WO2007108689A2 - Nouveau procede d'induction de la differenciation de cellules souches et progenitrices - Google Patents

Nouveau procede d'induction de la differenciation de cellules souches et progenitrices Download PDF

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WO2007108689A2
WO2007108689A2 PCT/NL2007/050120 NL2007050120W WO2007108689A2 WO 2007108689 A2 WO2007108689 A2 WO 2007108689A2 NL 2007050120 W NL2007050120 W NL 2007050120W WO 2007108689 A2 WO2007108689 A2 WO 2007108689A2
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cells
differentiation
stem cells
polyamine
progenitor
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WO2007108689A3 (fr
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Martinus Nicolaas Helder
Paulus Ignatius Jozef Maria Wuisman
Behrouz Zandieh Doulabi
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STICHTING SKELETAL TISSUE ENGINEERING
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells

Definitions

  • the invention relates to propagation and differentiation of stem cells and the chemical stimuli which are used in the environment of those cells to induce differentiation. Further, the invention relates to the prevention of dedifferentiation of already differentiated cells. 5
  • Stem cells are totipotential or pluripotential precursor cells capable of generating a variety of mature cell lineages, and precursor cells are cells capable of generating cells of specific cell 0 lineages. These abilities serve as the basis for the cellular differentiation and specialization necessary for organ and tissue development.
  • stem and progenitor cells have provided new clinical tools to reconstitute and/or supplement bone marrow after myeloablation due to disease, exposure to toxic chemical and/or radiation. 5 Further evidence exists that demonstrates that stem cells can be employed to repopulate many, if not all, tissues and restore physiologic and anatomic functionality. The application of stem cells in tissue engineering, gene therapy delivery and cell therapeutics is also advancing rapidly.
  • stem cells Many different types have 0 been characterized. For example, embryonic stem cells, embryonic germ cells, adult stem cells or committed stem cells or progenitor cells are known. Certain stem cells have not only been isolated and characterized but have also been cultured under conditions to allow differentiation to a limited extent. However, a basic problem remains; that is, it has been difficult to control or regulate the 5 differentiation of stem cells and progenitor cells, such as hematopoietic progenitor cells. Presently, existing methods of modulating the differentiation of these cells are crude and unregulatable, such that the cells differentiate into unwanted cell types, at unwanted times. Moreover, the yield of the product cells is typically low.
  • stem or progenitor cells are typically obtained from embryos or foetal tissue, including abortuses, due, in part, to the limited quantity of stem or progenitor cells found in blood or tissue, and the significant discomfort involved in obtaining bone marrow aspirates.
  • harvesting of stem or progenitor cells from alternative sources in adequate amounts for therapeutic and research purposes is generally laborious, involving, e.g., dissection, harvesting of cells or tissues from a donor subject or patient, culturing and/or propagation of cells in vitro, etc.
  • procurement of these cells from embryos or foetal tissue, including abortuses has raised religious and ethical concerns.
  • Hu et al. discloses human amniotic epithelial cells derived from placenta at delivery that are isolated, cultured, cryopreserved for future use, or induced to differentiate.
  • a placenta is harvested immediately after delivery and the amniotic membrane separated from the chorion, e.g., by dissection.
  • Amniotic epithelial cells are isolated from the amniotic membrane according to standard cell isolation techniques.
  • the disclosed cells can be cultured in various media, expanded in culture, cryopreserved, or induced to differentiate.
  • amniotic epithelial cells are multipotent (and possibly pluripotent), and can differentiate into epithelial tissues such as corneal surface epithelium or vaginal epithelium.
  • the drawback of such methods is that they are labor-intensive and the yield of stem cells is very low.
  • Currently available methods for the ex vivo expansion of cell populations are also labour-intensive.
  • Emerson et al. (Emerson et al., U.S. Pat. No. 6,326,198) discloses media conditions for ex vivo culturing of human stem cell division and/or the optimization of human hematopoietic progenitor stem cells.
  • human stem cells or progenitor cells derived from bone marrow are cultured in a liquid culture medium that is replaced, preferably perfused, either continuously or periodically, at a rate of 1 ml of medium per ml of culture per about 24 to about 48 hour period. Metabolic products are removed and depleted nutrients replenished while maintaining the culture under physiologically acceptable conditions.
  • Kraus et al. discloses that a predetermined target population of cells may be selectively expanded by introducing a starting sample of cells from cord blood or peripheral blood into a growth medium, causing cells of the target cell population to divide, and contacting the cells in the growth medium with a selection element comprising binding molecules with specific affinity (such as a monoclonal antibody for CD34) for a predetermined population of cells (such as CD34 cells), so as to select cells of the predetermined target population from other cells in the growth medium.
  • a selection element comprising binding molecules with specific affinity (such as a monoclonal antibody for CD34) for a predetermined population of cells (such as CD34 cells), so as to select cells of the predetermined target population from other cells in the growth medium.
  • 6,335,195 discloses methods for ex vivo culture of hematopoietic and mesenchymal stem cells and the induction of lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof or All AT2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.
  • the stem cells are derived from bone marrow, peripheral blood or umbilical cord blood.
  • Retinoids such as vitamin A and retinoic acid (RA) have been known to affect differentiation of stem cells.
  • retinoic acid has been shown to inhibit proliferation of abnormally committed (chronic myelogenous leukemia) hematopoietic stem cells (Nadkarni et al. 1984, Tumori 70:503-505) and to induce differentiation and loss of self-renewal potential in promyelocytic leukemia cells (Melchner et al., 1985, Blood 66(6): 1469-1472).
  • Retinoic acid has also been shown to induce differentiation of neurons from embryonic stem cells and to repress spontaneous mesodermal differentiation (Slager et al., Dev. Genet.
  • Retinoic acid has further been shown to induce differentiation of transformed germ cell precursors (Damjanov et al., 1993, Labor. Investig. 68(2):220-232), placental cell precursors (Yan et al., 2001, Devel. Biol. 235: 422-432), and endothelial cell precursors (Hatzopoulos et al, 1998, Development 125: 1457-1468).
  • the effect of retinoids on differentiation has yet to be completely understood such that it could be used as a regulatable means of controlling differentiation of stem cells.
  • folic acid analogues such as aminopterin and amethopterin (methotrexate)
  • folic acid analogues are used as chemotherapeutic agents in acute lymphoblastic anemias and other blood proliferation disorders and cancers, and have been shown to effect differentiation of stem cells by killing off certain populations of stem cells (DeLoia et al., 1998, Human Reproduction 13(4):1063-1069), and thus, would not be an effective tool for regulating differentiation of large quantities of stem cells for administration to a patient.
  • cytokines such as IL-I, IL-2, IL-3, IL-6, IL- 7, IL-Il, as well as proteins such as erythropoietin, Kit ligand, M-CSF and GM-CSF have also been shown to direct differentiation of stem cells into specific cell types in the hematopoietic lineage (Dushnik-Levinson et al., 1995, Biol. Neonate 67:77-83), however, these processes are not well understood and still remain too crude and imprecise to allow for a regulatable means of controlling differentiation of stem cells.
  • Murashov, A.K. et al. (FEBS Lett., 2004, 569:165-168) described the use of a poly-L-ornithine/fibronectin coating as support for differentiating embryonic stem cells and they observed an increased differentiation into neuronal cells after application of 17- ⁇ -oestradiol or NGF in comparison with a gelatin coating. However, they ascribed the morphogen inducing neuronal differentiation effect to the use of fibronectin in the coating.
  • Knippenberg, M. et al. (Biochem. Biophys. Res. Comm. 2006, 342:902-908) in one example used spermine in the culture of adipose tissue- derived mesenchymal stem cells, in which example the effects of bone morphogenetic proteins (BMP-2 and BMP- 7) on chondrogenic and/or osteogenic differentiation were investigated. Although these effects were observed for BMP-2 and BMP- 7 no effect of the spermine (alone or in combination with the test proteins) was observed or described. Because control over stem and precursor cell differentiation can produce cell populations that are therapeutically useful, there is a need for the ability to control and regulate the differentiation of those cells.
  • BMP-2 and BMP-7 bone morphogenetic proteins
  • the invention now provides a method for the ex vivo induction of differentiation of stem cells or progenitor cells comprising the steps of: a. providing a progenitor or stem cell suspension in a culture medium; b. adding a polyamine to the culture medium.
  • the polyamines are removed from the culture medium after a period of time; preferably 1 min - 2 hour; more preferably 15 — 60 min.
  • the polyamine compound is preferably selected from the group essentially consisting of ornithine and its derivatives putrescine, spermidine and spermine.
  • differentiation inducing factors may be added in parallel , preferably from the group essentially consisting of steroid hormones, vitamins and/or growth hormones, such as TGFB, BMP, Osf, or LMP-I, and combinations of these.
  • the stem cells are preferably mesenchymal stem cells, preferably from adipose tissue.
  • the method of the invention preferably provides in osteogenic or chondrogenic differentiation.
  • the cells are preferably combined with bioresorbable materials, such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids or polyglycolids, poly-caprolactones, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these.
  • bioresorbable materials such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids or polyglycolids, poly-caprolactones, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these.
  • the stem cells and/or bioresorbable materials will either be directly implanted, or further cultured in the proper culture medium prior to implantation.
  • Another embodiment of the invention is the use of a polyamine in a method according to the invention.
  • a further embodiment is a method for preventing dedifferentiation of cells comprising applying a polyamine and/or the use of a polyamine to prevent dedifferentiation of cells.
  • a next embodiment is a method for inducing transdifferentiation of cells comprising applying polyamine and/or use of a polyamine to induce transdifferentiation
  • FIG. 1 Effect of 1,25(OH) 2 D 3 on relative runx-2 gene expression (A), osteopontin gene expression (B), SSAT gene expression (C), and PMF-I gene expression (D) by goat ASCs.
  • 1,25(OH) 2 D 3 significantly up-regulated gene expression of the osteogenic markers runx-2 (A) and osteopontin (B) after 14 days.
  • the gene expression levels for the polyamine-regulated genes SSAT (C) and PMF-I (D) were significantly up-regulated, however, already at 7 days of 1,25(OH) 2 D 3 treatment and not any more at 14 days post-treatment.
  • T/C l,25(OH) 2 D 3 -treated-over-control ratios
  • Data are mean ⁇ SEM of T/C ratios.
  • ASCs adipose tissue stem cells;
  • SSAT spermidine/spermine N-acetyl transferase, PMF-I, polyamine modulated factor-1. * Significant effect of 1,25(OH) 2 D 3 , p ⁇ 0.05.
  • FIG. 1 Effect of spermine on SSAT gene expression in goat-derived AT- MSCs.
  • Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 and 14 days after treatment, SSAT gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test.
  • SSAT spermidine/spermine N(l) acetyltransferase. * Significant effect of spermine, p ⁇ 0.05.
  • Figure 3 Effect of spermine on runx-2 gene expression in goat-derived AT- MSCs. Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 (A) and 14 (B) days after treatment, runx-2 gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p ⁇ 0.05.
  • Figure 4 Effect of spermine on osteopontin gene expression in goat-derived AT-MSCs.
  • Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 (A) and 14 (B) days after treatment, osteopontin gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p ⁇ 0.05.
  • Figure 5. Effect of spermine on BMP-2 and l,25-dihydroxyvitamin-D3 (1,25(OH)2D3) induced osteopontin gene expression in AT-MSCs.
  • Goat-derived AT-MSCs were treated or not for 15 minutes with 10 ⁇ M spermine, followed by 15 minutes treatment with 10 ng/ml BMP-2 or 10 nM 1,25(OH)2D3 combined or not with 10 ⁇ M spermine.
  • Four days after treatment osteopontin gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p ⁇ 0.05.
  • polyamines spermidine, spermine and their precursors ornithine and putrescine are ubiquitous aliphatic poly cations with multiple cellular functions. Polyamines have been called essential for a number of functions in the cell, including proliferation and apoptosis, however, their explicit role in these cellular processes is mostly unknown.
  • ornithine is formed from L-glutamate in a series of reactions.
  • Ornithine is the starting point for the biosynthesis of proline and arginine and of the here mentioned polyamines.
  • all the polyamines mentioned here are derived from the same building block, ornithine.
  • ODC ornithine decarboxylase
  • the catabolic pathway is controlled predominantly by the action of spermine/spermidine Nl-acetyltransferase (SSAT).
  • SSAT spermine/spermidine Nl-acetyltransferase
  • the concentration of the polyamine(s) should be in the range of 1 nM tot 100 ⁇ M, preferably in the range of about 100 nM to about 10 ⁇ M. It has also appeared that the presence of polyamine(s) in the medium can be limited to a certain time of minimal 5 minutes, but preferably more than 15 minutes. Further, to prevent pleiotropic or even toxic effects of the added polyamine(s) it is preferable to remove the polyamine(s) from the medium after the induction. However, this is not strictly necessary and the polyamine(s) may stay in the medium even if the stem cells have differentiated. In such a case, the polyamine(s) might prevent the differentiated cells from dedifferentiation.
  • Dulbecco's Modified Eagles Medium (DMEM) (obtainable as Cat# 11965-084 from GibcoBRL) with the addition of foetal bovine serum (FBS) (Cat# 10437-028, Gibco-BRL) has appeared to be a suitable medium. If desired antibiotics and/or antimycotics may be added, and also ascorbate-2-phosphate and ⁇ -glycerophosphate to ensure proper collagen processing and mineralization] can be added. After inducing differentiation with polyamines, as discussed above, the culturing of the cells can continue in this medium without the polyamines for continued differentiation and proliferation.
  • DMEM Dulbecco's Modified Eagles Medium
  • FBS foetal bovine serum
  • polyamines can be co- introduced with compounds which are known to induce differentiation wherein the polyamines function to enhance or boost the inducing effects of these compounds.
  • Other differentiation factors which can be used in this respect are known to the person skilled in the art and can be compounds such as steroid hormones, vitamins (such as vitamin A, retinoic acid and 1,25- dihydroxyvitamin D3), cytokines (such as IL-I, IL-2, IL-3, IL-6, IL-7, IL-I), proteins (such as erythropoietin, Kit ligand, M-CSF and GM-CSF) and growth factors (such as TGFB, BMP, Osf and LMP-I)
  • vitamins such as vitamin A, retinoic acid and 1,25- dihydroxyvitamin D3
  • cytokines such as IL-I, IL-2, IL-3, IL-6, IL-7, IL-I
  • proteins such as erythropoietin, Kit ligand, M
  • the stem cells which can be used and induced according to this invention can be any stem cells, such as umbilical stem cells and foetal stem cells.
  • progenitor cells or mesenchymal stem cells are used, which can be derived from adipose tissue.
  • Mesenchymal stem cells (MSC) from human bone marrow are known to be able to differentiate into chondrocytes, adipocytes, myeloblasts and osteoblasts.
  • Stromal cells of human adipose tissue have been shown to have similar characteristics in vitro (Zuk et al, 2001 Tissue Eng 7:211-228) .
  • Adipose stem cells can be obtained relatively easy, without long purification procedures and with high yield through resection and from liposuction aspirates (either in a conventional way or through ultrasonic mediated liposuction). 300 cc Aspirate gives a yield of about 2 to 6 x 10 8 cells. This takes away the need for in vitro expansion. Preferably stem cells derived from resection or conventional liposuction are used.
  • the adipose stem cells can be maintained fro prolonged periods in vitro without apparent loss of multipotency and they are, being autologous stem cells, immunocompatible.
  • the aspiration of adipose tissue can be accomplished using any known method, e.g. through procedures that have been proven repeatedly, wherein also has been pointed at procedures for obtaining stem cells from these aspirates (Halvorsen et al., 2001, Tissue Eng 7:729-41; Mizuno et al., 2002, Plast Reconstr. Surg. 109:199-209; Zuk et al., 2001 Tissue Eng7:211-228).
  • the aspirate can be obtained from various parts of the body, e.g. buttock, thigh or abdomen.
  • Aspirates can be processed directly or can be stored for a short period. The person skilled in the art will be able to choose the storage conditions to retrieve sufficient amount and quality after storage. Preferably, the aspirate is processed directly.
  • a special embodiment of this invention is to direct the stem cells to osteogenic or chondrogenic differentiation routes. This can be done by adding growth factors which are able to specifically direct differentiation in the osteoblast-forming or chondroblast forming pathways.
  • growth factors which are able to specifically direct differentiation in the osteoblast-forming or chondroblast forming pathways.
  • inducers for osteoblast formation are: BMP-2, 1,25-dihydroxyvitaniin D3, dexamethasone, while examples of inducers for chondroblast formation are: TGF- ⁇ i-3 and BMP-7 .
  • polyamines can be used to boost the effect of such inducers.
  • stem cells can be obtained (e.g. by aspiration of adipose tissue), can be induced ex vivo, and can then be replaced in the body for tissue outgrowth (e.g. in constructive bone surgery).
  • the stem cells can be reintroduced in the body as such, but they can also be combined with filler and/or carrier material.
  • the carrier material can be comprised of, optionally processed, autologous or homologous bone tissue or a combination thereof.
  • synthetic materials can be applied, such as osteoconductive calciumphosphates, bioresorbable polymers, like polylactides and polyglycolides, collagens, fibrinogens, alginates, hyaluronic acid derivatives, chitins and/or a combination of these materials.
  • Such filler or carrier material can take any form (porous, amorphous, granules, fibrous, powder) as long as it, temporarily, can be a mixable, fluid or pliable substance, which has the ability to function as attachment substrate for bone, cartilage, vertebral disc or connective tissue forming cells or tissues.
  • the substance should be injectable, but it could also be used as a pliable or even solid substance in open surgery.
  • the carrier material is preferably applied as a scaffold, which is defined for the present invention, as an optionally porous, physiological support for individual cells or tissues.
  • These cells are preferably the stem cells or the differentiated stem cells according to the invention.
  • This scaffold preferably has an architecture which enhances the migration, maintenance and/or proliferation and differentiation of the cells according to the present invention or the tissues which develop from these cells.
  • the term 'porous' means, for the present invention, having holes (pores) of sufficient size to be occupied by a cell and being able of being penetrated by polyamines alone or in combination with other inducing factors as discussed above, and/or by a cell suspension.
  • Another embodiment of the present invention is a method for preventing cells, e.g. derived from cartilage, intervertebral disc, bone, and connective tissue, to dedifferentiate by the application of a polyamine, preferably selected from the group consisting of ornithine, putrescine, spermidine and spermine.
  • a polyamine preferably selected from the group consisting of ornithine, putrescine, spermidine and spermine.
  • the polyamines can be used in combination with other factors, such as vitamins, growth factors, hormones, and the like.
  • polyamines are added to the culture medium intermittently, i.e. with some intervals. This would allow for maintaining tissue specificity while continuous cell expansion or proliferation is maintained.
  • a further embodiment of the present invention is a method to provide transdifferentiation from cells of one tissue type to cells of another tissue type. It is envisaged that e.g. skin fibroblasts can be turned into e.g. chondrocytes by application of polyamines, optionally in combination with one of the above mentioned differentiation factors. Such a transdifferentiation would allow for both in vivo and in vitro regeneration of tissues from adjacent cells.
  • Polyamine levels in tissues are modulated by a number of polyamine-regulated genes, including polyamine-modulated transcription factor- 1 (PMF-I) and spermidine/spermine N-acetyl transferase (SSAT (14-16).
  • PMF-I polyamine-modulated transcription factor- 1
  • SSAT spermidine/spermine N-acetyl transferase
  • example 1 the regulation of polyamine-related genes after growth factor- induced differentiation was illustrated.
  • polyamine spermine can modulate polyamine-regulated genes (example: SSAT) as well as osteogenic differentiation markers (runx-2, osteopontin, ALP activity) in AT-MSCs.
  • SSAT polyamine-regulated genes
  • osteogenic differentiation markers runx-2, osteopontin, ALP activity
  • runx-2 As well as osteopontin gene expression were significantly upregulated, However, 14 days after treatment, although runx-2 and osteopontin gene expression both showed increased expression levels in particular with the higher spermine dosages, only runx-2 reached significance with the 10 ⁇ M dosage.
  • polyamines i.e. spermine in this study
  • BMP-2 Bone Morphogenic Protein-2
  • 1,25- dihydroxyvitamin-D3 1,25(OH)2D3
  • BMP-2 Bone Morphogenic Protein-2
  • 1,25(OH)2D3 1,25(OH)2D3-induced osteopontin gene expression.
  • Cells were either treated or not for 15 minutes with 10 ⁇ M spermine, followed by 15 minutes treatment with spermine combined with either BMP-2 (10 ng/ml) or 1,25(OH)2D3 (10 ⁇ M), or with BMP-2 or 1,25(OH)2D3 alone.
  • BMP-2 ng/ml
  • 1,25(OH)2D3 1,25(OH)2D3
  • treatment with neither spermine, nor BMP-2 or 1,25(OH)2D3 significantly affected osteopontin gene expression.
  • treatment with the combination of spermine and either BMP-2 or 1,25(OH)2D3 synergistically increased osteopontin gene expression.

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Abstract

La présente invention concerne la propagation et la différenciation des cellules souches et les stimuli chimiques qui sont utilisés dans l'environnement de ces cellules pour induire la différenciation. Plus particulièrement, l'invention concerne un procédé d'induction ex vivo de la différenciation de cellules souches mésenchymateuses, dérivées de préférence de tissu adipeux, ou de cellules progénitrices en ajoutant une polyamine aux cellules pendant une courte durée (1 min - 2 heures), puis en l'éliminant. D'autres facteurs d'induction de la différenciation peuvent être ajoutés en parallèle, ces facteurs appartenant de préférence au groupe comrenant essentiellement des hormones stéroïdiennes, des vitamines et/ou des hormones de croissance, la LMP-I et leurs combinaisons. Le procédé décrit assure préférentiellement une différenciation ostéogénique ou chondrogénique et les cellules sont donc combinées de préférence à des matières bio-résorbables telles que des phosphates de calcium ostéo-conducteurs, des polymères bio-résorbables tels que des polylactides ou des polyglycolides, des poly-caprolactones, des collagènes, des minéraux, des fibrinogènes, des alginates, des hyaluronases, du chitosan et/ou leurs combinaisons. Les cellules souches et/ou les matières bio-résorbables seront soit implantées directement, soit davantage cultivées dans un milieu de culture approprié avant implantation. De plus, l'utilisation d'une polyamine est décrite en tant que procédé permettant d'éviter la dé-différenciation des cellules et/ou d'induire la trans-différenciation des cellules.
PCT/NL2007/050120 2006-03-21 2007-03-21 Nouveau procede d'induction de la differenciation de cellules souches et progenitrices Ceased WO2007108689A2 (fr)

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Cited By (7)

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US20100119492A1 (en) * 2008-10-31 2010-05-13 Synthes Usa, Llc Method and device for activating stem cells
WO2011144785A1 (fr) 2010-05-18 2011-11-24 Universidade De Santiago De Compostela Utilisation de poloxamines comme inductrices de la différenciation ostéogénique de cellules mésenchymateuses
CN104877961A (zh) * 2015-04-15 2015-09-02 广州赛莱拉干细胞科技股份有限公司 一种无血清的人羊膜间充质干细胞培养基及其制备方法
CN104877963A (zh) * 2015-04-15 2015-09-02 广州赛莱拉干细胞科技股份有限公司 一种无血清的人脐带间充质干细胞培养基及其制备方法
CN104877962A (zh) * 2015-04-15 2015-09-02 广州赛莱拉干细胞科技股份有限公司 一种无血清的脂肪干细胞培养基及其制备方法
EP2843042A4 (fr) * 2012-04-25 2015-12-30 Riken Préparation cellulaire contenant une cellule engagée du myocarde
CN110832321A (zh) * 2017-06-29 2020-02-21 K·埃瑟 鉴定诱导未分化或去分化的实体肿瘤细胞(再)分化的制剂的方法

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AU6125396A (en) * 1995-06-07 1996-12-30 Novartis Ag Serum-free media for primitive hematopoietic cells and metho ds of use thereof
WO1999040783A1 (fr) * 1998-02-17 1999-08-19 Gamida Cell Ltd. Procede de lutte contre la proliferation et la differenciation de cellules souches et parentes
DK1411918T3 (da) * 2001-07-31 2012-04-23 Genzyme Global S A R L Fremgangsmåder til at mobilisere progenitor/stamceller
US20050058687A1 (en) * 2003-09-12 2005-03-17 Becton, Dickinson And Company Covalently attached collagen VI for cell attachment and proliferation

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100119492A1 (en) * 2008-10-31 2010-05-13 Synthes Usa, Llc Method and device for activating stem cells
US20160222351A1 (en) * 2008-10-31 2016-08-04 DePuy Synthes Products, Inc. Method and Device for Activating Stem Cells
US10494604B2 (en) 2008-10-31 2019-12-03 DePuy Synthes Products, Inc. Method and device for activating stem cells
WO2011144785A1 (fr) 2010-05-18 2011-11-24 Universidade De Santiago De Compostela Utilisation de poloxamines comme inductrices de la différenciation ostéogénique de cellules mésenchymateuses
EP2843042A4 (fr) * 2012-04-25 2015-12-30 Riken Préparation cellulaire contenant une cellule engagée du myocarde
US9644181B2 (en) 2012-04-25 2017-05-09 Riken Cell preparation containing myocardium-committed cell
CN104877961A (zh) * 2015-04-15 2015-09-02 广州赛莱拉干细胞科技股份有限公司 一种无血清的人羊膜间充质干细胞培养基及其制备方法
CN104877963A (zh) * 2015-04-15 2015-09-02 广州赛莱拉干细胞科技股份有限公司 一种无血清的人脐带间充质干细胞培养基及其制备方法
CN104877962A (zh) * 2015-04-15 2015-09-02 广州赛莱拉干细胞科技股份有限公司 一种无血清的脂肪干细胞培养基及其制备方法
CN110832321A (zh) * 2017-06-29 2020-02-21 K·埃瑟 鉴定诱导未分化或去分化的实体肿瘤细胞(再)分化的制剂的方法

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