adult and fetal tissues (P <0.05, total number of erroneous classifications (TNoM) = 0). The expression profiles of these genes showed those increased in adult tissues (n = 57 genes; Figure 12 c, upper part) and those decreased (n = 11 genes; Figure 12 c, lower part). Some examples of genes related to immunity with a more significant change include those that code for the molecules that participate in both the acquired and innate immune responses (Table 3).

Analysis: the results currently described demonstrate that Grafts derived from human renal tissue in a gestation phase of between 7 and 8 weeks, and Grafts derived from pig renal tissue in a gestation phase of between 20 and 28 days, transplanted in mice Immunodeflective, survive, grow and experience complete nephrogenesis, forming a functional organ capable of producing urine. Cells in an early gestation phase do not mature exclusively in differentiated renal structures, and form non-renal differentiated derivatives and disorganized cell clusters. Additionally, optimal renal organogenesis was achieved by transplanting grafts in an early stage of full organ gestation. In these early gestation phases, both human and porcine renal tissues contain renal mesenchymal stem cells and ramifications of urethral primordia, but no glomeruli, emphasizing their remarkable potential to differentiate after transplantation. A key observation of the results

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described above is that the growth and development of said renal tissues in an early gestation phase are facilitated by the vasculature derived from the host.

Table 3. Immunity-related genes, differentially expressed in human renal tissue in one phase.

of early pregnancy compared to an adult phase.

 Gen category

 Differentially expressed gene

 HLA

 MHC class I, C

 MHC class I, A

 MHC class I, E

 MHC class II, DP (31

 Chemokines / Adhesion

 RANTES

 monocyte chemotactic protein 1 (MCP-1)

 monocyte chemotactic protein 2 (MCP-2)

 E-selectin

 Cytokines

 osteopontin

 interleukin-15 (IL-15)

 prointerleukin-1 (3

 interleukin 1 receptor (IL-1)

 Innate immunity

 Clr complement component

 complement component 2

 complement control protein factor

 trickster receiver 1

 Apoptosis

 TNF 1 receptor-associated protein (TRADD)

 TNF-related apoptosis-inducing ligand (TRAIL)

 caspase 2 apoptosis regulatory protein

 apoptotic cysteine Mch 4

For more than four decades, embryonic tissues have been known to be less immunogenic compared to their adult counterparts (Medawar, P.B., 1953. Symp. Soc. Exp. Biol. 7, 320-323). Therefore, the currently described definition of the earliest time point in human or swine renal gestation during which normal differentiation is possible and subsequent renal function may also indicate the ideal time for tissue removal with less tendency to rejection. immune by alloimmune / xenoimmune responses. Consequently, graft acceptance may reflect the progressive development of a complex matrix of cell surface molecules and soluble factors that determine the immune recognition of the organ during pregnancy. In the results currently described, a microarray analysis established that the development of an immunological maturity in the human kidney is rather a late gestation episode, since renal tissues in an early gestation phase have the restricted expression of many of genes related to immunity. Therefore, it was unexpectedly found that 13 of the 57 genes were significantly regulated by increase in adult renal tissues with respect to those of a gestation phase belonging to the HLA systems of class I and class II. In addition, it was also unexpectedly found that molecules that mediate leukocyte trafficking, such as RANTES and MCP-1 chemokines (Nelson, PJ and Krensky, AM, 2001. Immunity 14, 377-386), the adhesion molecule E-selectin (Tedder, TF et al., 1995. FASEB J. 9, 866-873), proinflammatory cytokines such as osteopontin (O'Regan, AW et al., 2000. Immunol. Today 21, 475-478 ; Ashkar, S. et al., 2000. Science 287, 860-863; Xie, Y. et al., 2001. Kidney Int. 60, 1645-1657) complement genes, which are known to be associated with the innate immunity (Pratt, JR et al., 2002. Nature Med. 8, 582-587), were associated with reduced immunogenicity of renal tissues at an early gestation stage with respect to more mature tissues.

The immunogenicity of developing renal tissues was evaluated by using two different immunological models. In the first model, the grafts were implanted in immunodeficient reconstituted with human PBMC. The significant level of specific human immunity generated in this model after the infusion of human PBMCs has been well documented (Segall, H. ef al., 1996. Blood 88, 721-730; Reisner, Y. and Dagan, S. , 1998. Trends Biotechnol. 16, 242-246). In this model, both primary and secondary infusion of human PBMCs, obtained from different donors, and therefore representing two independent T lymphocyte repertoires, were not able to reject grafts derived from human or porcine renal tissues in one phase. of early gestation. While a global gene analysis indicated that the immunity tolerance of such tissue-derived grafts in an early gestation phase is probably associated with regulation by

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decrease of multiple immune routes, the reduction in the expression of CD40 and B7-1 observed in said grafts implies a possible absence or immaturity of the donor's hematopoietic antigen presenting cells. In addition, the reduced immunogenicity could also be related to the observed decrease in donor endothelial cells, which has recently been shown to function as antigen presenting cells and / or as targets for cytotoxicity mediated by T lymphocytes in direct allorecognition ( Kreisel, D. et al., 2002. Nature Med. 8, 233-239). It has been shown that human skin with genetically modified allogeneic tissue, devoid of donor endothelial cells, and therefore limited in its ability to present the antigen, functions similarly to renal tissues at an early gestation stage in a model Humanized skin rejection (Briscoe, DM et al., 1999. Transplantation 67, 1590-1599).

In the second model, kidney tissues were transplanted into normal immunocompetent hosts. The rejection in said hosts can be triggered by the donor antigen presenting cells transferred with the implant, or alternatively, by cross-sensitization against the host antigen presenting cells loaded with donor antigens, in a manner similar to the process of normal presentation of bacterial or viral antigens (Sayegh, MH and Turka, LA, 1998. N. Engl. J. Med. 338, 1813-1821; Benichou, G., 1999. J. Immunol. 162, 352-358). Because renal tissues at an early gestation stage possibly lack mature antigen presenting cells, in addition to a relative reduction in targeted migration receptors and in specific cytokines or chemokines, the hypothesis that sensitization blockade was tested cross-linking may be sufficient to alleviate the rejection observed in these implants. The results indicated that the immune rejection of renal tissue grafts in an early gestation phase could be avoided by a short costimulant block with CTLA4-lg, a protocol that failed to prevent the rejection of grafts derived from developed adult renal tissue. These results highlighted the reduced immunogenicity of tissues in an early gestation phase with respect to tissues in a late gestation phase. These results can be extrapolated and used for the design of immunosuppressive regimens for transplantation of both allogeneic and xenogenic developing renal organs / tissues in human subjects.

Finally, since renal tissues in an early gestation phase generate functional renal organs that produce urine independently of the host, such grafts can be used in conjunction with a urinary anastomosis with the urinary system of the host for the treatment of kidney diseases, for example, for correct biochemical aberrations in an uremic individual. An increase in the number of transplants and / or the administration of specific human growth factors can be used to favor functional replacement.

Conclusion: The results described above unexpectedly and convincingly demonstrated that grafts derived from human organs / tissues in a gestation phase between 7 and 8 weeks, or grafts derived from porcine organs / tissues in a gestation phase between 20 and 28 days, they are able to generate, in the absence of graft-derived teratomas, structurally and functionally differentiated organs / tissues, vascularized by the host that are optimally tolerated by human alloreactive / xenoreactive lymphocytes. In particular, these results unexpectedly and convincingly demonstrated that grafts derived from human or porcine renal tissue in the aforementioned respective phases of pregnancy have all these capabilities, including that of generating urine-producing renal organs.

As such, the above-described method of general organ / tissue transplantation is overwhelmingly superior to all such prior art methods, since it overcomes the critical limitations of the latter; namely: (i) use of organ / tissue grafts unsatisfactory tolerated by allogeneic / xenogenic human lymphocytes; (ii) inability of organ / tissue grafts to generate structurally and functionally differentiated organs / tissues, in particular the inability of renal grafts to differentiate into renal organs producing urine; and / or (iii) use of organ / tissue grafts not available in sufficient quantities.

REFERENCE EXAMPLE 2

Treatment of human kidney diseases by transplanting human or porcine renal organs / tissues at an early stage of pregnancy without immunosuppression of graft recipients or with minimal immunosuppression

As shown in Example 1 of the previous section of Examples, grafts derived from human organs / tissues in a gestation phase between 7 and 8 weeks, or grafts derived from porcine organs / tissues in a gestation phase between 20 and 28 days, transplanted in a host, are capable of generating structurally and functionally differentiated organs / tissues optimally tolerated by human alloreactive / xenoreactive lymphocytes. In particular, as shown therein, that human or porcine renal transplants in the aforementioned respective phases of gestation show all these capacities, including that of generating urine-producing renal organs. Therefore, in the conception of the present invention, the hypothesis was developed that the transplantation of grafts derived from human or porcine organs / tissues could be used in the aforementioned respective phases of gestation, for the treatment of diseases of said organs / tissues in humans. In particular, the hypothesis that it could

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the transplantation of the aforementioned renal grafts for the treatment of kidney diseases in humans, as described below, be used.

Materials and methods:

An adequate amount of human renal tissue is extracted in a gestation phase of between 7 and 8 weeks, or of porcine renal tissue in a gestation phase of between 20 and 28 days, as described in Example 1 above, and implants a therapeutically effective number of grafts in an anatomical location suitable for kidney transplantation in a human subject with a kidney disease treatable by a kidney transplant. Optionally, the subject is given a short-term costimulation block treatment in the form of the administration of CTLA4-lg, as described in the previous Example 1. The growth and differentiation of the graft (s) in the renal organ (s) funclonal (s) are controlled until urine production is detected, at which time an anastomosis is performed between the Graft and the subject's urinary system, so that allow the drainage of urine produced by the graft through the subject's urinary system. Alternatively, or together with urinary anastomosis, urine drainage produced by the graft is performed through a catheter, as described above in Example 1 of the previous Examples section.

REFERENCE EXAMPLE 3

Transplantation of human and animal pancreatic grafts in an early gestation phase in a host generates pancreatic organs / tissues that show 10-fold growth

As described in Example 1 of the previous section of Examples, human or porcine organs / tissues at an early gestation stage transplanted into a host are capable of generating structurally and functionally differentiated structurally / functionally integrated organs / tissues in the host. by human alloreactive / xenoreactive lymphocytes. Therefore, in the conception of the present invention, the hypothesis was developed that the transplantation of human or animal pancreatic organs / tissues at an early stage of gestation in a host will generate pancreatic organs / tissues that show significant development, as follows.

Materials and methods:

Donor pancreatic tissues: human pancreatic tissues were obtained in a gestation phase between 12 and 16 weeks after scraping, with a warm ischemia time of less than 30 minutes. After dissection, the pancreatic tissues were stored at 4 ° C in a UW solution for less than 45 minutes under sterile conditions. The study protocol was approved by the hospital committee (Kaplan Medical Center, Rehovot, Israel) of Helsinki.

Animal pancreatic tissues in a gestation phase of 12 to 14 days were microdissected from mouse embryos under the optical microscope. The tissues were stored at 4 ° C in a solution of PMRI 1640 medium before transplantation.

Transplant procedure: The transplantation of human and animal pancreatic organs / tissues in early stages of gestational development was carried out as described in Example 1 of the previous section of Examples, with modifications. For transplantation under the renal capsule, the host kidney is exposed through a left lateral incision, a 1.5 mm incision is made at the caudal end of the renal capsule and the donor's pancreatic tissues are grafted under the capsule. renal fragments of [1 - 2] x [1 - 2] millimeters.

Transplant development analysis: The growth and development of the transplanted pancreatic organs / tissues was analyzed as described in Example 1 of the previous Examples section.

Experimental results:

Four grafts derived from human pancreatic organs / tissues were transplanted in a gestation phase between 12 and 16 weeks under the renal capsule of 4 normal and 4 SCID mice. The size of each fragment in the transplant was 1 - 2 millimeters in diameter. In all immunocompetent mice a rejection was detected that began 5 days after transplantation, as determined by histological analysis, which indicated a graft necrosis and tissue destruction. In all immunodeficient mice, graft acceptance was observed, as determined by graft growth and the absence of rejection signs after histological and macroscopic analysis. In a graft derived from human pancreatic tissue in a 12-week gestation phase removed 8 weeks after transplantation, the graft size had increased 10 times (2x2 millimeters before transplantation versus 8 x 5 millimeters in the extraction; Figure 13) .

Mouse pancreatic organs / tissues were transplanted in a gestation phase of 14, 13 and 12 days under the renal capsule in immunodeficient syngeneic mice (Balb / c). In a tissue derived graft in the phase of

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12-day gestation taken 2 weeks after the transplant, the graft size had increased 10 times (1 x 1 millimeter before the transplant versus 5 x 3 millimeters after the transplant).

Conclusion: human or animal pancreatic organs / tissues in an early gestation phase transplanted into hosts generate pancreatic organs / tissues that show significant development.

REFERENCE EXAMPLE 4

Generation of diabetic mice

Methods and Materials: Diabetes is induced in host mice by treatment with streptozotocin, as previously described (Soria et al., 2000. Diabetes 49, 1-6). In summary, diabetes is induced in host mice through a single intraperitoneal injection of 200 mg of streptozotocin (Sigma) freshly dissolved in citrate buffer (pH = 4.5) per kilogram of body weight. Then the onset of diabetes is confirmed and controlled by the presence of weight loss, polyuria and blood glucose levels of less than 500 mg / dl. Blood is obtained for glucose tests by a tail cut and measured between 9 and 11 a.m. in conditions without fasting, and analyzed with a portable glucometer. Two weeks after the Streptozotocin Injection, the donor pancreatic tissues are implanted to the diabetic recipients and glucose levels are monitored as described above, in order to check the restoration of glycemic control.

REFERENCE EXAMPLE 5

Treatment of diabetes by transplanting human or porcine pancreatic tissue at an early gestation stage in diabetic human recipients without immunosuppression of the receptors or with minimal immunosuppression

Diabetes is a disease with a tremendous medical and economic impact, however the treatment of this disease through a daily Insulin Injection, the usual therapy in the prior art, does not prevent or satisfactorily relieve its debilitating or fatal consequences. A methodology that has been attempted to overcome this limitation has been the treatment of diabetes by transplanting adult pancreatic islets from a cadaver donor. However, this strategy cannot be routinely implemented due to the insufficient number of pancreas from immunologically compatible allogeneic donors from which to isolate the sufficient number of islets needed. As shown in Example 1 of the previous section of Examples, grafts derived from human organs / tissues in a gestation phase between 7 and 8 weeks, or grafts derived from porcine organs / tissues in a gestation phase between 20 and 28 days, transplanted in the hosts, generate organs / tissues of the graft type structurally and functionally differentiated optimally tolerated by human alloreactive / xenoreactive lymphocytes. As shown in Example 3 of the previous Examples section, the transplantation of human or animal pancreatic organs / tissues at an early gestation stage in a host generates pancreatic organs / tissues that show significant development. Therefore, in the conception of the present invention, the hypothesis was developed that human or porcine pancreatic organ / tissue transplantation could be used in the aforementioned respective phases of pregnancy for the treatment of diabetes in humans, as described below.

Materials and methods:

An adequate amount of pancreatic islets is isolated from human pancreatic tissue in a gestation phase of between 7 and 8 weeks, or of pig pancreatic tissue in a gestation phase of between 20 and 28 days, and transplanted into a human recipient diabetic according to prior art techniques, as previously described [National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK;
http://www.niddk.nih.gov)]. In summary, ultrasound is used to guide the placement of a small catheter through the upper abdomen and the liver of the subject. The pancreatic islets are then injected through the catheter into the liver. The recipient receives a local anesthetic, however if the recipient cannot tolerate local anesthesia, general anesthesia is used and the transplant is carried out through a small incision. Normally, for a 70 kilogram receiver, approximately one million pancreatic islets are administered. It takes some time for the administered cells to adhere to the new blood vessels and begin to release insulin, and therefore after transplantation, the blood glucose levels of the recipient are tightly controlled and endogenous insulin is administered as needed until needed. glycemic control is achieved. Optionally, to prevent graft rejection, the recipient is temporarily immunosuppressed by blocking short-term costimulation in the form of administration of CTLA4-lg, as described in the previous Example 1.

REFERENCE EXAMPLE 6

Transplantation of human liver grafts in a 7-week gestation phase, or swine in a 28-day gestation phase, generates, in the absence of graft-derived teratomas, liver organs / tissues

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structurally and functionally differentiated that will be optimally tolerated by human alloreactive / xenoreactive lymphocytes: basis for the optimal treatment of liver diseases

Liver organ / tissue transplantation of an allogeneic donor remains the optimal therapeutic option in the case of liver failure. However, therapeutic transplantation of liver organ / tissue grafts derived from an allogeneic donor is often impossible to implement due to haplocompatibility barriers. In addition, even when a compatible donor is found, in order to prevent graft rejection, such a transplant requires permanent immunosuppression of the graft recipient, usually through the administration of toxic immunosuppressive drugs, such as cyclosporine A. Such treatments! Immunosuppressants contribute to the disadvantages of allogeneic transplantation, since these are often unsuccessful to prevent graft rejection in the short term and are usually unable to prevent graft rejection indefinitely. An alternative to allograft transplantation involves the transplantation of xenografts, particularly porcine grafts, which are considered the optimal animal alternative to human grafts. However, xenografts generally cannot be used for transplantation due to a high suboptimal tolerance of such grafts by human lymphocytes. Therefore, liver organs / tissues suitable for therapeutic transplantation in humans and tolerated by non-syngeneic human lymphocytes, and suitable sources of such organs / tissues are highly desired. A potential strategy for providing liver organs / tissues for transplantation involves the use of grafts of said organs / tissues at early stages of development, since it has been shown that the earlier the stage of development of an organ / tissue, the better tolerated will be when transplanted into a non-syngeneic host. However, to date, satisfactory growth and differentiation of non-syngeneic hepatic organ / tissue grafts and satisfactory immunological tolerance of these grafts by human lymphocytes in the absence of graft-derived teratomas has not been achieved.

In the conception of the present invention, the hypothesis that there is a developmental phase during which the liver organs / tissues are differentiated enough to engage in a specific liver development in the absence of graft-derived teratomas was developed, while that are undifferentiated enough to be optimally tolerated when transplanted into a non-syngeneic host. In carrying out the present invention, the existence of specific gestation phases was unexpectedly discovered during which human and porcine liver organs / tissues can be transplanted into a host so that they generate, in the absence of graft-derived teratomas, Structurally and functionally differentiated liver organs / tissues that will be optimally tolerated by human alloreactive / xenoreactive lymphocytes, as described below.

Materials and methods:

Removal of human liver organs / tissues during pregnancy: human liver organs / tissues during pregnancy for transplantation were obtained by extracting fragments of the organ / tissue after voluntary abortions mechanically carried out by aspiration in one phase 7 weeks gestation, after obtaining informed consent. The warm ischemia time of the collected samples was kept below 30 minutes and after dissection, the organ precursors were stored at 40 degrees Celsius in a UW or PBS solution for less than 45 minutes under conditions sterile The study protocol was approved by the hospital committee (Kaplan Medical Center, Rehovot, Israel) of Helsinki.

Extraction of the porcine liver organs / tissues in the gestation phase: the porcine liver organs / tissues in the gestation phase for transplantation were obtained with the help of Lahav Instltute for animal research, Kibbutz Lahav. Developing tissues were collected in a 28-day gestation phase from pregnant sows operated under general anesthesia. The study protocol was approved by the local Institute of Ethics Committee. Tissues for transplantation were removed under an optical microscope and stored under sterile conditions at 40 degrees Celsius for approximately two hours in RPMI 1640 (Biological Industries, Bet HaEmek, Israel) before transplantation.

Transplant procedures: Transplants were performed in NOD / SCID receptors under general anesthesia induced by an intraperitoneal injection of 2.5% Avertlna in PBS (10 ml / kg). For transplantation under the renal capsule, the host's kidney was exposed through a left lateral incision. A 1.5 mm incision was made at the caudal end of the renal capsule and 1-2 mm diameter fragments of liver tissue grafts were implanted during pregnancy under the renal capsule. For intrasplenic transplantation, the liver tissue in the gestation phase was cut into 1 mm fragments in sterile PBS. The host spleen was exposed through a left lateral incision and a suspension of 1 mm diameter fragments of liver was injected during pregnancy at the lower pole of the spleen. The hemostasis was achieved by suture ligation below the injection site.

Histological analysis: the tissues were fixed by overnight incubation in 4 percent paraformaldehyde in PBS, the fixed tissues were processed through graduated alcohols, through x-fills and included in paraffin. Four micrometer thick sections of the included tissues were cut and mounted on positively charged glass slides. Tlsular sections mounted on the slides were

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dewaxed with xylene after rehydration with graduated alcohols. Endogenous peroxidase was inactivated with 0.6 percent hydrogen peroxide in 70 percent methanol for 20 minutes. When necessary, antigen recovery was applied by microwave boiling or protease pretreatment. For immunostaining, the slides were incubated in a humidified chamber for 60 minutes with primary antibody, after application of the DAKO Envision TM + system, horseradish peroxidase (HRP). As chromogens, the reagents diaminobenzidine (DAB) or aminoethylcarbasol (AEC) were used. The slides were counterstained with hematoxylin and mounted.

The swine anti albumin antibody was obtained from Betil Laboratories; the anti K.67 antibody was used as a marker of cell proliferation, and periodic acid-Schiff dye (PAS) was used as a marker of glycogen synthesis.

Additional details regarding the experimental protocols described in this Example can be provided in Materials and Methods, in Example 1 of this section, above.

Experimental results:

Porcine hepatic xenografts in a 28-day gestation phase, but not 21 days, are incorporated and show functional and structural liver differentiation: Porcine liver derived grafts in a 21-day gestation phase transplanted in NOD / recipient mice SCID carriers of xenorrhea-active human PBMC showed a clear development of teratomas (Figure 14 a) with a wide differentiation of cartilage (Figure 14 b) when examined 7 weeks after transplantation, clearly indicating for the first time that the optimal gestation phase for Transplantation of these tissues to obtain adequate hepatic differentiation in the absence of teratomas is greater than day 21 of pregnancy. In contrast, porcine liver-derived grafts in a 28-day gestation phase transplanted in the spleens of these mice that were examined 6 weeks after transplantation showed a specific liver and functional structural differentiation, 6 weeks after transplantation. All tissue sections of the graft stained with H&E, with periodic acid-Schiff or with anti-porbumin albumin antibody (Figures 15 a, 15by15c, respectively) showed a remarkable differentiation of hepatic lobular structures. Hepatic functionality was demonstrated by glycogen synthesis / storage and by albumin synthesis (Figures 15b and 15c, respectively). Additionally, staining the sections with an antibody specific for the Ki67 proliferation marker demonstrated the clear proliferative capacity of hepatocytes derived from the graft Similarly, said grafts transplanted under the renal capsule of said receptors, analyzed 6 weeks after transplantation also showed liver function, evidenced by glycogen synthesis / storage and albumin synthesis, as determined by staining the tissue sections of the graft through the use of periodic Schiff acid and swine anti albumin antibody (Figures 16 a and 16 b, respectively).

Human liver allografts in a 7-week gestation phase are incorporated and show a functional and structural liver differentiation: grafts derived from human liver in a 7-week gestation phase transplanted under the renal capsule of NOD / SCID mice showed a differentiation Specific structural and functional liver, 6 weeks after transplantation. Tissue sections of the graft stained with H&E or with periodic acid-Schiff showed a remarkable differentiation of the bile ducts and a glycogen synthesis / storage (Figures 17 a and 17 b, respectively).

Conclusion: The results described above convincingly demonstrate that grafts derived from human liver in a gestation phase of 7 weeks, or grafts derived from pig liver in a gestation phase of 28 days, but not 21 days, are capable of generating , in the absence of graft-derived teratomas, structurally and functionally differentiated hepatic organs / tissues that will be optimally tolerated by allogeneic / xenogenic human lymphocytes. As such, the results described above demonstrate that grafts derived from human liver can be used in a 7-week gestation phase, or grafts derived from porcine liver for 28 days, to optimally carry out therapeutic transplantation of liver tissues / organs. allogeneic and xenogeneic, respectively, with respect to all prior art methods.

REFERENCE EXAMPLE 7

Transplantation of human pancreatic grafts in a gestation phase of 8 weeks, or pigs in a gestation phase of between 27 and 28 days generates, in the absence of graft-derived teratomas, insulin-producing pancreatic organs / tissues that will be optimally tolerated by Alloreactive / xenorreactive human lymphocytes: basis for the optimal treatment of diabetes

Donor allogeneic pancreatic organ / tissue transplantation remains the optimal therapeutic option in the case of pancreatic insufficiency. However, therapeutic transplantation of pancreatic organ / tissue grafts derived from an allogeneic donor is often impossible to implement due to haplocompatibility barriers. Furthermore, even when a compatible donor is found, in order to prevent graft rejection, such a transplant requires permanent immunosuppression of the graft recipient, usually at

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through the administration of toxic immunosuppressive drugs, such as cyclosporin A. Such immunosuppressive treatments contribute to the inconveniences of allogeneic transplantation, since these are often unsuccessful to prevent graft rejection in the short term and are usually unable to prevent indefinitely graft rejection. An alternative to allograft transplantation involves the transplantation of xenografts, particularly porcine grafts, which are considered the optimal animal alternative to human grafts. However, xenografts generally cannot be used for transplantation due to a high suboptimal tolerance of such grafts by human lymphocytes. Therefore, pancreatic organs / tissues suitable for therapeutic transplantation in humans and tolerated by non-syngeneic human lymphocytes, and suitable sources of such organs / tissues are highly desired. A potent strategy for providing pancreatic organs / tissues for transplantation involves the use of grafts of said organs / tissues at early stages of development, since it has been shown that the earlier the stage of development of an organ / tissue, the better tolerated will be when transplanted into a non-syngeneic host. However, to date, the generation of tissues / organs derived from pancreatic grafts that show satisfactory growth and differentiation in the absence of graft-derived teratomas or a satisfactory immunological tolerance by human alloreactive / xenoreactive lymphocytes has not been achieved, without immunosuppression or with minimal immunosuppression.

In the conception of the present invention, the hypothesis that there is a development phase during which the pancreatic organs / tissues are sufficiently differentiated to engage in specific pancreatic development in the absence of graft-derived teratomas was developed, while that are undifferentiated enough to be optimally tolerated when transplanted into a non-syngeneic host. In carrying out the present invention, the existence of specific gestation phases was unexpectedly discovered during which human or porcine pancreatic organs / tissues can be transplanted into a host so that they generate, in the absence of graft-derived teratomas, structurally and functionally differentiated insulin-producing organs / tissues that will be optimally tolerated by human alloreactive / xenoreactive lymphocytes, as described below.

Materials and methods:

Removal of the human pancreatic organs / tissues in the gestation phase: the human pancreatic organs / tissues in the gestation phase for transplantation were obtained by extracting fragments of the organ / tissue after voluntary abortions mechanically carried out by aspiration in one phase 8 weeks gestation, after obtaining informed consent. The warm ischemia time of the collected samples was kept below 30 minutes and after dissection, the organ precursors were stored at 40 degrees Celsius in a UW or PBS solution for less than 45 minutes under conditions sterile The study protocol was approved by the hospital committee (Kaplan Medical Center, Rehovot, Israel) of Helsinki.

Extraction of the porcine pancreatic organs / tissues in the gestation phase: the porcine pancreatic organs / tissues in the gestation phase for transplantation were obtained with the help of the Lahav Institute for animal research, Kibbutz Lahav. Developing tissues were collected in a gestation phase of 27-28 days from pregnant sows operated under general anesthesia. The study protocol was approved by the local Ethics Committee. Tissues for transplantation were removed under an optical microscope and stored under sterile conditions at 40 degrees Celsius for approximately two hours in RPMI 1640 (Biological Industries, Bet HaEmek, Israel) before transplantation.

Transplant procedures: Transplants were performed in Balb / c x NOD / SCID chimeras or in NOD / SCID mice under general anesthesia induced by an intraperitoneal injection of 2.5% Avertine in PBS (10 ml / kg). For transplantation under the renal capsule, the host's kidney was exposed through a left lateral incision. A 1.5 mm incision was made at the caudal end of the renal capsule and 1-2 mm diameter fragments of pancreatic tissue were implanted during pregnancy under the renal capsule.

Histological analysis: the anti-cytokeratin MNF 116 antibody clone (without cross-reactivity with mouse tissues) was used for immunostaining of the porcine epithelium; and the anti-insulin antibody and the human anti-vimentin antibody clone V9 (without cross-reactivity with mouse tissues; used for staining human mesenchymal cells) were obtained in DAKO. The tissues were fixed by overnight incubation in 4 percent paraformaldehyde in PBS, the fixed tissues were processed through graded alcohols, through xylenes and included in paraffin. Four micrometer thick sections of the included tissues were cut and mounted on positively charged glass slides. The tissue sections mounted on the slides were deparaffinized with xylene after rehydration with the graduated alcohols. Endogenous peroxidase was quenched with 0.6 percent hydrogen peroxide in 70 percent methanol for 20 minutes. When necessary, antigen recovery was applied by microwave boiling or protease pretreatment. For immunostaining, the slides were incubated in a humidified chamber for 60 minutes with primary antibody, after application of the DAKO Envision TM + system, horseradish peroxidase (HRP). As chromogens, the reagents diaminobenzidine (DAB) or aminoethylcarbasol (AEC) were used. The slides were counterstained with hematoxylin and mounted.

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Experimental results:

Transplants of porcine pancreatic xenografts between 27 and 28 days gestation phase are incorporated and show a functional and structural pancreatic differentiation: grafts derived from porcine pancreas in a gestation phase of between 27 and 28 days transplanted under the renal capsule of mice NOD / SCID clearly showed a specific pancreatic structural and functional differentiation, 6 weeks after transplantation. Porcine liver derived grafts in a 28-day gestation phase transplanted in the spleens of these mice that were examined 5 weeks after transplantation showed significant pancreatic growth, as can be seen from the photograph of the entire graft (Figure 18 a ). The pancreatic structural differentiation was clearly evident 6 weeks after transplantation in a graft derived from porcine pancreatic tissue at a 27-day gestation phase, as determined through tissue sections of the graft stained with H&E, which showed the differentiation of the structures Pancreatic lobes with ductal structures and pancreatic acinars (Figures 18 b - c). Functional pancreatic differentiation was also evident 6 weeks after transplantation into tissue sections of a tissue-derived graft in a 27-day gestation phase in the form of insulin and pancreatic peptide synthesis (Figures 19 a and 19 b, respectively). As can be seen further in Figure 19 c, immunostaining of a graft derived from porcine pancreatic tissue in a 28-day gestation phase with anti cytokeratin antibody showed a clear differentiation of the graft-derived pancreatic ductal epithelium.

Human pancreatic allografts in an 8-week gestation phase are incorporated and show functional and structural pancreatic differentiation: grafts derived from human pancreatic tissue in an 8-week gestation phase transplanted under the renal capsule of NOD / SCID mice carrying Alloreactive human lymphocytes clearly showed a specific structural and functional differentiation of the pancreas, 6 weeks after transplantation. The graft pancreatic functionality was convincingly demonstrated by the differentiation of insulin-positive beta cells in the pancreatic islets (Figures 20 a-b). In addition, grafts derived from human pancreatic tissue in an 8-week gestation phase transplanted under the renal capsule of chimeras Balb / cx NOD / SCID carrying alloreactive human PBMCs also clearly showed a specific structural and functional differentiation of the pancreas, as shown through the differentiation of human mesenchymal cells positive for vimentin (Figures 20 c-d).

Conclusion: the results described above convincingly demonstrate that grafts derived from human pancreatic tissue in a gestation phase of 8 weeks, or pigs in a gestation phase of between 27 and 28 days, are capable of generating, in the absence of teratomas derived from graft, structurally and functionally differentiated pancreatic insulin-producing organs / tissues that will be optimally tolerated by human alloreactive / xenoreactive lymphocytes. As such, the results described above demonstrate that grafts derived from human pancreatic tissue can be used in an 8 week gestation phase, or porcine pancreatic tissue in a gestation phase between 27 and 28 days, to optimally perform the therapeutic transplantation of allogeneic / xenogenic pancreatic tissues / organs, respectively, with respect to all prior art methods.

REFERENCE EXAMPLE 8

Transplantation of grafts derived from human cardiac tissue in a 9-week gestalon phase generates, in the absence of graft-derived teratomas, cells / tissues that show a proliferative cardiac phenotype that will be optimally tolerated by alloreactive human lymphocytes: basis for treatment optimal heart disease

Allogeneic donor organ / tissue transplantation remains the optimal therapeutic option in the case of heart failure. However, therapeutic transplantation of cardiac organ / tissue grafts derived from an allogeneic donor is often impossible to implement due to haplocompatibility barriers. In addition, even when a compatible donor is found, in order to prevent graft rejection, such a transplant requires permanent immunosuppression of the graft recipient, usually through the administration of toxic immunosuppressive drugs, such as cyclosporine A. Such immunosuppressive treatments they contribute to the disadvantages of allogeneic transplantation, since these are often unsuccessful to prevent graft rejection in the short term and are usually unable to prevent graft rejection indefinitely. Therefore, cardiac organs / tissues suitable for therapeutic transplantation in humans and tolerated by non-syngeneic human lymphocytes, and suitable sources of such organs / tissues are highly desired. A powerful strategy for providing cardiac organs / tissues for transplantation involves the use of grafts of said organs / tissues at early stages of development, since it has been shown that the earlier the stage of development of an organ / tissue, the better tolerated will be when transplanted into a non-syngeneic host. However, to date, satisfactory growth and differentiation of non-syngeneic tissue / cardiac organ grafts or satisfactory immunological tolerance of these grafts by lymphocytes in the absence of graft-derived teratomas has not been achieved.

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In the conception of the present invention, the hypothesis that there is a developmental phase during which the cardiac organs / tissues are differentiated enough to engage in a specific cardiac development in the absence of teratomas derived from the Graft was developed, while that are undifferentiated enough to be optimally tolerated when transplanted into a non-syngeneic host. In carrying out the present invention, the existence of specific gestation phases was unexpectedly discovered during which human cardiac organs / tissues can be transplanted into a host so that they generate, in the absence of teratomas derived from the Graft, organs / tissues that show a prollferatlvo cardiac phenotype that will be optimally tolerated by human alloreactive lymphocytes, as described below.

Materials and methods:

Removal of human cardiac organs / tissues during pregnancy: human cardiac organs / tissues during pregnancy for transplantation were obtained by extracting organ / tissue fragments after voluntary abortions mechanically performed by aspiration in one phase 9 weeks gestation, after obtaining informed consent. The warm ischemia time of the collected samples was kept below 30 minutes and after dissection, the organ precursors were stored at 40 degrees Celsius in a UW or PBS solution for less than 45 minutes under conditions sterile The study protocol was approved by the hospital committee (Kaplan Medical Center, Rehovot, Israel) of Helsinki.

Transplant procedures: Transplants were performed in NOD / SCID receptors under general anesthesia induced by a 2.5% intraperitoneal injection of Avertine in PBS (10 ml / kg). For transplantation under the renal capsule, the host's kidney was exposed through a left lateral incision. A 1.5 mm incision was made at the caudal end of the renal capsule and 1-2 mm diameter fragments of cardiac tissue were implanted during pregnancy under the renal capsule.

Histological analysis: anti-sarcomeric alpha actin antibody and anti-neurofilament protein antibody, respectively, were used to stain cardiomyocytic cells and basal ganglion cells. The tissues were fixed by overnight incubation in 4 percent paraformaldehyde in PBS, the fixed tissues were processed through graduated alcohols, through xylenes and included in paraffin. Four micrometer thick sections of the included tissues were cut and mounted on positively charged glass slides. The tissue sections mounted on the slides were deparaffinized with xylene after rehydration with the graduated alcohols. Endogenous peroxidase was quenched with 0.6 percent hydrogen peroxide in 70 percent methanol for 20 minutes. When necessary, antigen recovery was applied by microwave boiling or protease pretreatment. For immunostaining, the slides were incubated in a humidified chamber for 60 minutes with primary antibody, after application of the DAKO Envlslon TM + system, horseradish peroxidase (HRP). As chromogens, the reagents diaminobenzidine (DAB) or amlnoetllcarbasol (AEC) were used. The slides were counterstained with hematoxylin and mounted.

Experimental results:

Allografts of human cardiac tissue in a 9-week gestation phase are incorporated and show a proliferative cardiac phenotype: grafts derived from human cardiac tissue in a 9-week gestation phase transplanted under the renal capsule of alloreactive human PBMC-bearing mice They clearly showed a proliferative cardiac phenotype 6 weeks after transplantation. Cardiac differentiation of grafts in the form of a differentiation of cardiomyocytic cells and basal ganglion cells was clearly seen in tissue sections of the graft stained with anti-sarcomeric alpha actin antibody or with anti-neurofilament protein (Figures 21 a and 21 b, respectively ).

Conclusion: The results described above convincingly demonstrate that grafts derived from human cardiac tissue in a 9-week gestation phase are capable of generating, in the absence of graft-derived teratomas, graft-derived cells / tissues that show a proliferative cardiac phenotype that they will be optimally tolerated by allogeneic human lymphocytes. As such, the results described above demonstrate that grafts derived from human cardiac tissue can be used in a gestation phase of 9 weeks, to optimally carry out the therapeutic transplantation of allogeneic cardiac tissues / organs, respectively, with respect to all methods of the prior art.

EXAMPLE 9

The transplantation of porcine lymphoid organ / tissue derived grafts in a 28-day gestation phase generates, in the absence of graft-derived teratomas, well-differentiated and vascularized mesenchymal / stromal lymphoid tissues that will be optimally tolerated by xenorreactive human lymphocytes: basis for the optimal treatment of genetic and / or hematological diseases

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Transplantation of lymphoid organs / tissues capable of generating a lymphoid stroma of xenogeneic origin, particularly of porcine origin, which are considered the optimal animal alternative to human grafts, is a potentially optimal therapeutic option for hematological and / or genetic diseases, Including those related to a coagulation disorder / a deficiency / defect in a coagulation factor, such as hemophilia, those related to a deficiency / defect in an enzyme, such as Gaucher's disease, and / or those related to a defect in the lymphoid stroma. However, xenografts cannot generally be used for transplantation due to a high suboptimal tolerance of such grafts by human lymphocytes. Therefore, lymphoid organs / tissues suitable for therapeutic transplantation in humans and tolerated by xenogeneic human lymphocytes, and suitable sources of such organs / tissues are highly desired. A potent strategy to provide such lymphoid organs / tissues for transplantation involves the use of grafts of said organs / tissues at an early stage of development, since it has been shown that the earlier the stage of development of an organ / tissue, Better tolerated will be when transplanted into a non-syngeneic host. However, to date, satisfactory growth and differentiation of developing xenogeneic lymphoid tissue / organ grafts and satisfactory immunological tolerance of such grafts by human xenograft lymphocytes in the absence of graft-derived teratomas has not been achieved.

In the conception of the present invention, the hypothesis that there is a developmental phase during which the lymphoid organs / tissues are sufficiently differentiated to engage in a specific lymphoid development in the absence of graft-derived teratomas was developed, while that are undifferentiated enough to be optimally tolerated when transplanted into a non-syngeneic host. In carrying out the present invention, the existence of specific gestation phases was unexpectedly discovered during which the porcine lymphoid organs / tissues can be transplanted into a host so as to generate, in the absence of graft-derived teratomas, organs / Well-differentiated and vascularized lymphoid mesenchymal / stromal tissues that will be optimally tolerated by xeno-reactive human lymphocytes, as described below.

Materials and methods:

Extraction of the porcine splenic organs / tissues in the gestation phase: the porcine splenic organs / tissues in the gestation phase for transplantation were obtained with the help of the Lahav Institute for animal research, Kibbutz Lahav. Developing tissues were collected in a 28-day gestation phase from pregnant sows operated under general anesthesia. The study protocol was approved by the local Institute of Ethics Committee. Tissues for transplantation were removed under an optical microscope and stored under sterile conditions at 40 degrees Celsius for approximately two hours in RPMI 1640 (Biological Industries, Bet HaEmek, Israel) before transplantation.

Transplant procedures: Splenic tissue graft transplants were performed in NOD / SCID mice under general anesthesia induced by an intraperitoneal injection of 2.5% Avertine in PBS (10 ml / kg). A 1.5 mm incision was made at the caudal end of the renal capsule and 1-2 mm diameter fragments of splenic tissue grafts were implanted during pregnancy under the renal capsule.

Histological analysis: an H&E stain was used for the identification of splenic differentiation. The tissues were fixed by overnight incubation in 4 percent paraformaIdehyde in PBS, the fixed tissues were processed through graded alcohols, through xylenes and included in paraffin. Four micrometer thick sections of the included tissues were cut and mounted on positively charged glass slides. The tissue sections mounted on the slides were deparaffinized with xylene after rehydration with the graduated alcohols. Endogenous peroxidase was quenched with 0.6 percent hydrogen peroxide in 70 percent methanol for 20 minutes.

Experimental results:

Porcine splenic xenografts in a 28-day gestation phase are incorporated and show a splenic differentiation: the analysis, 6 weeks after transplantation, of the H&E stained sections of the grafts derived from porcine splenic tissue in a 28-gestation phase days transplanted under the renal capsule of mice, it clearly showed the generation of well-differentiated and vascularized mesenchymal / stromal lymphoid tissues that will be optimally tolerated by xenogenic human lymphocytes (Figure 22).

Conclusion: the results described above convincingly demonstrate that porcine lymphoid organ / tissue grafts in a 28-day gestation phase are capable of generating, in the absence of graft-derived teratomas, well-differentiated and vascularized mesenchymal / stromal lymphoid tissues derived from the graft which will be optimally tolerated by xeno-reactive human lymphocytes. As such, the results described above demonstrate that porcine lymphoid organ / tissue grafts can be used in a 28-day gestation phase to optimally perform therapeutic transplantation of xenogeneic lymphoid grafts with respect to all prior art methods, for example, for the treatment of hematological and / or genetic diseases, including those related to a coagulation disorder / a

deficiency / defect in a coagulation factor, such as hemophilia, those related to a deficiency / defect in an enzyme, such as Gaucher's disease, and / or those related to a lymphoid stromal defect.

It will be appreciated that certain features of the Invention, which for clarity have been described in the context of Individual embodiments, may also be provided combined in a single embodiment. On the contrary, several features of the invention, which are briefly described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination.

Claims (13)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 1. A graft of a splenic organ or of porcine splenic tissue during pregnancy for use in the treatment of a disease or disorder selected from the group consisting of a coagulation disorder, a deficiency in a factor of coagulation, a defect in the stroma llnfolde, a disorder related to Immunity and a lysosomal storage disease / enzyme deficiency, said graft being selected from a splenic organ or porcine splenic tissue so that it does not express or present at least one molecule capable of stimulate or enhance an immune response in a xenogeneic subject, in which said at least one molecule is a lymphocyte co-receptor or a ligand of a lymphocyte co-receptor, and in which said phase of differentiation of said graft from a splenic organ or of a porcine splenic tissue corresponds to a pig between 20 and 63 days of gestation. 2. The graft of a splenic organ or splenic tissue for use according to claim 1, wherein said subject is a human being. 3. The graft of a splenic organ or splenic tissue for use according to claim 1, wherein said differentiation phase corresponds to between 20 and 56 days of gestation. 4. The graft of a splenic organ or splenic tissue for use according to claim 3, wherein said differentiation phase corresponds to between 20 and 42 days gestation. 5. The graft of a splenic organ or splenic tissue for use according to claim 4, wherein said differentiation phase corresponds to 42 days gestation. 6. The graft of a splenic organ or splenic tissue for use according to claim 1, which includes the use of an immunosuppressive drug, thereby favoring the establishment of said graft of a splenic organ or splenic tissue in said subject. 7. The graft of a splenic organ or splenic tissue for use according to claim 6, wherein said immunosuppressive drug is capable of blocking the binding of a lymphocyte correceptor with a ligand of said lymphocyte correceptor. 8. The graft of a splenic organ or splenic tissue for use according to claim 7, wherein said immunosuppressive drug is CTLA4-lg. 9. The graft of a splenic organ or splenic tissue for use according to claim 1, wherein said lymphocyte correceptor or said lymphocyte correceptor ligand is chosen from the group consisting of B7-1, CD40 and CD40L. 10. The grafting of a splenic organ or splenic tissue for use according to claim 1, wherein said graft selection of a splenic organ or splenic tissue is carried out through an analysis by an RT -PCR of said graft of a splenic organ or of splenic tissue. 11. The graft of a splenic organ or splenic tissue for use according to claim 1, wherein said deficiency in a coagulation factor comprises hemophilia. 12. The graft of a splenic organ or splenic tissue for use according to claim 1, wherein said lysosomal storage disease / enzyme deficiency comprises Gaucher's disease. 13. The graft of a splenic organ or splenic tissue for use according to claim 1, wherein said defect in the lymphoid stroma comprises a defect in the lymphoid stroma resulting in a deterioration in growth and / or hematological cell differentiation.