[go: up one dir, main page]

WO1996017627A9 - Systeme destine a l'entretien, la croissance et la differentiation de cellules de moelle osseuse pluripotentes souches, parentes et a maturite, d'origine humaine ou provenant de primates - Google Patents

Systeme destine a l'entretien, la croissance et la differentiation de cellules de moelle osseuse pluripotentes souches, parentes et a maturite, d'origine humaine ou provenant de primates

Info

Publication number
WO1996017627A9
WO1996017627A9 PCT/US1995/015986 US9515986W WO9617627A9 WO 1996017627 A9 WO1996017627 A9 WO 1996017627A9 US 9515986 W US9515986 W US 9515986W WO 9617627 A9 WO9617627 A9 WO 9617627A9
Authority
WO
WIPO (PCT)
Prior art keywords
cells
human
mouse
bone marrow
stem cells
Prior art date
Application number
PCT/US1995/015986
Other languages
English (en)
Other versions
WO1996017627A3 (fr
WO1996017627A2 (fr
Filing date
Publication date
Application filed filed Critical
Priority to AU44193/96A priority Critical patent/AU4419396A/en
Priority to EP95943042A priority patent/EP0796110A2/fr
Priority to JP8517803A priority patent/JPH10510702A/ja
Publication of WO1996017627A2 publication Critical patent/WO1996017627A2/fr
Publication of WO1996017627A3 publication Critical patent/WO1996017627A3/fr
Publication of WO1996017627A9 publication Critical patent/WO1996017627A9/fr

Links

Definitions

  • the present invention relates to the use of an immortalized human cell line to create a human icroenvironment in mice for the study of human disease states.
  • cells such as human and non-human primate pluripotent stem cells can be maintained and differentiated.
  • the present invention relates to the injection of an immortalized human stromal cell line (such as Lof (11-10)) into mice to create a human microenvironment supplying human cytokines that are necessary to support the engraftment, maintenance and differentiation of human and non-human primate CD34+ stem cells.
  • an immortalized human stromal cell line such as Lof (11-10)
  • Such a human microenvironment provides a valuable animal model for the study of human hematopoiesis. Description of the Prior Art
  • mice have been used to investigate Alzheimer's disease (Wi et al. , Chung Kuo Chung Hsi I Chieh Ho Tsa Chih (China) 12:622, 1992), staphylococcal infection (Bel'skii et al. , Zh. Mikrobiol Epidemiol. Immunobiol. (USSR) 3:16, 1989), encephalopathy (Ross et al.
  • Hematopoiesis the formation of mature blood cells, involves a complex scheme of multilineage differentiation (Metcalf, Nature 339:27-30, 1989) .
  • pluripotent stem cells proliferate and differentiate into progenitor cells which then develop into different types of mature blood cells (Gordon et al. , Bone Marrow Transplant 4:335, 1989; Dexter et al. , Ann. Rev. Cell Bio. 3:423, 1987).
  • the pluripotent stem cell identified by the presence of the CD34 hematopoietic cell surface antigen, is capable of differentiating and giving rise to cells of all hematopoietic lineages (Sprangruide et al. , Science 241:58, 1988).
  • the second system involves the surgical implantation of human fetal tissues into SCID mice (McCune et al. , Science 241:1632-1639 (1988); and Kaneshima et al., Nature 348:561-562 (1990)).
  • human fetal liver, human fetal thymus and/or human fetal lymph nodes are implanted into SCID mice. These mice support the differentiation of mature human T cells and B cells.
  • the McCune system has also succeeded in sustaining active human hematopoiesis in SCID mice implanted with human fetal bone (Kyoizumi et al. , Blood 79:1704-1711 (1992)).
  • the surgical procedures, however, which must be conducted on each mouse with the McCune system are time consuming and costly.
  • the availability of fetal tissue is limited and its use makes this approach controversial.
  • the present invention relates to a method of engrafting human or non-human primate stem cells in a mouse to create an animal model useful for the study of hematopoietic cells.
  • the cells are primitive stem cells that have the capability of differentiating into all hematopoietic cells.
  • the stem cells are CD34+.
  • the method comprises irradiating the mouse with a lethal or non-lethal dose of radiation; administering to the irradiated mouse immortalized human or non-human primate bone marrow stromal cells which express cytokines supporting growth of stem cells; and administering to the irradiated mouse purified human or non-human primate stem cells.
  • the present invention relates to a method of testing the effectiveness of a gene therapy protocol against an infectious agent such as HIV.
  • the method comprises irradiating a first and a second mouse with a lethal or non-lethal dose of radiation; administering to the irradiated mice immortalized human or non-human primate bone marrow stromal cells which express cytokines supporting growth of CD34+ stem cells; introducing a proposed therapeutic gene segment into a first group of human CD34+ stem cells; purifying the cells from the first group of CD34+ stem cells expressing the gene segment; introducing a control gene segment into a second group of human CD34+ stem cells; administering the purified CD34+ stem cells expressing the gene segment to the first irradiated mouse and administering the control cells to the second irradiated mouse; challenging both irradiated mice with the retrovirus; and comparing the survival of the human CD34+ stem cells from the two mice.
  • the present invention relates to an animal model for the study of human hematopoiesis.
  • This model comprises a rodent into which human or non-human primate CD34+ stem cells have been engrafted in the present of immortalized human or non-human primate bone marrow stromal cells.
  • the rodent is a mouse, such as a SCID mouse.
  • Figure 1 shows the relative expression of cytokine mRNA transcripts in the human stromal cell line Lof (11-10) .
  • Figure 2 shows the survival of lethally irradiated SCID mice reconstituted with the human stromal cell line Lof (11-
  • Figure 3 shows the detection of Lof(11-10) cells in SCID mice by DNA polymerase chain reaction (PCR) .
  • Figure 4 shows the detection of human and monkey CD34 derived cells in the bone marrow compartment of SCID mice by reverse transcriptase (RT) -dependent RNA PCR. Lanes 1-4 show
  • Lane 5 shows mice receiving l x
  • Lanes 6-8 show mice receiving 1 x 10 Lof (11-10) cells i.p. plus 2 x 10 monkey CD34+ cells i.v.
  • the present invention relates to a method of engrafting human or non-human primate pluripotent stem cells in a rodent.
  • SCID severe combined immunodeficiency
  • the inventors developed a method for engrafting human or non- human primate pluripotent CD34+ stem cells into the SCID mouse.
  • any rodent may be used.
  • Preferred rodents include SCID mice; these mice have mutated scid genes which prevent production of mature T cells and B cells by the mice. All other hematopoietic lineages are produced as normal in these mice.
  • Pluripotent stem cells can be defined functionally as well as phenotypically. Functionally, pluripotent stem cells are those hematopoietic cells having the capability of multilineage differentiation and self-renewal. Pluripotent stem cells can also be defined phenotypically by cell surface markers. For example, human stem cells have been characterized as CD34+ CD38-, HLA-DR-, THY+, and Rhl23-.
  • human and non-human primate pluripotent stem cells are engrafted in a rodent, preferably a mouse.
  • the mouse is first irradiated with a lethal or non- lethal dose of radiation.
  • a lethal or non- lethal dose of radiation For example, each mouse can be exposed to 200 or 400 rads of gamma irradiation.
  • the mice are given a dose of immortalized cells which support the growth of stem cells.
  • the supportive cells are immortalized bone marrow stem cells which express cytokines.
  • the mice are given an injection of purified human or non-human primate CD34+ cells.
  • the cells engrafted into the rodent are preferably CD34+ stem cells obtainable from bone marrow, peripheral blood or cord blood. These cells can be isolated from human or non- human primate bone marrow or blood using methods known in the art. Other cells useful in this engraftment method include, but are not limited to, fetal liver and fetal thymus.
  • Immortalized bone marrow stromal cells are administered to the irradiated mice to provide, within the mice, a substratum on which later administered primitive hematopoietic cells, such as CD34+ stem cells, can adhere. These stromal cells also nurture such cells by producing the cytokines necessary for their growth and differentiation.
  • the immortalized bone marrow stromal cells serve as a microenvironment within the mouse where stem cells engraftment, differentiation, and self-renewal can occur.
  • the immortalized stromal cells preferably produce human cytokines.
  • Suitable immortalized bone marrow stromal cells include, but are not limited to, the Lof(11-10) cell line.
  • the Lof (11-10) cell line is preferred because these cells naturally produce human cytokines and, therefore, can support the growth of CD34+ cells in vi tro .
  • the Lof (11-10) cell line produces Il-l ⁇ , IL-13, IL-6, IL-8, GM-CSF, M-CSF, G-CSF, and SCF (stem cell factor) .
  • these cells are immortalized using the SV40 large T-cell antigen and, unlike transformed cells, do not produce tumors when administered to mice.
  • the immortalized bone marrow stem cells employed in the present invention may naturally produce the desired cytokines.
  • the bone marrow stem cells can be genetically engineered, using standard methods known in the art, to produce the desired cytokines or other desired supporting components.
  • the Lof (11-10) cell could be modified to further produce such human cytokines as IL-3, additional SCF, IL-7 or
  • the immortalized bone marrow stromal cells and the purified CD34+ cells are administered in amounts sufficient to promote engraftment of the CD34+ cells.
  • each rodent 7 5.0 x 10 bone marrow stromal cells are administered to each rodent, more preferably each rodent is given 0.1, 0.5, 1.0 or
  • each rodent is given a dosage of 0.1, 0.5, 1.0 or 5.0 x 10 5 CD34+ cells.
  • the immortalized bone marrow stromal cells are administered first intraperitoneally (i.p.), followed by sequential administration of the purified CD34+ cells intravenously (i.v.).
  • the purified CD34+ cells can be administered about 10 days after the immortalized bone marrow stromal cells are administered, preferably 2-7 days after the immortalized bone marrow stromal cells are administered.
  • the present invention is an inexpensive, straight forward, and versatile system for engrafting human and non- human primate CD34+ cells in the mouse animal model.
  • the method is relatively inexpensive as the immortalized bone marrow stromal cells are capable of growth in cell cultures.
  • Lof (11-10) cells can be grown to 1 x 10 per T225 flask, which is enough cells to introduce into one mouse.
  • the method is straight forward in that the injection of the immortalized bone marrow stromal cells is easy to perform and provides a continuous supply of human cytokines in the mouse.
  • the immortalized bone marrow stromal cells can be made to express additional human growth factor, surface receptors and/or cytokine genes.
  • mice engrafted with human stem cells such as CD34+ cells produce all lineages of human blood cells.
  • the engrafted CD34+ cells are able to proliferate and differentiate into all blood cell lineages including human T cells and human B cells, thereby establishing a human hematopoietic microenvironment in the mouse.
  • This animal model for human hematopoiesis has several uses.
  • the mice of the present invention can be used as a model for studying infectious diseases in vivo.
  • This model can also be used to screen compounds for antiviral activity in vivo .
  • This mouse model can also be used to assay for hematopoietic growth factors, immune modulators and/or immune toxins.
  • mice engrafted with human CD34+ cells can be used in the production of human monoclonal antibodies and human cytolytic T cell clones.
  • the present invention also relates to a method of testing the effectiveness of gene therapy protocols against infectious agents such as human immunodeficient virus (HIV) , human cytomegalovirus (CMV) , human papilloma virus (HPV) , and Epstein-Barr virus (EBV) .
  • HIV human immunodeficient virus
  • CMV human cytomegalovirus
  • HPV human papilloma virus
  • EBV Epstein-Barr virus
  • putative therapeutic genetic material is introduced into the human CD34+ cell and the CD34+ cells are then engrafted in a rodent to determine whether the introduction of the genetic material prolongs the survival of the CD34+ cells and their progeny in vivo .
  • the genetically engineered CD34+ cells are engrafted in, for example, a mouse as described above. The mouse is then challenged with the infectious agent and survival of the human CD34+ cells and their progeny is determined.
  • the present testing method can be used to determine the effectiveness of a gene therapy protocol against HIV, the causative agent of AIDS.
  • An effective gene therapy protocol against HIV infection would confer resistance against HIV to cells in vivo . If resistance to HIV infection can be conferred upon cells in vivo using a particular gene therapy protocol, further studies would be warranted to optimize the therapeutic benefits of the molecular intervention in AIDS patients.
  • a proposed anti-HIV gene is introduced into human CD34+ cells and the cells expressing the gene are then purified.
  • Introduction of the proposed anti-HIV gene into CD34+ cells can be accomplished by techniques standard in the art such as a viral-mediated vector system. Purification of the expressing CD34+ cells can be easily done by techniques in the art such as either drug selection or surface expression of genes co-introduced with the anti-HIV gene.
  • a control gene is introduced into a separate aliquot of human CD34+ cells.
  • the control gene for example, contains the proposed anti-HIV gene but has a frameshift which abrogates expression of the proposed anti- HIV gene.
  • Effects of the proposed anti-HIV gene and the control anti-HIV gene can be analyzed using either one or two populations of mice that have been irradiated with a lethal or non-lethal dose of radiation and given a dose of immortalized human bone marrow stromal cells which express cytokines supporting the growth of CD34+ cells. If two mice populations are used, one population is administered the human CD34+ cells expressing the proposed anti-HIV gene and the other is given the human CD34+ control cells. The mice are then challenged with HIV. The half-life and survival of the human CD34+ cells and their progeny which express the proposed anti-HIV gene are compared with the CD34+ control cells.
  • the present system can also be used to assay for the successful introduction of a therapeutic gene into the true stem cells, which are capable of self-renewal and life-long expression. This can be determined by PCR analyses of human spleen colonies growing in SCIDs 14 days after CD34+ cells injection. These colonies represent the earliest assayable cells derived from the pluripotent stem cell . Introduction of an effective therapeutic into this cell population would ensure a one-time gene therapy in recipient patients.
  • the expression of a therapeutic gene in the CD34+ population does not necessarily mean that the therapeutic is in the stem cell. Although the expression of a therapeutic would be useful in the mature cells in the blood, the life-span of these mature cells is finite. Introduction of the therapeutic into the pluripotent self-renewing stem cell insures a continuous supply of committed progenitors which in turn insures a continuous supply of mature cells in the blood armed with the therapeutic.
  • the anti-HIV gene would be present in the mature T cells and monocytic cells of an HIV infected patient, potentially allowing that individual to lead a normal life in spite of harboring the AIDS virus.
  • a functional assay for the introduction of a therapeutic into the stem cell is nonexistent and would have to wait for the outcome of a clinical trial or a bone marrow transplant in primates.
  • the use of the spleen colony assay of the present invention for human cells within the SCID spleen would greatly facilitate the development of the procedures to assay for the introduction of the therapeutic gene into human stem cells, insuring the continuous supply of committed progenitors and mature cells armed with the antiviral gene.
  • the present invention further relates to a method of treating patients exposed to radiation to reduce recovery time.
  • Radiation is currently used to treat different forms of cancer including leukemia and to treat patients undergoing bone marrow transplantations.
  • all of their bone marrow is destroyed, including their progenitor cells (which give rise to the mature cells in the blood) and the stromal cells (which nurture the stem and progenitor cells to renew, grow and differentiate) .
  • the window of time (1 to 2 months) between whole body irradiation and the growth/differentiation of the "newly" introduced bone marrow cells into the patient is the most critical time in bone marrow transplantation. This is the time when the patient is most susceptible to either rejection (host versus graft disease) or opportunistic infections (due to an incompetent immune system) .
  • Cytokine therapy has shown promise when used during the period of time between whole body irradiation and growth/ differentiation of the new cells, suggesting that the establishment of a functional stromal layer is essential to supply the cytokines needed to allow stem and progenitor cell growth.
  • the "newly" introduced bone marrow cells will, in time, establish their own stromal layer; however, the introduction of a stromal cell line expanded in vi tro and introduced with the "new" marrow may reduce this window of uncertainty.
  • the stromal cells can be obtained from the patients' own marrow or from their donor by procedures similar to those used to establish the Lof(11-10) cell line.
  • the immortalized human stromal cell line Lof (11-10) was used to reconstitute SCID mice with human and monkey CD34+ cells. It is, however, to be understood that the discussion generally applies to the reconstitution of any mouse with human cells capable of producing cytokines.
  • Example 1 Lof (11-10) Cells Produce Cytokines Which Support In Vi tro CD34+ Cell Growth
  • Lof (11-10) is a human immortalized stromal cell line established from human bone marrow by transfection of a plasmid containing a SV40 large T antigen (pSV3gt) as described by S.W. Gartner (Establishment and Characterization of Human Bone Marrow Stroma-Derived Cell Lines .Transformed Following Transfection with Cloned SV40 Early Region DNA (1984) (Ph.D. dissertation, Stanford University) . To determine whether Lof (11-10) cells produce cytokines which are required to support cell growth, Lof (11-10) cells were grown in T162 tissue culture flasks for 10 days. The expression of cytokine mRNA transcripts in the Lof (11-10) cultures was then detected using reverse transcriptase (RT) - dependent RNA PCR analysis.
  • RT reverse transcriptase
  • RNA was isolated from the cultured Lof (11- 10) cells using the RNAzol B RNA isolation solvent (Tel-Test) according to manufacturer's instructions.
  • Ten micrograms of purified RNA were incubated at 37° C for 30 minutes with 100 units of RNase-free DNase in 0.1 M sodium acetate, 5 mM MgClnet in a 50 ⁇ l reaction, followed by phenol/chloroform extraction and ethanol precipitation.
  • Samples of 5 ⁇ g of DNase-treated RNA were converted to cDNA by primer extension with 0.1 mg/ml random primers, and 600 units of Moloney urine leukemia virus RT (Bethesda Research Laboratories) .
  • RNA and the random primers were heated to 70° C for 10 minutes followed by quick cooling on ice.
  • the mixture was then incubated for 1 hour at 37° C in a 40 ⁇ l reaction volume containing 50 mM Tris-HCl (pH 8.3) , 40 mM KC1, 6 mM MgCl 2 , 10 mM DTT and 750 ⁇ M dNTPs.
  • the RT reaction was terminated by heating at 90° C for 5 minutes before PCR amplificatio .
  • IL-l ⁇ 5' -ATGGCCAAAGTTCCAGACATGTTTG (SEQ ID NO.l)
  • IL-6 5' -ATGAACTCCTTCTCCACAAGCGC (SEQ ID NO:5)
  • IL-8 5' -ATGACTTCCAAGCTGGCCGTGCT (SEQ ID NO:7)
  • TGF ⁇ 5' -ATGGTCCCCTCGGCTGGACAG (SEQ ID NO:15)
  • the PCR profile consisted of 94° C for 1 minute, 60° C for 1 minute, and 72° C for 3 minutes, for the denaturation, annealing, and extension reactions, respectively.
  • the samples were incubated at 94° C for 3 minutes before the start of the PCR profile and a final extension time of 20 minutes at 72° C was provided after the 28th PCR cycle.
  • amplified products were separated on 1.2% agarose gels in the presence of 0.5 ⁇ g/ml ethidium bromide, transferred to nylon membrane, and probed with 32P end- labeled oligodeoxyribonucleotides having the following sequences:
  • IL-le. CCTTCTATCATGTAAGCTATGGCCCACTCC (SEQ ID NO:19)
  • IL-13 GGTCCATATGAACTGAAAGCTCTCCACCTC (SEQ ID NO:20)
  • IL-6 GAAAAAGATGGATGCTTCCAATCTGG (SEQ ID NO:21)
  • IL-8 GAACTGAGAGTGATTGAGAGTGGACCACAC (SEQ ID NO:22)
  • GM-CSF CGCCTGGAGCTGTACAAGCAGGGCCTGCGG (SEQ ID NO:23)
  • G-CSF CTGCAGGCCCTGGAAGGGATCTCCCCCGAG (SEQ ID NO:24)
  • M-CSF GAGGACACCATGCGCTTCAGAGATAACACC (SEQ ID NO:25)
  • TGF ⁇ GGAACCTGCAGGTTTTTGGTGCAGGAGGAC (SEQ ID NO:26)
  • SCF CCAAAAGACTACATGATAACCCTCAAATATG (SEQ ID NO:27) .
  • the probe sequences represented regions internal to, and not overlapping with, primers used for amplification.
  • Hybridization signals were quantified directly from the nylon membrane using a Phosphorlmager scanner with ImageQuant software form Molecular Dynamics (Sunnyvale, CA) .
  • Lof (11- 10) cells produce cytokines, such as IL-1B, IL-8, M-CSF and B-ACTIN, which support the growth of cells, such as the CD34+ stem cell. Additional cytokines with short-lived mRNAs like 11-6, GM-CSF, and G-CSF are also expressed in the present of protein synthesis inhibitors.
  • Cytokine expression by Lof (11-10) cells, 5637 cells (a human bladder primary carcinoma) and GCT cells (a human fibrous histicytoma) were quantitated using ELISA immunoassays.
  • the 5637 and GCT cell lines are available from American Type Culture Collection (ATCC) , Rockville, MD. Briefly, measurement of cytokines was performed with culture supernatant using ELISA kits from R & D Systems (Minneapolis, MN) according to the manufacturer's specifications. The results of the ELISA are shown below in Table 1. TABLE 1
  • Example 2 Lof (11-10) Cells Support In Vi tro CD34+
  • the Lof (11-10) cells supported colony formation similar to the conditioned media obtained from either the 5637 or the GCT cell lines (Tables 2 and 3) . Both the 5637 and GCT lines cannot be used in the SCID mouse because of their transformed phenotype, which causes tumor formation.
  • the Lof (11-10) cells are immortalized and will not form tumors in mice.
  • Table 2 Human CD34+ Bone Marrow Derived Cells
  • each mouse was gamma irradiated with either 200 rads (non- lethal) or 400 rads (lethal) .
  • the mice were irradiated to provide an internal space within which the CD34+ cells could repopulate. Four hours after irradiation, the mice were
  • each mouse was given an intravenous (i.v.) injection of either 3 to 5 purified human CD34+ cells or 2 to 5 purified monkey CD34+ cells.
  • the bone marrow-derived CD34+ cells were obtained from the posterior iliac crest under local anesthesia from healthy donors with informed consent. Briefly, bone marrow aspiration was performed into a 20 ml syringe containing EDTA as an anticoagulant. Bone marrow cells were diluted 1:2 with PBS and were subjected to ficoll- hypaque density gradient centrifugation.
  • Interphase mononuclear cells were washed twice in PBS and were further purified using magnetic beads linked with the high affinity CD34 monoclonal antibody K6.1.
  • the monoclonal antibody K6.1 was produced by fusing SP-2/0-AG14 plasmacytoma cells ( (ATCC) , Rockville, MD) with splenocytes from a BALB/cByJ mouse (Jackson Laboratory, Bar Harbor, ME) which had been immunized with viable KG-la cells (ATCC, Rockville, MD) . After magnetic selection, beads were chemically dissociated from the cells and the cells were referred to as CD34- positive cells (Kirshenbaum et al . , J. Immunol . 14:1410, 1991) .
  • the time interval between the injection of the two cell populations was to allow engraftment of the Lof (11-10) cells before the introduction of the CD34+ cells.
  • human cytokines needed to nourish the CD34+ hematopoietic pluripotent stem, progenitor and blood lineage precursor cells were supplied.
  • the Lof (11-10) cells served as a substratum for cell "homing" and the early attachment of the CD34+ cells (1 to 10 days) during reconstitution.
  • Lof (11-10) cells were confirmed using DNA PCR. Briefly, DNA was purified from both the spleen and bone marrow cells from either control or Lof (11-10) -injected mice. Aliquots (1 ⁇ g) were amplified with 0.5 ⁇ M sequence-specific primer pairs for either T- antigen or mouse ribosomal 28S gene, in a 50 ⁇ l reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2 mM MgCl 2 , 200 mM dNTPs, and 1.5 units of Taq DNA polymerase for 28 cycles. The specific primer pairs used were as follows:
  • the PCR profile consists of 94° C for 1 minute, 60° C for 1 minute, and 72° C for 3 minutes, for the denaturation, annealing, and extension reactions, respectively.
  • the amplified products were separated on 1.2% agarose gels in the presence of 0.5 ⁇ g/ml ethidium bromide, transferred to nylon membranes, and probed with 32P end- labeled oligonucleotides for either T-antigen or mouse 28S gene, respectively.
  • the probes used were as follows:
  • the probe sequences represented regions internal to, and not overlapping with, primers used for amplification.
  • Hybridization signals were quantified directly from the nylon membranes using a Phosphorlmager scanner with ImageQuant software form Molecular Dynamics (Sunnyvale, CA) .
  • the results of the DNA PCR experiments are shown in Figure 3.
  • Lof (11-10) reconstituted 2 39 (200 rad, 4-6 wks)
  • the CD34+ cells detected in the bone marrow of the mice were derived from the human CD34+ cells as indicated by the fact that they are class 2 positive, expressing HLA-DR alpha transcripts by reverse transcriptase dependent RNA PCR. Lof (11-10) cells are class 1 positive, but class 2 negative. Additional evidence that the PCR signals observed in the bone marrow are CD34+ cells derived comes from the lack of detection of SV40 T-antigen by DNA-PCR in these preparations. Further characterization of state of differentiation of the CD34+ cells within the bone marrow will be done by following the expression of various cell surface receptors using reverse transcriptase dependent RNA PCR.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

La présente invention concerne un procédé permettant de favoriser la prise de greffe de cellules souches pluripotentes d'origine humaine ou provenant de primates, telles que des cellules CD34+, chez une souris. Ce procédé consiste à administrer, à des souris irradiées, des cellules du stroma de moelle osseuse d'origine humaine ou provenant de primates qui expriment des cytokines assurant la croissance des cellules, telles que des cellules CD34+, et à administer, de manière séquentielle ou simultanée des cellules CD34+ purifiées d'origine humaine ou provenant de primates. On peut administer les cellules du stroma et les cellules CD34+ par injections intraveineuses ou intrapéritonéales. Cette invention peut servir à évaluer des protocoles de thérapie génique pour les cellules CD34+ humaines et peut également avoir des applications relatives au traitement de patients soumis à une irradiation comme cela se produit lors d'une greffe de moelle osseuse. Cette inventin concerne également un modèle animal destiné à l'étude de l'hématopoïèse humaine.
PCT/US1995/015986 1994-12-09 1995-12-08 Systeme destine a l'entretien, la croissance et la differentiation de cellules de moelle osseuse pluripotentes souches, parentes et a maturite, d'origine humaine ou provenant de primates WO1996017627A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU44193/96A AU4419396A (en) 1994-12-09 1995-12-08 System for the maintenance, growth and differentiation of human and non-human primate pluripotent stem, progenitor and mature bone marrow cells
EP95943042A EP0796110A2 (fr) 1994-12-09 1995-12-08 Systeme destine a l'entretien, la croissance et la differentiation de cellules de moelle osseuse pluripotentes souches, parentes et a maturite, d'origine humaine ou provenant de primates
JP8517803A JPH10510702A (ja) 1994-12-09 1995-12-08 ヒトおよび非ヒト霊長類の多能性幹、前駆および成熟骨髄細胞の維持、増殖および分化のための系

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35295794A 1994-12-09 1994-12-09
US08/352,957 1994-12-09

Publications (3)

Publication Number Publication Date
WO1996017627A2 WO1996017627A2 (fr) 1996-06-13
WO1996017627A3 WO1996017627A3 (fr) 1996-10-31
WO1996017627A9 true WO1996017627A9 (fr) 1996-12-05

Family

ID=23387167

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/015986 WO1996017627A2 (fr) 1994-12-09 1995-12-08 Systeme destine a l'entretien, la croissance et la differentiation de cellules de moelle osseuse pluripotentes souches, parentes et a maturite, d'origine humaine ou provenant de primates

Country Status (5)

Country Link
EP (1) EP0796110A2 (fr)
JP (1) JPH10510702A (fr)
AU (1) AU4419396A (fr)
CA (1) CA2207216A1 (fr)
WO (1) WO1996017627A2 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1197699A (en) 1997-10-23 1999-05-10 Geron Corporation Methods and materials for the growth of primate-derived primordial stem cells
US7410798B2 (en) 2001-01-10 2008-08-12 Geron Corporation Culture system for rapid expansion of human embryonic stem cells
US6667176B1 (en) 2000-01-11 2003-12-23 Geron Corporation cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells
AU5403100A (en) * 1999-06-08 2000-12-28 Novartis Ag Enforced expression of bcl-xl
US7455983B2 (en) 2000-01-11 2008-11-25 Geron Corporation Medium for growing human embryonic stem cells
CA2417356A1 (fr) 2000-08-01 2002-02-07 Yissum Research Development Company Differentiation dirigee de cellules embryonnaires

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5399493A (en) * 1989-06-15 1995-03-21 The Regents Of The University Of Michigan Methods and compositions for the optimization of human hematopoietic progenitor cell cultures

Similar Documents

Publication Publication Date Title
CN107922925B (zh) 用于自然杀伤细胞扩增的方法
JP2002516085A (ja) 哺乳動物における造血幹細胞集団に影響を及ぼす組成物および使用法
JP2002516562A (ja) Ｔリンパ球のインビトロ増殖のための改変された迅速な拡大方法（「改変ｒｅｍ」）
TW200804599A (en) Isolation and purification of hematopoietic stem cells from post-liposuction lipoaspirates
Brenner Gene marking
Ogata et al. Long-term repopulation of hematolymphoid cells with only a few hemopoietic stem cells in mice.
CN101580817A (zh) 含有细胞因子诱导杀伤细胞的细胞群的制造方法
Lemoli et al. Expression and functional role of c‐kit ligand (SCF) in human multiple myeloma cells
US11299709B2 (en) Pluripotent stem cell and T cell differentiated therefrom and application thereof
Nienhuis et al. Gene transfer into hematopoietic stem cells
Watts et al. Hematopoietic stem cell expansion facilitates multilineage engraftment in a nonhuman primate cord blood transplantation model
WO1996017627A9 (fr) Systeme destine a l'entretien, la croissance et la differentiation de cellules de moelle osseuse pluripotentes souches, parentes et a maturite, d'origine humaine ou provenant de primates
AU4419396A (en) System for the maintenance, growth and differentiation of human and non-human primate pluripotent stem, progenitor and mature bone marrow cells
CN118853582A (zh) 基因编辑的肿瘤浸润淋巴细胞及其在免疫细胞治疗中的应用
JP2010533486A (ja) 幹細胞の生体外増殖培地
Andrews et al. Differential engraftment of genetically modified CD34+ and CD34− hematopoietic cell subsets in lethally irradiated baboons
AU694430B2 (en) Use of modified tall-104 cells to treat cancer and viral diseases
JP2012507997A (ja) 細胞、核酸構築物、前記構築物を含む細胞、及び疾患の治療において前記細胞を利用する方法
JPWO2006022330A1 (ja) 霊長類動物胚性幹細胞から樹状細胞の製造方法
US20100209396A1 (en) Method of Enhancing Proliferation and/or Hematopoietic Differentiation of Stem Cells
WO2007008309A2 (fr) Expansion de cellules souches et progenitrices par evi, des genes de type evi et setbp1
JP2003527096A (ja) Ｃ−ｍｙｃアンチセンス処理した造血幹細胞組成物および方法
JP5089389B2 (ja) 移植における免疫応答の予防および治療のための肝臓間質細胞
Kaushal et al. Providing a microenvironment for the development of human CD34+ hematopoietic cells in SCID mice
JPH04502853A (ja) 原始細胞コロニー刺激因子およびリンパ造血始原細胞