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  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors

Definitions

• This invention relates to isolated, mammalian, stem cells. More particularly the invention is related to lineage negative hematopoietic stem cell (Lin ' HSC) populations derived from bone marrow and methods of preserving cone cells in a retina of a mammal suffering from an ocular degenerative disease by treating the eye of the mammal with the isolated Lin " HSC populations.
• Lin ' HSC lineage negative hematopoietic stem cell
• HSC lineage negative hematopoietic stem cell
• HSCs have been used in transplants for over 40 years.
• advanced methods of harvesting purified stem cells are being investigated to develop therapies for treatment of leukemia, lymphoma, and inherited blood disorders.
• Clinical applications of stem cells in humans have been investigated for the treatment of diabetes and advanced kidney cancer in limited numbers of human patients.
• the present invention provides a method of ameliorating cone cell - A - degeneration in the retina of a mammal that suffers from an ocular disease.
• the method comprises the step of administering to the retina of the mammal a mammalian bone marrow-derived, isolated, lineage negative hematopoietic stem cell population, which comprises hematopoietic stem cells and endothelial progenitor cells.
• the cells are administered in an amount sufficient to retard cone cell degeneration in the retina.
• the isolated, Lin " HSCs of the present invention include a gene encoding a neurotrophic peptide.
• the neurotrophic Lin " HSCs are useful for promoting neuronal rescue in ocular diseases involving retinal neural degeneration, such as glaucoma, retinitis pigmentosa, and the like.
• (F) Linear correlations between the length of vasculature (X axis) and the number of cell nuclei in the ONL (Y axis) at P30 (left), P60 (middle), and Pl 80 (right) of Lin " HSC or control cell injected retinas.
• FIGURE 17 demonstrates that retinal function is rescued by Lin " HSC injection. Electroretinographic (ERG) recordings were used to measure the function of Lin “ HSC or control cell (CD31 " ) injected retinas . (A and B) ,
• CD31- HSC treated eyes are identical to non-injected rd/rd retinas, without any staining for cone (E) or rod (H) opsin.
• Lin-HSC treated contralateral eyes exhibited a markedly reduced, but clearly present ONL that is predominantly comprised of cones, as evidenced by positive immunoreactivity for cone red/green opsin (F). A small number of rods were also observed (I).
• the cells are isolated from adult human bone marrow and are further separated by CD133 lineage.
• One preferred method of isolating human Lin " HSCs includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody and recovering a CD133 positive, Lin " HSC population. Typically, less than about 30% of such cells express CD31 and less than about 30% of such cell express integrin a ⁇ .
• the human Cdl33 positive, Lin " HSC populations of the present invention can target sites of peripheral ischemia-driven neovascularization when injected into eyes that are not undergoing angiogenesis.
• Bone marrow cells were extracted from B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB/NJ (rd/rd mice) or Balb/cBYJ adult mice (The Jackson Laboratory, ME). Monocytes were then separated by density gradient separation using HISTOPAQUE ® polysucrose gradient (Sigma, St.
• Murine Lin HSC Population
• Bone marrow cells were extracted from Balb/C, ACTbEGFP, and C3H mice by the General Procedure described above.
• the Lin " HSC cells were analyzed for the presence of cell surface markers (Sca-1, KDR, c-kit, CD34, CD31 and various integrins: ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4, cc5, ⁇ 6, ⁇ M , ⁇ v , ⁇ x , ⁇ IIb ,, ⁇ l5 ⁇ 4 , ⁇ 3 , ⁇ 4 , ⁇ 5 and ⁇ 7 ). The results are shown in Table 1.
• Lineage negative HSC Population A of the present invention (approximately 10 5 cells in about 0.5 ⁇ l to about 1 ⁇ l of cell culture medium) was then injected intravitreally using a 33-gauge (Hamilton,
• T2-TrpRS The amino acid sequence of His 6 -tagged T2-TrpRS is shown as SEQ ID NO: 2, FIG. 8.
• the human Lin " HSC population was further separated into two sub-populations based on CD133 expression.
• the cells were labeled with biotin- conjugated CD133 antibodies ans separated into CD133 positive and CD 133 negative sub-populations.
• the fiat-mounted retinas were re-embedded for cryostat sections. Retinas were placed in 4% PFA overnight followed by incubation with 20% sucrose. The retinas were embedded in optimal cutting temperature compound (OCT: Tissue-Tek; Sakura FineTech, Torrance, CA). Cryostat sections (10 ⁇ m) were re-hydrated in PBS containing the nuclear dye DAPI (Sigma-Aldrich, St.
• mice were anesthetized by intraperitoneal injection of 15 ⁇ g/gm ketamine and 7 ⁇ g/gm xylazine.
• Electroretinograms were recorded from the corneal surface of each eye after pupil dilation (1 % atropine sulfate) using a gold loop corneal electrode together with a mouth reference and tail ground electrode.
• Stimuli were produced with a Grass Photic Stimulator (PS33 Plus, Grass Instruments, Quincy, MA) affixed to the outside of a highly reflective Ganzfeld dome.
• a filled histogram shifted to the right of the outlined (control) histogram represents an increased fluorescent signal and expression of the antibody above background level. Comparing the position of the peaks of the filled histograms between the two cell populations represents the difference in protein expression on the cells.
• CD31 is expressed above background on both CD1334- and CD133- cells of the invention; however, there are more cells expressing lower levels of CD31 in the CD133 + cell population than there are in the CD 133- population. From this data it is evident that CD31 expression varies between the two populations and that the alpha 6 integrin expression is largely limited to cells in the Lin- population, and thus may serve as a marker of cells with vasculo- and neurotrophic rescue function.
• Example 10 Intravitreal Administration of Murine Cells in Murine Models for Oxygen Induced Retinal Degeneration. New born wild-type C57B16 mice were exposed to hyperoxia
• FIG. 23 illustrates that the Lin " HSC populations of the present invention can reverse the degenerative effects of high oxygen levels in the developing mouse retina.
• Fully developed superficial and deep retinal vasculature was observed at P17 in the treated eyes, whereas in the control eyes showed large avascular areas with virtually no deep vessels (FIG. 24).
• Approximately 100 eyes of mice in the OIR model were observed. Normal vascularization was observed in 58% of the eyes treated with the Lin " HSC populations of the invention, compared to 12% of the control eyes treated with CD31 " cells and 3 % of the control eyes treated with PBS.
• FIG. 1 (a and b) depicts schematic diagrams of developing mouse retina.
• Panel (a) depicts development of the primary plexus (dark lines at upper left of the diagram) superimposed over the astrocyte template (light lines) whereas, (b) depicts the second phase of retinal vessel formation.
• GCL stands for ganglion cell layer
• IPL stands for inner plexus layer
• the neonatal mouse retinal angiogenesis model is useful for studying the role of HSC during ocular angiogenesis for several reasons.
• a large astrocytic template exists prior to the appearance of endogenous blood vessels, permitting an evaluation of the role for cell-cell targeting during a neo vascular process.
• this consistent and reproducible neonatal retinal vascular process is known to be hypoxia-driven, in this respect having similarities to many retinal diseases in which ischemia is known to play a role.
• EPC Endothelial Progenitor Cells
• Lm + or Lin " HSC from Balb/c mice were injected into rd/rd mice intravitreally at P6.
• P33 after injection with Lin + cells, vessels of the deepest retinal layer were nearly completely absent (FIG. 4 (a and b)).
• most Lin " HSC-injected retinas by P33 had a nearly normal retinal vasculature with three parallel, well-formed vascular layers (FIG. 4 (a and d)). Quantification of this effect demonstrated that the average length of vessels in the deep vascular plexus of Lin " injected rd/rd eyes was nearly three times greater than untreated or Lm + cell-treated eyes (FIG. 4 (e)).
• Intravitreally injected Lin " HSC populations localize to retinal astrocytes, incorporate into vessels, and can be useful in treating many retinal diseases. While most cells from injected HSC compositions adhere to the astrocytic template, small numbers migrate deep into the retina, homing to regions where the deep vascular network will subsequently develop. Even though no GFAP-positive astrocytes were observed in this area prior to 42 days postnatally, this does not rule out the possibility that GFAP-negative glial cells are already present to provide a signal for Lin " HSC localization. Previous studies have shown that many diseases are associated with reactive gliosis. In DR, in particular, glial cells and their extracellular matrix are associated with pathological angiogenesis.
• Lin " HSC compositions of the present invention can be used to target pre-angiogenic lesions in the retina. For example, in the ischemic retinopathies such as diabetes, neovascularization is a response to hypoxia. By targeting Lin " HSC compositions to sites of pathological neovascularization, developing neovasculature can be stabilized preventing abnormalities of neovasculature such as hemorrhage or edema
• angiostatic proteins such as T2-TrpRS can be delivered to sites of pathological angiogenesis by using transfected Lin " HSC compositions and laser-induced activation of astrocytes. Since laser photocoagulation is a commonly used in clinical ophthalmology, this approach has application for many retinal diseases. While such cell-based approaches have been explored in cancer therapy, their use for eye diseases is more advantageous since intraocular injection makes it possible to deliver large numbers of cells directly to the site of disease.
• Rescued rd/rd retinal cell types are predominantly cones.
• Rescued and non-rescued retinas were analyzed immunohistochemically with antibodies specific for rod or cone opsin.
• the same eyes used for the ERG recordings presented in FIG. 17 were analyzed for rod or cone opsin.
• rod or cone opsin In wild type mouse retinas, less than about 5% of photoreceptors present are cones (Soucy et al. 1998, Neuron 21: 481-493) and the immunohistochemical staining patterns observed with red/green cone opsin as shown in FIG. 25 (A) or rod rhodopsin as shown in FIG. 25 (B), were consistent with this percentage of cone cells.
• HSCs human bone marrows
• hLin " HSCs These cells were labeled ex-vivo with fluorescent dye and injected into C3SnSmn.CB17-iWc SCID mouse eyes.
• the injected hLin " HSCs migrated to, and targeted, sites of retinal angiogenesis in a fashion identical to that observed when murine Lin " HSCs were injected (FIG. 18 (A)).
• the human Lin ' HSCs also provided a robust rescue effect on both the vascular and neuronal cell layers of the rdl/rdl mice (FIG. 18 (B and C)). This observation confirms the presence of cells in human bone marrow that target retinal vasculature and can prevent retinal degeneration.
• HSCs were hybridized to human specific Affymetrix U133A microarray chips. After stringent analysis, a number of genes were found whose mRNA expression was human specific, above background, and significantly higher in the human Lin " HSC rescued retinas compared to the murine Lin " HSC rescued retinas and the human control cell-injected non-rescued retinas (FIG. 20, panel C).
• CD6 a cell adhesion molecule expressed at the surface of primitive and newly differentiated CD34+ hematopoietic stem cells, and interferon alpha 13, another gene expressed by hematopoietic stem cells, were both found by the microarray bioinformatics technique, validating the evaluation protocol.
• several growth factors and neurotrophic factors were expressed above background by human Lin " HSC rescued mouse retina samples (FIG. 20, panel D).
• Markers for lineage-committed hematopoietic cells were used to negatively select a population of bone marrow-derived Lin " HSC containing EPC.
• Inherited retinal degenerative diseases are often accompanied by loss of retinal vasculature. Effective treatment of such diseases requires restoration of function as well as maintenance of complex tissue architecture. While several recent studies have explored the use of cell-based delivery of trophic factors or stem cells themselves, some combination of both may be necessary. For example, use of growth factor therapy to treat retinal degenerative disease resulted in unregulated overgrowth of blood vessels resulting in severe disruption of the normal retinal tissue architecture. The use of neural or retinal stem cells to treat retinal degenerative disease may reconstitute neuronal function, but a functional vasculature will also be necessary to maintain retinal functional integrity.
• the precise molecular basis of the neurotrophic rescue effect remains unknown, but is observed only when there is concomitant vascular stabilization/rescue.
• the presence of injected stem cells, per se, is not sufficient to generate a neurotrophic rescue and the clear absence of stem cell-derived neurons in the outer nuclear layer rules out the possibility that the injected cells are transforming into photoreceptors.
• Data obtained by microarray gene expression analysis demonstrated a significant up-regulation of genes known to have anti- apoptotic effects. Since most neuronal death observed in retinal degenerations is by apoptosis, such protection may be of great therapeutic benefit in prolonging the life of photoreceptors and other neurons critical to visual function in these diseases.
• HSC protected retinas includes members of the crystallin family (Fig 20, panel B). Similar to heat-shock and* other stress-induced proteins, crystalline may be activated by retinal stress and provide a protective effect against apoptosis. Abnormally low expression of ⁇ A-crystallin is correlated with photoreceptor loss in a rat model of retinal dystrophy and a recent proteomic analysis of the retina in the rdlrd mouse demonstrated induction of crystalline up-regulation in response to retinal degeneration. Based on our microarray data of EPC-reseued rd/rd mouse retinas, upregulation of crystalline appear to play a key role in EPC mediated retinal neuroprotection.
• liver endothelial cells can be induced to produce, after VEGFRl activation, growth factors critical to hepatocyte regeneration and maintenance in the face of hepatic injury (LeCouter et al. 2003, Science 299:890-893).

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• 2005
  • 2005-09-02 CA CA002579292A patent/CA2579292A1/en not_active Abandoned
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