EP1128836A2 - Transplantation intra-uterine de cellules embryonnaires mesenchymateuses humaines - Google Patents

Transplantation intra-uterine de cellules embryonnaires mesenchymateuses humaines

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Publication number
EP1128836A2
EP1128836A2 EP99972103A EP99972103A EP1128836A2 EP 1128836 A2 EP1128836 A2 EP 1128836A2 EP 99972103 A EP99972103 A EP 99972103A EP 99972103 A EP99972103 A EP 99972103A EP 1128836 A2 EP1128836 A2 EP 1128836A2
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European Patent Office
Prior art keywords
human
cells
mesenchymal stem
stem cells
organ
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EP99972103A
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German (de)
English (en)
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Mark Thiede
Alan Flake
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Osiris Therapeutics Inc
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Osiris Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)

Definitions

  • the present invention relates to the field of cell therapy, and more particularly to the field of in vivo gene therapy by administering mesenchymal stem cells.
  • Certain diseases including inherited metabolic diseases, may produce irreversible damage to the fetus before birth.
  • In utero hematopoietic stem cell transplantation is a potential therapy for a large number of immunodeficiency diseases, hemoglobinopathies and others.
  • Advantages for the fetal recipient include potential immunologic tolerance for transplanted cells, and rapid expansion of the hematopoeitic compartment with formation of new "niches" for the competitive engraftment of donor cells.
  • the existence of natural models of hematopoietic chimerism in dizygotic twins which share cross placental circulation during development supports the potential to achieve high levels of donor cell chimerism with prenatal transplantation in recipients with normal hematopoiesis. Experimental work in sheep and other animal models has.
  • Mesenchymal stem cells are the formative pluripotential cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiation into any of the mesenchymal or connective tissues, for example, bone, cartilage, muscle, stroma, tendon, and fat.
  • This homogeneous population of cells can be passaged in culture and may be characterized by the lack of hematopoietic cell markers and by the presence of a unique set of surface antigens. Under specific conditions they have been induced to form bone, cartilage, adipose tissue, tendon, and muscle, and in their undifferentiated state, resemble roughly stromal fibroblasts and can support hematopoiesis as evidenced by the support of LT-CIC in long term bone marrow culture. Preliminary in vivo studies suggest that these cells home to bone marrow of post-natal recipients after intravenous administration and can accelerate constitution after myeloablative conditioning regimes.
  • Another object of the invention is to increase donor cell engraftment in fetal recipients. Another object is to regenerate damaged or diseased tissue by providing to a fetal recipient donor cells which can differentiate in situ. Yet another object is to prepare chimeric organs and tissues. Still another object is to treat a recipient by administering mesenchymal stem cells ii which , has. been ' modified "to express a therapeutic gene product.
  • MSCs mesenchymal stem cells transplanted into a fetus in utero then were distributed throughout the fetus.
  • the MSCs remained viable and differentiated into cellular types appropriate for the tissue or organ in which they engrafted. Surprisingly, the MSCs and their differentiated progeny were not rejected by immunocompetent hosts.
  • the MSCs can be used for cellular therapy and tissue engineering.
  • Normal MSCs express and secrete a number of cytokises, including G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11. (Haynesworth, et al, J. Cell Physiol., Vol. 166, No. 3, pgs. 585-592 (March 1996)).
  • cytokises including G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11.
  • transduced mesenchymal stem cells which have been modified to carry exogenous genetic material of interest, are induced to differentiate, the progeny cells also carry the new genetic material.
  • These transduced cells are able to express the exogenous gene product.
  • transduced mesenchymal stem cells and the cells differentiated therefrom can be used for applications where treatment using such modified cells is beneficial.
  • these modified cells can be used as a delivery system for therapeutic proteins encoded by the exogenous gene for treatment of inherited and/or acquired disorders of blood coagulation and wound healing.
  • the present invention provides a method of obtaining genetically modified mesenchymal stem cells, comprising transducing mesenchymal stem cells with exogenous genetic material and placing the transduced cells under conditions suitable for differentiation of the mesenchymal stem cells into lineages which then contain the exogenous genetic material.
  • the present invention is directed to a method for effecting gene therapy in vivo by administrating human mesenchymal stem cells in utero.
  • the mesenchymal stem cells can differentiate into specific ' cell liheagesiidepen ⁇ -ing ⁇ i-uthe. environment and integrate with like tissue to effect repair of tissue defects.
  • the mesenchymal stem cells can be transduced with exogenous genetic material such that a gene product will be expressed by the mesenchymal stem cell or its differentiated progeny in vivo to provide a desired therapeutic effect.
  • the present invention involves a method for treating a subject in need thereof by administering human mesenchymal stem cells in an amount effective to enhance the in vivo distribution and engraftment of mesenchymal stem cells.
  • the mesenchymal stem cells can be transduced with exogenous genetic material such that a gene product will be expressed by the mesenchymal stem cell or its differentiated progeny in vivo to provide a desired therapeutic effect.
  • Figure 1 is a photograph of liver tissue at 9 weeks after mesenchymal stem cells were injected intraperitoneally at 65 days gestation.
  • Figure 2 is a photograph of bone marrow at 9 weeks after mesenchymal stem cells were injected intraperitoneally at 85 days gestation.
  • Figure 3 is a photograph of heart tissue at 9 weeks after mesenchymal stem cells were injected intraperitoneally at 65 days gestation.
  • Figure 4 is a photograph of thymus tissue at 9 weeks after mesenchymal stem cells were injected intraperitoneally at 85 days gestation.
  • Figure 5 shows results of PCR analysis for human-specific ' ⁇ -2 sculpturecr ⁇ glob ⁇ iri performed on tissue samples of liver (Lv), spleen (Sp), bone marrow (BM), thymus (Th), lung (Lg), brain (Br), and blood (Bd).
  • Figure 6A is a photograph of a gel showing the presence of human ⁇ -2 microglobulin DNA in liver, spleen, lung, bone marrow, thymus, brain, and blood isolated from sheep fetuses at 65 or 85 days gestation, wherein the sheep fetuses were given human mesenchymal stem cells in utero.
  • Figures 6B, 6C, 6D, 6E, and 6F are photographs of slides showing the presence of human cells in sheep fetal liver, spleen, bone marrow, thymus, and lung, respectively.
  • Figure 7A is a photograph of a slide showing human mesenchymal stem cells, in the cardiac muscle of sheep fetuses stained with anti-human ⁇ -2 microglobulin.
  • Figures 7B and 7C are photographs of slides showing human cells in the cardiac muscle of sheep fetuses stained with anti-human ⁇ -2 microglobulin and
  • Figures 8A and 8B are photographs of slides showing the presence of human ⁇ -2 microglobulin through nickel chloride staining in cartilage lacunae of lambs given human mesenchymal stem cells in utero at 65 days gestation, and wherein the cartilage was harvested at 2 months and 5 months after transplantation, respectively.
  • Figure 9 shows photographs of slides of human cells, contacted with human- specific anti-CD23 antibody, found in the bone marrow of sheep at 5 months after in utero transplantation of human mesenchymal stem cells.
  • Figure 9A is a control showing normal ovine tissue contacted with human-specific anti-CD23 antibody.
  • Figures 9B through 9D show CD23+ human cells in ovine bone marrow at increasing magnification.
  • Figure 10 shows photographs of slides of human cells, contacted with human-specific anti-CD74 antibody, found in the bone marrow of sheep at 5 months after in utero transplantation of human mesenchymal stem cells.
  • Figure 10A is a control showing normal ovine tissue contacted with human-specific anti-CD74 antibody.
  • Figures 10B through 10D show CD74+ human " : cells . ⁇ h.Ovihe%ymus;.a ⁇ increasing magnification.
  • Figures 11A and 11B are photographs of slides showing human ⁇ -2 microglobulin positive cells in the central nervous system of sheep at 5 months after the sheep were given mesenchymal stem cells in utero.
  • the human/sheep model is a unique model of widely disparate xenogeneic chimerism in which human hematopoietic stem cells survive for years in the sheep bone marrow after in utero transplantation. These cells can establish multilineage long term engraftment after retransplantation in utero into second generation recipients proving the engraftment of pluripotent hematopoietic stem cells.
  • the system is limited by species specificity of hematopoietic cytokines.
  • the sheep microenvironment can support the viability of human HSCs and progenitors but human cytokine administration is required to drive the cells toward differentiation and peripheral expression. Human cells can be detected in this system using a variety of sensitive methodologies including flow cytometry, fluorescence in situ hybridization, immunohistochemistry, and PCR.
  • the present invention relates generally to the use of human mesenchymal stem cells and to compositions comprising human mesenchymal stem cells for in utero administration.
  • mesenchymal stem cells can be recovered from other cells in the bone marrow or other mesenchymal stem cell source.
  • Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullary spaces.
  • Other sources of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, and blood.
  • the presence of mesenchymal stem cells in the culture colonies may be verified by specific cell surface markers which are identified with unique monoclonal antibodies, see, e.g., U.S. Patent No.
  • the mesenchymal stem cell populations can be autologous, allogeneic, or xenogeneic to the recipient.
  • mesenchymal stem cells are from the same species as the recipient.
  • the MSCs are human in origin.
  • a method of isolating mesenchymal stem cells comprises the steps of providing a tissue specimen containing mesenchymal stem cells, preferably bone marrow; isolating the mesenchymal stem cells from the specimen, for example by density gradient centrifugation: adding the isolated cells to a medium which contains factors that stimulate mesenchymal stem cell growth without differentiation and allows, when cultured, for the selective adherence of only the mesenchymal stem cells to a substrate surface; culturing the specimen-medium mixture; and removing the non-adherent matter from the substrate surface.
  • the isolated mesenchymal stem cells are culture expanded in appropriate media, i.e. cultured by methods which favor cell growth of the enriched cell populations.
  • the cells are plated at a density of 0.05-2xl0 5 cells/cm 2 , preferably at a density of 0.5 -10 x 10 4 cells/cm 2 .
  • the cells may be characterized prior to, during, and after culture to determine the composition of the cell population, for example by flow cytometric analysis (FACS).
  • FACS flow cytometric analysis
  • the human mesenchymal stem cells can be stained with human mesenchymal stem cell-specific monoclonal antibodies.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the cells utilized in this invention and will be apparent to one of skill in the art.
  • the mesenchymal stem cells produced according to the methods described herein can be used to provide a reliable and constant source of mesenchymal stem cells for individuals in need thereof, e.g. those in need of cellular therapy, tissue engineering or regeneration and gene therapy.
  • Another aspect of the present invention relates to the introduction of genes into the mesenchymal stem cells such that the mesenchymal stem cells and progeny of the cells carry the new genetic material.
  • the mesenchymal stem cells can be modified with genetic material of interest.
  • the mesenchymal stem cells are able to express the product of the gene expression and, with a signal sequence, secrete the expression product. These modified cells can then be administered to a target, i.e., in need of mesenchymal stem cells or the gene expression product, where the expressed product will have a beneficial effect.
  • mesenchymal stem cells may be genetically engineered to express therapeutic proteins. Those that may be mentioned include providing continuous delivery of insulin, which at present must be isolated from the pancreas and extensively purified or manufactured in vitro recombinantly and then injected into the body by those whose insulin production or utilization is impaired. Genetically engineered human mesenchymal stem cells can also be used for the production of clotting factors. Persons suffering from hemophilia A lack a protein called Factor VIII, which is involved in clotting.
  • a recombinant Factor VIII product is now available and is administered by injection (Kogenate®, Bayer, Berkeley, CA)Jncorporation of genetic material of interest into human stem cells and other types of cells is particularly valuable in the treatment of inherited and acquired disease.
  • Inherited disorders that could be treated in this way include disorders of amino acid metabolism, such as Fabry's Disease, Gaucher's Disease, histidinurea or familial hypocholesterolemia; and disorders of nucleic acid metabolism, such as hereditary orotic aciduris.
  • MSCs express exogenous genes at high levels for long periods. This expression can continue through and after terminal differentiation.
  • the mesenchymal stem cells may be genetically modified by incorporation of genetic material into the cells, for example using recombinant expression vectors.
  • recombinant expression vector refers to a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • Structural units intended for use in eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • the mesenchymal stem cells thus may have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid.
  • Cells may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, for example.
  • Cells may be engineered by procedures known in the art :;by ' ,use .of a retroviral particle ' , containing RNA encoding a polypeptide.
  • Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is MGIN, derived from murine embryonic stem cells. Generally regarding retroviral mediated gene transfer, see McLachlin et al.(1990).
  • a preferred retroviral packaging cell line is described in U.S. Patent No. 5,910,434, the contents of which are incorporated herein by reference. Such cell line permits very high levels of transfection, i.e., greater than 80%.
  • the nucleic acid sequence encoding the polypeptide is under the control of a suitable promoter.
  • suitable promoters which may be employed include, but are not limited to, TRAP promoter, adeno viral promoters, such as the adeno viral major late promoter; the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral long terminal repeats (LTRs); ITRs; the ⁇ -actin promoter; and human growth hormone promoters; the GPIIb promoter.
  • TRAP promoter adeno viral promoters, such as the adeno viral major late
  • the promoter also may be the native promoter that controls the gene encoding the polypeptide. These vectors also make it possible to regulate the production of the polypeptide by the engineered progenitor cells. The selection of a suitable promoter will be apparent to those skilled in the art.
  • the retroviral LTR is preferred.
  • vehicles other than retroviruses to genetically engineer or modify the mesenchymal stem cells.
  • Genetic information of interest can be introduced by means of any virus which can express the new genetic mater ⁇ afin such cells.
  • SV40 herpes virus, adenovirus, adeno-associated virus, and human papillomavirus can be used for this purpose.
  • the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed cells such as dihydrofolate reductase, neomycin resistance or green fluorescent protein (GFP).
  • selectable marker genes such as dihydrofolate reductase, neomycin resistance or green fluorescent protein (GFP).
  • the mesenchymal stem cells may be transfected through other means known in the art.
  • Such means include, but are not limited to transfection mediated by calcium phosphate or DEAE-dextran; transfection mediated by the polycation Polybrene (Kawai and Nishizawa 1984; Chaney et al. 1986); protoplast fusion (Robert de Saint Vincent et al. 1981; Schaffher 1980; Rassoulzadegan et al. 1982); electroporation (Neumann et al. 1982; Zimmermann 1982; Boggs et al. 1986); liposomes (see, e.g.
  • the present invention further makes it possible to genetically engineer mesenchymal stem cells in such a manner that they produce, in vitro or in vivo polypeptides, hormones and proteins not normally produced in human mesenchymal stem cells in biologically significant amounts or produced in small amounts but in situations in which increased expression would lead to a therapeutic benefit. These products then would be secreted into the surrounding media or purified from the cells.
  • the human mesenchymal stem cells formed in this way can serve as continuous short-term or long-term production systems of the expressed substance.
  • the cells could be modified such that a protein normally expressed will be expressed at much lower levels. This can be accomplished with antisense nucleic acid technology, catalytic enzyme expression, single chain antibody expression, and the like.
  • This technology may be used to produce additional copies of essential geheS to allow augmented expression by the mesenchymal stem cells of certain gene products in vivo.
  • genes can be, for example, hormones, matrix proteins, cell membrane proteins, cytokines, adhesion molecules, "rebuilding" proteins important in tissue repair.
  • the expression of the exogenous genetic material in vivo is often referred to as "gene therapy”.
  • Disease states and procedures for which such treatments have application include genetic disorders and diseases of blood and the immune system.
  • Cell delivery of the transformed cells may be effected using various methods and includes intravenous or intraperitoneal infusion and direct depot injection into periosteal, bone marrow and subcutaneous sites.
  • the mesenchymal stem cells of the present invention may be administered to the fetus using methods generally known in the art.
  • the present invention provides a method of modifying fetal organs of a first species by administering mesenchymal stem cells of a second species to a fetus of the first species in utero.
  • human mesenchymal stem cells are administered to a non-human fetus in utero.
  • the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, it is believed that the non-human fetal organs may be less immunogenic, in the context of subsequent transplants into humans than unmodified organs. (See, for example, published PCT Application No. WO99/47163.) Thus, the modified non-human organs may be more suitable for transplantation.
  • This aspect of the present invention is applicable particularly to the transplantation of animal organs into humans.
  • organs include, but are not limited to, the heart, pancreas, kidney, skin, liver, thymus, spleen, bone marrow, cartilage, and bone.
  • human mesenchymal stem cells are administered to a non-human animal fetus.
  • the MSCs preferably are autologous to the human patient. After birth, the non-human animal is raised to an appropriate age and/or size such that the size of the organ or organs to be transplanted approximates the size of the organ or organs required by the human.
  • the modified organ(s) then is (are) harvested and transplanted into the human patient.
  • mesenchymal stem cells from a pediatric patient with a severe congenital heart defect are administered to a pig fetus. After birth, the pig would be raised to an appropriate age and/or size such that its heart approximates the size of the patient's heart. The modified heart then would be removed and transplanted into the pediatric patient.
  • MSCs mesenchymal stem cells
  • the MSCs were cultured in Control Media at 37°C in a humidified atmosphere containing 5% CO 2 . Upon reaching near- confluence, the cells were detached with 0.25% trypsin containing ImM EDTA (Gibco/BRL) for 5 minutes at 37°C. The cells were washed with Control Media and resuspended at 5 x 10 6 MSCs/mL in Control Media containing 5% DMSO (Sigma Chemical Co., St. Louis, MO). The cells were then stored in liquid nitrogen for use in the subsequent experiments.
  • MSC's human mesenchymal stem cells
  • the fetal liver remains the primary hematopoietic organ but stromal elements can be seen in the marrow with a few hematopoietic cells.
  • both fetal liver and bone marrow hematopoiesis are both active.
  • the day 65 fetus does not mount an immune response to non-self hematopoietic cells, whereas the day 85 fetus is immune competent and rejects hematopoietic cell transplants routinely.
  • Recipients were sacrificed at 7 or 14 days after transplantation and the liver, spleen, bone marrow, thymus, lung, brain and blood were analyzed by PCR for human specific ⁇ -2 microglobulin. Tissues positive for human sequence were confirmed by immunohistochemistry for morphologic assessment by staining for human ⁇ -2 microglobulin with secondary staining with horseradish peroxidase for visualization.
  • Fetal lambs at 65 or 85 days gestational age underwent transuterine-intraperitoneal or intravascular injection of 5 x 10 6 , 50 x 10 6 MSCs/fetus, respectively.
  • the ewes underwent horizontal hysterotomy with the use of electrocautery and Babcock clamps to control bleeding at the cut uterus margin.
  • the hind limbs and base of the umbilical cord were exteriorized and intravascular injection performed.
  • the fetal hind limbs and umbilical cord were returned to the amniotic cavity. Amniotic fluid volume was restored with warm, sterile lactated Ringer's solution and the hysterotomy closed with a TA-90 stapler.
  • the maternal abdomen was closed in layers and dressed with colloidin.
  • the ewe was placed in a movable cage where she was monitored until completely recovered from anesthesia. The ewes were continuously monitored until completely alert, and were able to stand, eat and drink. At this time, the animal was transported back to her holding room.
  • Buprenorphine was administered to alleviate postoperative pain. The animals were checked by the investigator's team 4-8 hours later, and then once or twice daily. Further doses of buprenorphine were administered every 8 hours as needed for pain. Also, antibiotic (liquamycin) was given daily for 5 days.
  • Fetal tissues were fixed overnight in 10% neutral buffered formalin at 4°C and paraffin embedded. For isolation of total cellular DNA, samples from each tissue were snap frozen in liquid nitrogen, and stored at -80°C for later DNA extraction.
  • Immunohistochemistry Serial 5um sections were obtained from each of the paraffin embedded tissues using a 30/50 microtome. Sections were deparaffinated, dehydrated, and rehydrated and then subjected to microwave antigen retrieval. Sections were then stained immunohistochemically for human class I antigen and SH2/SH3 antigen. The latter two antigens are found on MSCs. (See U.S. Patent No. 5,837,539.)
  • Total cellular DNA from the organs mentioned above were isolated using DNAzol. Specific primers for human class I antigen were selected based on the published human class I sequence.
  • lug DNA were added to each 0.65 mL microcentrifuge tube and placed on ice. A master mix was prepared and added on ice such that the final concentration of reagents for each sample was 2.5U Amplitaq Gold DNA polymerase (Perkin Elmer, Norwalk, CT). 200 uM deoxytriphosphates (dNTP's, Pharmacia, Piscataway, NJ), 50mM KC1, lOmM Tris-Cl (pH 8.3 at 22°C), 1.5mM MgCl 2 , 0.01% gelatin, and luM upstream and downstream primers.
  • thermocycler block reached 94°C, when the samples were immediately placed into the block for 9 minutes.
  • Samples were amplified for 40 cycles of 30 seconds at 94°C followed by 30 seconds of primer annealing followed by 1 minute of extension at 72°C.
  • samples were incubated for 5 minutes at 72°C.
  • PCR products were subjected to electrophoresis through a 2.5% NuSieve/1% Seakem agarose gel containing 0.5ug ethidium bromide/mL in IX Tris acetate running buffer. The gels were illuminated with UV 280-nm light and photographed with type 55 positive/negative Polaroid film. The negative was scanned transmissively and the intensities of the bands determined using the Intelligent Quantifier densitometer. Band intensities was compared to standard curves generated with known concentrations of human DNA.
  • the fetal sheep were sacrificed at 1 or 2 weeks or 2 or 5 months after injection and the liver, spleen, lung, bone marrow, thymus, brain, heart, skeletal muscle, cartilage, and blood were harvested and analyzed for the presence of human cells.
  • the fetal tails were docked at the time of MSC injection and the tail wounds harvested at 1 week or 2 months after wounding for DNA isolation and immunohistochemistry.
  • DNA Isolation Total cellular DNA from the organs mentioned above was isolated using DNAzol (Molecular Resource Center, Inc., Cincinnati, OH). In brief, approximately 100 mg of tissue was homogenized in lmL of DNA zol. The DNA was precipitated with 0.5mL of 100% ethanol. The DNA precipitate was pelleted by centrifugation and then washed twice with 95% ethanol. The DNA pellet was then dissolved in sterile water.
  • PCR Analysis To screen the ovine tissues for the presence of human cells, total cellular DNA was subjected to PCR analysis for human specific ⁇ -2 microglobulin using a modification of previously described methods (Gilliland, et al., Proc. Nat. Acad. Sci., Vol. 87, pgs.
  • PCR products were subjected to electrophoresis through a 2.5% NuSieve/1% Seakern agarose gel containing 0.5ug ethidium bromide/mL in IX Tris acetate running buffer. The gels were illuminated with UV 280-nm light and photographed with type 55 positive/negative Polaroid film.
  • Immunohistochemistry To verify the PCR results, inrmunohistochemistry for human ⁇ -2 microglobulin was performed as previously described.
  • Tissue Unmasking Fluid Ted Pella, Redding, CA
  • Blocking for 30 minutes at room temperature (RT) was performed using non- immune serum from the species in which the primary antibody was raised (1 :20 dilution), followed by a 12 hour incubation with the specific primary antibody.
  • the primary antibody dilutions used were as follows: human ⁇ -2 microglobulin (Pharmingen International, San Diego, CA, 1 :200); human CD74 (Pharmingen, 1 : 10); or human CD23 (Vector Laboratory, Burlingame, CA, 1:10).
  • the slides were then washed with PBS followed by a second blocking step with methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature. Slides were then rinsed with deionized water, then PBS, followed by incubation with biotinylated secondary antibody (1 :200 dilution) for 30 min. at RT. The slides were washed with PBS and avidin-biotin complex added for 45 min. at RT. The slides were then rinsed well in PBS, developed with the chromagen 3,3'-diaminobenzidine. For sections stained for human ⁇ -2 microglobulin, CD74, and CD23 the slides then were lightly counterstained with hematoxylin.
  • the human ⁇ -2 microglobulin was developed first using nickel chloride as the chromagen, and then subjected to a secondary immunohistochemical staining for SERCA-2 (Vector Laboratory, 1 :50 dilution) or GFAP (Vector Laboratory, 1 :100 dilution), respectively, as previously described (Van Der Loos, et al., Histochem. J., Vol. 25, pgs. 1-1 1 (1993); VanDer Loos, et al., J. Hist. Cyto., Vol. 42, pgs. 289-294 (1994)). Secondary staining was developed using Vector VIP substrate kit (Vector Laboratory). No counterstaining was performed on these double-stained slides.
  • PCR for human specific ⁇ -2 microglobulin DNA sequences was performed on DNA isolated from liver, spleen, lung, bone marrow, thymus, brain, heart, skeletal muscle, and blood from fetuses transplanted at either 65 or 85 days gestation. Tissue was harvested at 2 weeks, 2 months, or 5 months after in utero transplantation. Two weeks after transplantation human ⁇ -2 microglobulin DNA was detected in all tissues examined in fetal sheep transplanted at 65 and 85 days gestation ( Figure 6A and Table 1), with the exception of skeletal muscle. Cartilage was not examined.
  • Human MSCs could often be appreciated in a single high-power field ( Figures 6B and 6C) in these tissues.
  • Human cells were also identified in non-lymphohematopoietic sites including the heart, skeletal muscle, cartilage, perivascular areas of the CNS, and lung ( Figure 6F). Five months after transplantation, human cells continued to be present in multiple tissues including the bone marrow, thymus, cartilage, heart, skeletal muscle, and brain.
  • Differentiation of human MSCs in various tissues following transplantation was assessed by one of three techniques: 1) characteristic morphology on anti- human ⁇ -2 microglobulin staining; 2) immunohistochemical double-staining for anti-human ⁇ -2 microglobulin and a second non-human specific differentiation marker; or 3) when available, positive staining with human specific differentiation markers proven to not cross-react with sheep cells.
  • site- specific differentiation was confirmed for human cardiomyocytes, chondrocytes, bone marrow stromal cells, thymic stromal cells, and skeletal myocytes. Human cells were identified in the CNS.
  • Cardiomyocyte Differentiation To assess differentiation of human MSCs found in cardiac muscle, double- staining immunohistochemistry was performed using anti-human ⁇ -2 microglobulin and anti-SERCA-2 (Smooth Endoplasmic Reticulum ATPase). At 2 and 5 months after in utero transplantation, human cells were detected in the cardiac muscle of fetuses transplanted at 65 and 85 days gestation. These cells had similar morphology to the surrounding ovine cardiomyocytes and also double-stained with human ⁇ -2 microglobulin and SERCA-2, consistent with human cardiomyocyte differentiation ( Figures 7B and 7C).
  • Chondrocyte differentiation was identified by the finding of human ⁇ -2 microglobulin positive cells in cartilage lacunae of lambs transplanted at 65 days and harvested at 2 months or 5 months after transplantation. Immunohistochemistry was performed using a nickel chloride-based developing technique giving the particulate appearance observed ( Figures 8A and 8B). The immunohistochemical identification of human cells within the lacunae of cartilage specimens that were DNA PCR positive for human ⁇ -2 microglobulin sequences represents clear evidence of human chondrocyte differentiation.
  • Bone Marrow Stromal Differentiation To assess differentiation of human MSCs found in the bone marrow immunohistochemistry was performed using a human specific anti-CD23 antibody (Pharmingen, San Diego, CA, mouse IgGi, Clone M-L233).
  • CD23 is the low affinity IgE receptor and has been shown to be expressed on a variety of cell types including bone marrow stromal cells (Huang, et al, Blood, Vol. 85, pgs. 3704-3712 (1995); Fourcade, et al., European Cytokine Network, Vol. 3, pgs. 539-543 (1992)).
  • tail wounds were created in five 65 day gestation fetal sheep at the time of MSC injection. One animal was sacrificed at one week and four animals at 2 months. Human ⁇ -2 microglobulin DNA was detected by PCR in the one tail wound at 1 week and in one of four tail wounds at 2 months. The PCR results were verified by human ⁇ -2 microglobulin immunihistochemistry (data not shown). The cells expressing human ⁇ -2 microglobulin in the tail wound appeared in the dermis and dermal appendages and had the morphologic appearance of fibroblasts consistent with participation in the wound healing response.
  • Mesenchymal stem cells are of increasing interest to the emerging fields of tissue engineering, cellular transplantation, and gene therapy because of their availability in bone marrow, their relative ease of expansion in culture, their amenability to genetic manipulation, and most importantly, their capacity for differentiation into multiple mesenchymal tissues. These properties support potential clinical applications of: 1) large scale tissue engineering particularly for repair of musculoskeletal injury; 2) cellular therapy for diseases of mesenchymal origin such as muscular dystrophy, osteoporosis, osteogenesis imperfecta, and collagen disorders; 3) bone marrow conditioning to facilitate engraftment of autologous or allogeneic hematopoietic stem cells; and 4) gene therapy. Prenatal MSC transplantation may provide a "reservoir" of normal stem cells to replace defective cells as they become damaged in degenerative diseases with progressive cellular and organ damage.
  • mice There have been two studies in mice in which cultured mouse adherent cell populations have been transplanted and documented to persist following transplantation.
  • cells from transgenic mice expressing a human mini-gene for collagen I were used as mesenchymal progenitor donors and the fate of the cells followed after transplantation into irradiated mice (Pereira, et al., Proc. Nat. Acad. Sci., Vol. 92, pgs. 4857-4861 (1995)).
  • Donor cells were detected in bone marrow, spleen, bone cartilage, and lung up to 5 months later by PCR for the human mini-gene, and a PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma.
  • Reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner.
  • a second study transplanted either cultured adherent cells or whole bone marrow into irradiated mice with a phenotype of fragile bones resembling osteogenesis imperfecta caused by expression of the human minigene for type I collagen (Pereira, et al., Proc. Nat. Acad. Sci., Vol. 95, pgs. 1142-1147 (1998)).
  • fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 months, donor male cells accounted for 4-19% of the fibroblasts or fibroblast- like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin.
  • MSCs although very large cells, can be transplanted, and are capable of homing to and engrafting in multiple tissues, even when transplanted into the fetal peritoneal cavity. This requires the transplanted MSC to cross endothelial barriers, integrate into host tissue microenvironments, and survive with available growth factors and regulatory signals.
  • This may be a function of the ability of specific microenvironments to support the engraftment and differentiation of MSCs, or alternatively, the loss of engraftment from some tissues may be due to heterogeneity, of me transplanted population with respect to differentiation potential or replicative capacity. Immune mediated rejection is less likely since the pattern of engraftment was not limited to immune privileged sites, and an immune mechanism should result in eradication of donor cells.
  • MSCs are capable of site specific, multipotential differentiation and tissue integration following transplantation.
  • Human MSCs have been shown in vitro to differentiate into adipocytic, chondrocytic, or osteocytic lineages (Pittenger, supra). Less well characterized MSC populations from other species have been induced in vitro toward myocytic differentiation.
  • This study confirms in vivo chondrocytic differentiation and for the first time clearly demonstrates in vivo cardiomyocytic and myocytic differentiation of a defined human MSC population.
  • MSCs derived from bone marrow from multiple species have been demonstrated to support hematopoiesis with equal or greater efficacy than stromal layers formed in long term Dexter cultures.
  • CD23 has been identified as a low affinity IgE receptor as well as a functional CD21 ligand (Huang, et al., 1995; Aubry, et al., Cell, Vol.
  • CD74 is a cell surface MHC class Il-associated invariant chain molecule that is expressed on B- cells, Langerhans cells, dendritic cells, activated T-cells, and thymic epithelium (Schlossman, et al., 1995).
  • the mo ⁇ hology of CD74+ cells in this study appears similar to the ovine thymic epithelial cells in the surrounding thymus.
  • the precursor of thymic dendritic cells is thought to be the hematopoietic stem cell, whereas the origin of the thymic epithelial cell is unknown.
  • Human MSCs are known to express Class I HLA antigen but do not express Class II, which may limit immune recognition.
  • thymic stromal cells are known to participate in thymocyte positive and negative selection and host thymic antigen presenting cells are capable of facilitating clonal deletion of donor reactive lymphocytes after in utero HSC transplantation (Kim, et al., J. Pediatr. Surg., Vol. 34, pgs.

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Abstract

La présente invention se rapporte à des cellules embryonnaires mésenchymateuses destinées à une transplantation intra-utérine. Ces cellules embryonnaires mésenchymateuses peuvent être utilisées dans une méthode de traitement d'un foetus, ou dans une méthode de greffe de cellules embryonnaires mésenchymateuses, ou encore dans une méthode de préparation d'un organe en vue de sa transplantation.
EP99972103A 1998-11-13 1999-11-12 Transplantation intra-uterine de cellules embryonnaires mesenchymateuses humaines Withdrawn EP1128836A2 (fr)

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EP1290444B1 (fr) 2000-06-14 2009-10-07 Vistagen, Inc. Typage de toxicite grace a des cellules embryonnaires de foie
KR20030009531A (ko) * 2000-06-14 2003-01-29 비스타겐 인코포레이티드 중간엽 줄기 세포를 사용하는 독성 타이핑
US20020129392A1 (en) * 2000-11-15 2002-09-12 Osiris Therapeutics, Inc. Immunocompetent animals including xenogeneic implants of mesenchymal stem cells
TW200301132A (en) * 2001-12-06 2003-07-01 Sankyo Co Pharmaceutical compositions containing cells derived from human caul
US20090186004A1 (en) * 2006-04-24 2009-07-23 Stemcell Institute Inc. Method For Preparing An Organ For Transplantation
WO2012048298A2 (fr) 2010-10-08 2012-04-12 Caridianbct, Inc. Procédés et systèmes de culture et de récolte de cellules dans un système de bioréacteur à fibres creuses avec conditions de régulation
EP3068867B1 (fr) 2013-11-16 2018-04-18 Terumo BCT, Inc. Expansion de cellules dans un bioréacteur
WO2015148704A1 (fr) 2014-03-25 2015-10-01 Terumo Bct, Inc. Remplacement passif de milieu
CN106715676A (zh) 2014-09-26 2017-05-24 泰尔茂比司特公司 按计划供养
WO2017004592A1 (fr) 2015-07-02 2017-01-05 Terumo Bct, Inc. Croissance cellulaire à l'aide de stimuli mécaniques
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
CN106806947A (zh) * 2017-01-13 2017-06-09 宁夏医科大学总医院 一种低免疫原性组织工程皮肤构建方法
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
WO2018184028A2 (fr) 2017-03-31 2018-10-04 Terumo Bct, Inc. Expansion cellulaire
WO2019040747A1 (fr) * 2017-08-23 2019-02-28 Wake Forest University Health Sciences Transplantation in utero de cellules exprimant le facteur viii pour le traitement de l'hémophilie
EP3946440A4 (fr) * 2019-04-02 2023-04-19 The General Hospital Corporation Procédés pour améliorer la régénération de lymphocytes t
CN113941032B (zh) * 2020-07-16 2023-04-07 北京中卫医正科技有限公司 一种间充质干细胞复合补片及其制备方法与应用
JP2024511064A (ja) 2021-03-23 2024-03-12 テルモ ビーシーティー、インコーポレーテッド 細胞捕獲及び増殖
CN113215084B (zh) * 2021-06-11 2023-04-07 中国农业科学院兰州兽医研究所 一种羊胎儿皮肤成纤维细胞、其分离方法与应用

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