WO1997022708A1 - Cellules souches hematopoietiques embryonnaires de primate, formant des groupements - Google Patents

Cellules souches hematopoietiques embryonnaires de primate, formant des groupements Download PDF

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Publication number
WO1997022708A1
WO1997022708A1 PCT/US1996/020716 US9620716W WO9722708A1 WO 1997022708 A1 WO1997022708 A1 WO 1997022708A1 US 9620716 W US9620716 W US 9620716W WO 9722708 A1 WO9722708 A1 WO 9722708A1
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cells
population
hematopoietic
cell
subject
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PCT/US1996/020716
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English (en)
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Bruno Peault
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Systemix, Inc.
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Priority to AU14309/97A priority Critical patent/AU1430997A/en
Publication of WO1997022708A1 publication Critical patent/WO1997022708A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow

Definitions

  • the hematopoietic stem cell which is capable of giving rise to all the blood cell lineages.
  • the stem cell divides to give rise to cells which are committed to a specific lineage, or to produce more stem cells by sel -regeneration.
  • the stem cell population constitutes only a small percentage of the total number of hematopoietic cells.
  • Fetal and adult hematopoietic stem cells have been characterized by the absence or presence of markers on the cell surface. The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-.
  • the blood cell lineages include lymphoid, myeloid and erythroid cells.
  • Cells of the lymphoid lineage, B cells and T cells produce antibodies, regulate the cellular immune system and detect foreign antigens and cells.
  • the myeloid lineage which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies in the blood stream, provides protection against neoplastic cells, scavenges foreign materials from the blood stream and produces platelets.
  • the erythroid lineage provides red blood cells, which act as oxygen carriers.
  • stem cell population There are a number of clinical uses for a purified stem cell population. Gene therapy may rely on transformation of a self-renewing population such as the stem cell. Bone marrow transplantation is currently used m conjunction with chemotherapy and radiation for the treatment of leukemia and other cancer patients. The isolation of factors and receptors required for the maintenance of stem cell properties is also of interest .
  • Cells of interest include very early hematopoietic cell populations, which may be isolated from embryonic tissues .
  • U.S. Patent no. 5,061,620 describes the characterization of human stem cells. The phenotype of stem cells with rhodamme staining is discussed m Spangrude and Johnson (1990) P.N.A.S. 87:7433-7437.
  • This invention relates to a novel population of cluster-forming embryonic hematopoietic ste cells.
  • the use of such cells m medical and research applications, and novel stem cell growth factor (s) obtained or obtainable from such cell populations is also described.
  • a dense population of cells marked by the surface expression of the CD34 glycoprotein and associated with the ventral wall of the dorsal aorta m early human embryos, is provided.
  • This novel population of human lntraembryonic hematopoietic cells appears to develop independently of the yolk sac. It is a very early stage population of stem cells and indeed appears to be the real stem of the whole blood system. It is, moreover, the first example of a population of cluster-forming stem cells.
  • These cluster-forming stem cells are isolated from embryos m the early stages of gestation, e . g. from about 10-60 days, preferably from about 4-6 weeks, most preferably about five weeks from conception.
  • the cells of the invention are associated with and may be isolated from endothelial tissue obtained from such embryos, e . g. from developing lntraembryonic blood vessels, most preferably from the ventral aspect of the dorsal aorta in the pre- umbilical region.
  • These stem cells are capable of self-regeneration and, like known fetal and adult stem cells, will m the natural state or in the presence of the requisite cytokines become lineage-committed progenitors that are dedicated to differentiation and expansion into a specific lineage.
  • Primate, e . g . human, stem cells identified to date have been found m vivo only in highly dispersed form.
  • the cells of the invention exist in vi vo as clusters of undifferentiated stem cells.
  • the invention thus provides for the first time an isolated population of primate, e . g . human, cluster-forming stem cells.
  • the cells are substantially free of other cell types, usually at at least about 90% free, preferably at least about 95% free, more preferably at least about 99% free.
  • CD34 glycoprotein is a convenient indicator of hematogenous cells in both the developing and the adult human organism.
  • CD34 * cells are found m fetal liver, umbilical cord or adult mobilized peripheral blood and m both fetal and adult bone marrow.
  • CD34 + cells also include the earliest ultipotential hematopoietic stem cells, which do not express known differentiation-associated molecules but display the Thy-1 antigen at their surface. CD34 is also expressed in the adult by most vascular endothelial cells.
  • CD34 expression accompanies the early ontogeny of the human vascular system, since it was detected on endothelial cells in the yolk sac and embryo at 23 days of gestation, the earliest stage tested.
  • CD34 is uniformly expressed at the lummal aspect of endothelial cells m developing mtraembryonic blood vessels.
  • a CD34-labeled thickening of the ventral aspect of the dorsal aorta is also observed m the pre-umbilical region of the embryo at that stage and seen at a higher magnification to be composed of packed, CD34-positive, round cells in close apposition to the endothelium.
  • Intraaortic CD34-positive cell clusters were observed m six different human embryos ranging from 30 to 37 days of gestation. In each case, these clusters were localized in the pre-umbilical region, ⁇ ust underneath the anterior limb rudiment. More rostral or more caudal sections of the dorsal aorta did not contain them.
  • the cells of the invention can be differentiated from cells of the aortic endothelium.
  • the lectin from the gorse plant, Ulex europaeus which specifically binds to murine and human adult vascular endothelial cells, marks most of the cells of the endothelial lining of the 35-day human dorsal aorta, but does not show affinity for the above-described intraaortic cell clusters.
  • a reverse pattern is observed following lmmunostaining of the same region for the pan-leukocyte CD45 marker, which was expressed by the endothelium-associated cells, but not by the endothelium itself.
  • both the aortic endothelium and the associated hematopoietic CD34+ cells are seen to express CD31, the platelet-endothelial cell adhesion molecule-1 (PECAM-l) which is displayed at the surface of both vascular endothelial cells and early hematopoietic precursor cells.
  • PECAM-l platelet-endothelial cell adhesion molecule-1
  • the cells of the invention are characterized as round adherent cells locally accumulated m the pre- umbilical region of the primate, e . g. human, dorsal aorta during the early stages of gestation, usually between about 10 to 60 days, more usually between about 4 to 6 weeks, preferably about 5 weeks post-fertilization for human cells.
  • the cells are furtner characterized as being positive for CD34, characteristic of hematogenous cells (CD34 + ) ; and negative for the receptor recognized by Ulex europaeus lectin, characteristic of endothelial cells (Ulex
  • the cells of the invention are optionally further characterized as lacking at least one or more of characteristic lineage markers (Lin) for committed cells, e.g., CD15 or CD33 for myeloid pathways, CD10 or CD19 for B-cell lineage, or glycophonn A or Ulex lectin for erythroid lineage.
  • Lin characteristic lineage markers
  • a cell which is identified as lacking one or more of these lineage markers is referred to herein as being Lin .
  • the cells are identified as lacking CD38, a global indicator of committed progenitors (CD38 ) .
  • the cells of the invention can optionally be selected for by the expression of the adhesion molecules CD43, CD44 and ICAM-1.
  • the cells of the invention can also optionally be identified by their low affinity for the supravital dye rhodamine 123 (Rho l ⁇ ) .
  • the cells of the invention are thus suitably isolated and characterized as being CD34 * , CD31 + , CD45 * and Ulex . They may be further characterized as Lm and/or CD38 and/or Rho lD .
  • the cells of the invention can also be segregated from the associated endothelial cells by the use of probes for the c-myb (hematopoietic-specific) and ets - 1 (endothelium-specific) oncogenes, used for in si tu hybridization.
  • the invention comprises the progeny of the cells of the invention, when the progeny is generated m ex vivo cell culture.
  • Serial, CD34-immunostained, transverse embryo sections were used to computerize a spatial image of the pre-umbilical region of a 35-day human dorsal aorta.
  • CD 34-positive, non-endothelial intraaortic cells appear to be densely gathered on the floor of the blood vessel, and virtually absent from its dorsal aspect. Their number has been estimated, from computer data, at 831 cells on a distance spanning one to two somites m the immediate pre-umbilical area.
  • the cells of the invention are forerunners of the fetal and post-natal human blood system. Rare scattered non-endothelial CD45+ CD34+ cells from the fifth week of gestation m the liver rudiment were also detected, indicating simultaneous hepatic hematopoietic development. Based on CD 34 detection m embryonic, fetal and adult human tissues, the intraaortic cell clusters described herein represent the densest local accumulation of hematopoietic CD34+ cells encountered throughout the development of the human blood system.
  • the cells are further purified by affinity separation techniques.
  • Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
  • the antibodies may be attached to solid support to allow for crude separation.
  • the separation techniques employed should maximize the viability of the fraction to be collected.
  • Various techniques of different efficacy may be employed to obtain "relatively crude” separations. Such separations are where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present not having the marker may remain with the cell population to be retained.
  • the particular technique employed will depend upon efficiency of separation, cytotoxicity of the methodology, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
  • Procedures for separation may include, but are not limited to, magnetic separation, using antibody-coated magnetic beads, flow cytometry, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used m conjunction with a monoclonal antibody, including, but not limited to, complement and cytotoxms, and "panning" with antibody attached to a solid matrix, e . g . plate, elut ⁇ ation or any other convenient technique.
  • separation techniques include, but are not limited to, those based on differences m physical (density gradient centrifugation and counter-flow centrifugal elut ⁇ ation) , cell surface (e . g . lectin and antibody affinity) , and vital staining properties (e . g. mitochondria-binding dye rhodamme 123 and DNA-bmdmg dye Hoechst 33342) .
  • Techniques providing accurate separation include, but are not limited to, FACS, which can have varying degrees of sophistication, e . g . a plurality of color channels, low angle and obtuse light scattering detecting channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • FACS Fluorescence Activated Cell Sorting
  • One procedure that may be used is first incubating the cells for a short period of time at reduced temperatures, generally about 4'C, with saturating levels of antibodies specific for a particular committed cell type, including, but not limited to, CD3 and CD8 for T cell determinants, and then washing the cells with a FCS cushion.
  • the cells may then be suspended m a buffer medium and separated on the basis of the antibodies for the particular determinants, using various protein (s) specific for the antibodies or antibody-antigen complex.
  • the antibodies are conjugated with markers, including, but not limited to, magnetic beads, which allow for direct separation, biotin, which can be removed with avidm or streptavidin bound to a support, fluorochromes, which can be used with a FACS, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.
  • the antibody for CD34 may be labeled with one fluorochrome, while the antibodies for the various dedicated lineages may be conjugated to a different fluorochrome .
  • Fluorochromes which find use in a multi-color analysis include, but are not limited to, phycobiliproteins, e . g . phycoerythnn and allophycocyanms; fluorescein, Texas red, etc.
  • the separation can be performed m any order, but generally, the sequence of the procedure is a negative selection step to remove undesired, e . g . endothelial cells and lineage-committed cells, m combination with positive selection for CD34 and optionally CD31 and/or CD45.
  • the cells may be selected against dead cells, by employing dyes associated with dead cells, including but not limited to, propidium iodide (PI) .
  • the cells are collected in a medium comprising 2% FCS.
  • the method should permit the separation from a residual amount of the non-stem cell populations.
  • Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens.
  • the invention provides a method for isolation or identification of the cells of the invention, comprising subjecting a population of cells obtained or derived from cells locally accumulated m the pre-umbilical region of the dorsal aorta of a human embryo during the early stages of gestation, to the following steps, m any order: (i) a negative selection step whereby the population of cells is contacted with one or more antibodies, e . g .
  • the method may utilize flow cytometry techniques or any of the selection techniques described above wherein the antibodies are conjugated to a label, bead, or solid support .
  • the subject methods include the use of an antibody recognizing the cells of the invention, e .
  • CD34, CD31, and/or CD45 antibody and/or of antibody or lectin recognizing undesired cells but not the cells of the invention, e . g . antibody to a Lm marker or CD38 antibody or Ulex lectin, in a method for identifying or isolating the cells of the invention as described herein, or in a kit or device for isolating the cells of the invention.
  • kits comprising reagents, parts or devices for isolation or identification of the cells of the invention may be assembled.
  • the kit may comprise at least one positive selection antibody capable of recognizing the cells of the invention as described herein; at least one negative selection antibody which recognizes undesired cells but not the cells of the invention as described herem, and optionally, instructions for use in a method as described above.
  • hematopoietic cells in vi tro are known in the art, including IDDM, Iscove's medium, DMEM, RPMI, etc.
  • Culture medium will contain serum, e . g. FCS, bovine serum, autologous serum, etc. , or a suitable serum-free replacement. See, for example, Ponting et al . (1991) Growth Factors 4:165-173.
  • Media may also contain such additives as b-mercaptoethanol , antibiotics, vitamins, and growth factors that support the maintenance of stem cells. Growth factors may be supplied as defined factors, e.g.
  • IL- 1, IL-3, IL-6, G-CSF, GM-CSF, c-kit ligand, LIF, etc. generally at final concentrations of from about 1 ng per ml to as high as about 1 mg/ml .
  • Growth factors may be added alone, or m combinations, depending on the desired effect.
  • growth factors may be supplied by stromal or endothelial cells that secrete the necessary growth factors.
  • Medium may be conditioned medium from stromal cells, such as stromal cells that can be obtained from bone marrow, fetal thy us or fetal liver, and are known to provide for the secretion of growth factors associated with stem cell maintenance.
  • the stem cells may be co-cultured with such cells, or m medium comprising maintenance factors supporting the proliferation of stem cells.
  • the stromal cells may be allogeneic or xenogeneic. Before using m the co-culture, the mixed stromal cell preparations may be freed of hematopoietic cells employing appropriate monoclonal antibodies for removal of the undesired cells, e . g .
  • stromal cell lines may be used where the stromal lines are be autologous, allogeneic or xenogeneic.
  • the cells of the invention find use as therapeutic agents by transplantation to regenerate the hematopoietic system of a host deficient in stem cells.
  • Conditions where such therapy is used include rescuing a subject that is diseased, e . g . suffering from lymphoma, leukemia, or other neoplastic condition, and can be treated by removal or destruction of bone marrow and hematopoietic tissue by irradiation or chemotherapy, followed by engrafttnent with the cells of the invention.
  • immunodeficiency disorders or diseases e . g . combined immunodeficiency (CID) , acquired immunodeficiency syndrome (AIDS) , and congenital immunodeficiencies are treated or alleviated by engraftment of the cells of the invention m a subject suffering from such diseases or disorders.
  • Damage to the hematopoietic system e . g. as a result of radiation, chemotherapy, immunosuppressive drugs, surgery or trauma, is repaired or alleviated by transplation of the subject cells.
  • the cells of the invention may be used for the treatment of genetic diseases.
  • Genetic diseases associated with hematopoietic cells may be treated by genetic modification of autologous or allogeneic stem cells to correct the genetic defect.
  • diseases including, but not limited to, -thalassemia, sickle cell anemia, adenosine deammase deficiency, recombmase deficiency, recombmase regulatory gene deficiency, etc. may be corrected by introduction of a wild-type gene into the cells of the invention, either by homologous or random recombination.
  • Suitable drug resistance genes include, but are not limited to, the gene encoding the ultidrug resistance (MDR) protein.
  • Diseases other than those associated with hematopoietic cells may also be treated, where the disease is related to tne lack of a particular secreted product including, but not limited to, hormones, enzymes, interferon, growth factors, or the like.
  • an appropriate regulatory initiation region inducible production of the deficient protein may be achieved, so that production of the protein will parallel natural production, even though production will be in a different cell type from the cell type that normally produces such protein. It is also possible to insert a ribozyme, antisense or other message to inhibit particular gene products or susceptibility to diseases, particularly hematolymphotropic diseases.
  • T-cell receptor Alternatively, one may wish to remove a particular variable region of a T-cell receptor from the T-cell repertoire.
  • homologous recombination, or antisense or ribozyme sequence which prevents expression the expression of the particular T-cell receptor may be inhibited.
  • the stem cells could be genetically modified to introduce an antisense sequence or ribozyme which would prevent the proliferation of the pathogen m the stem cell or cells differentiated from the stem cells.
  • Methods for recombination n mammalian cells may be found m Molecular Cloning, A Laboratory Manual (1989) Sambrook, Fritsch and Maniatis, Cold Spring Harbor, NY.
  • the cells of the invention are naive and undifferentiated. As they have not yet become specific for a particular individual, they will not mature into white blood cells which attack the cells of the recipient, as may occur m graft vs. host disease following bone marrow transplant. Maturation, proliferation and differentiation of the cells of the invention into one or more selected lineages is preferably accomplished by employing a variety of hematopoietic factors and cytokines, including, but not limited to erythropoietm, leukemia inhibitory factor (LIF) , colony stimulating factors, e . g.
  • LIF leukemia inhibitory factor
  • GM-CSF GM-CSF
  • G-CSF or M- CSF interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, etc., or the like, Steel factor (MGF or c-kit ligand) , or stromal cells associated with the stem cells becoming committed to a particular lineage, or with their proliferation, maturation and differentiation.
  • MMF Steel factor
  • c-kit ligand stromal cells associated with the stem cells becoming committed to a particular lineage, or with their proliferation, maturation and differentiation.
  • the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused.
  • the cells will usually be stored in 5% DMSO and 95% fetal calf serum.
  • the cells may be expanded by use of growth factors or stromal cells associated with stem cell proliferation and differentiation.
  • the cells may optionally be modified to have at least one non-autologous gene, as described above for use m gene therapy, and such genetically altered populations and their progeny are embraced within the scope of this invention.
  • compositions comprising the cells of the invention m a form suitable for administration, e . g. by injection or infusion, to a patient m need thereof, in combination with a suitable carrier medium for use m any of the foregoing treatments. Also provided are the cells of the invention for pharmaceutical use, and use of the cells of the invention m the manufacture of a pharmaceutical preparation, e.g., for use m any of the foregoing treatments.
  • Dosages of the cells of the invention for pharmaceutical uses such as reconstitution of a the hematopoietic system of a patient m need thereof, will vary depending on the nature of the condition to be treated and the other aspects of the patient's treatment, e . g . prior radiation or chemotherapy, or co-therapy with agents having an influence on hematopoiesis, e . g. cytokines, as well as on the purity and viability of the cell population to be administered. Because, m principle, a single cell of the invention is capable of regenerating the entire hematopoietic system of a patent, the precise dosage of cells to be administered is not critical.
  • At least IO 3 cells are administered by infusion, and the patient preferably also receives cytokines, e.g., GM-CSF and IL-3. Subsequent infusions may be performed as required.
  • cytokines e.g., GM-CSF and IL-3. Subsequent infusions may be performed as required.
  • the cells of the invention are also of use as research tools in producing various hematopoietic cell lines; detecting and evaluating growth factors relevant to stem cell self-regeneration; developing hematopoietic cell lines and assaying for factors associated with hematopoietic development; providing animal models engrafted with the cells of the invention, e . g.
  • SCID mice or other immunocompromised animals which are engrafted with cells of the invention, thereby developing a model human hematopoietic system thatcan be usefully employed to study the human hematopoietic and immune systems and diseases thereof, and to test drugs which modulate or affect such systems and treat or alleviate such diseases; identifying support cells (endothelial, stromal, fibroblast) in the developing embryo that allow for this localized concentration of CD34 + cells, which support cells can then be examined for novel cytokine production or novel adhesion molecules important for regulating the growth of these cells.
  • support cells endothelial, stromal, fibroblast
  • the cells may also be used m the isolation and evaluation of factors associated with the differentiation and maturation of hematopoietic cells.
  • the cells may be used m assays to determine the activity of media, such as conditioned media, evaluate fluids for growth factor activity, involvement with dedication of lineages, or the like.
  • the subject cells may also be used m the identification of supportive cells for the isolation and evaluation of factors associated with the self-renewal of hematopoietic cells.
  • the stem cells of the invention may be used m assays to determine either autocrine or paracrine regulatory signals and evaluate responses to growth factor either from external or intrinsic protein sources; and to determine the activity of media, such as conditioned media, evaluate fluids for cell growth activity, involvement with dedication of particular lineages, or the like.
  • the subject cells produce and/or are responsive to a factor that permits replication of stem cells without differentiation.
  • This factor may be isolated from media or cell extracts of supportive cells m which a population of the cells of the invention are growing or a supernate of a population of the cells, by separating or fractionating the fluid, e . g . chromatographically.
  • the active fraction containing the desired factor is identified by measuring the growth and differentiation of stem cells in the presence and absence of such fractions, or alternatively, using comparative analysis of fluid obtained from a population of adult stem cells.
  • cDNA libraries of the cells of the invention may be prepared and compared to cDNA libraries from stem cells isolated from adults, and the gene for the factor (or its receptor) identified thereby. Growth factor or receptor genes in the cDNA libraries may optionally be amplified and identified using oligonucleotide primers based on conserved sequences within known growth factor or receptor families.
  • RNA is isolated from the subject cells. Residual DNA may be removed m accordance with conventional techniques and the polyadenylated RNA purified further, on oligo-dT sepharose, gel chromatography, etc. cDNA may then be prepared in accordance with conventional techniques using reverse transcriptase (see Sambrook, et al . , supra) . The polymerase chain reaction may be used to amplify the amount of cDNA that is produced. The cDNA is then introduced into an appropriate cloning system. The cDNA may be used for further probing of the cDNA library for a complete transcript.
  • cDNA sequence may be used to probe a genomic library to identify the genomic gene encoding the subject proteins (See, for example, Sambrook et al . supra . )
  • cDNA libraries will generally include complete or partial copies of at least about 10 2 different DNA species, more usually at least about 10 ⁇ different species, and may comprise as many as 10 4 .
  • Each cDNA may be represented from 1 to 10 3 times in the initial library.
  • the nucleic acid compositions of the subject invention may be genomic or cDNA sequences encoding all or a part of the subject adhesion and homing molecules. Fragments may be obtained of the cDNA or genomic sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, fragments will be of at least 12 nt, more usually at least 18 nt . Preferably fragments will include a functional epitope.
  • the DNA may also be used to identify cells or organs which are expressing the subject genes.
  • the manner in which one probes cells for the presence of particular nucleotide sequences, particularly as DNA, mRNA or cDNA, is well-established in the literature and does not require elaboration here.
  • mRNA may be isolated free of DNA, and by using reverse transcriptase and PCR with primers specific for the various allergens, the subject cDNAs cf may be expanded, separated on gel electrophoresis and then probed using Southern blotting or sequencing. Other techniques may also find use.
  • the DNA sequences may be inserted into an appropriate expression vector, where the native transcriptional initiation region may be employed or an exogenous transcriptional initiation region, i . e . a promoter other than the promoter which is associated with the gene in the normally occurring chromosome.
  • the promoter may be introduced by recombinant methods in vi tro, or as the result of homologous integration of the sequence into a chromosome.
  • a wide variety of transcriptional initiation regions are known for a wide variety of expression hosts, where the expression hosts may involve prokaryotes or eukaryotes, particularly E. coli , B .
  • subtili s mammalian cells, such as CHO cells, COS cells, monkey kidney cells, lymphoid cells, particularly human cell lines, and the like.
  • the promoter may be operably linked to the coding sequence of the genes of interest so as to produce a translatable mRNA transcript.
  • Expression vectors have convenient restriction sites located near the promoter sequence so as to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
  • the promoters m suitable expression vectors may be either constitutive or inducible.
  • the cDNA clones may be introduced into a variety of vectors, where the vectors will normally be characterized by the ability to provide selection of cells comprising the expression vectors .
  • the vectors may provide for extrachromosomal maintenance, particularly as plasmids m bacteria or viruses m eukaryotic cells, or for integration, particularly m mammalian cells. Where extrachromosomal maintenance is desired, an origin sequence will be provided for the replication of the plasmid, which may be a low- or high-copy plasmid.
  • markers are available for selection, particularly those which protect against toxins, more particularly against antibiotics.
  • the particular marker which is chosen will be selected m accordance with the nature of the host, where m some cases, complementation may be employed with auxotrophic hosts, e . g. yeast.
  • Introduction of the DNA construct may be by any convenient means, e . g. calcium- precipitated DNA, electroporation, fusion, transfection, infection with viral vectors, etc.
  • polyclonal and/or monoclonal antibodies capable of specifically binding to a protein(s) or fragments thereof are provided wherein the cells of the invention are used as antigen to provide an antibody recognizing an epitope characteristic of the cells of the invention.
  • antibody is used to refer both to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Monoclonal or polyclonal antibodies specifically reacting with the protein(s) may be made by methods known m the art, see for example, Antibodies: A Laboratory Manual , CSH Laboratories; Monoclonal Antibodies : Principles and Practice, 2d ed, Academic Press, New York.
  • recombinant immunoglobulins may be produced by methods known m the art, including, but not limited to, the methods described m US Patent No. 4,816,567.
  • Monoclonal antibodies with affinities of 10 s M l preferably IO 9 to 10 10 or more are preferred for cell identification or negative selection; lower affinities are preferred for positive cell selection.
  • Such antibodies using the cells of the invention are useful, e . g. m identifying and purifying populations of stem cells.
  • the invention provides for a stem cell growth factor, characterized as being non-differentiatmg, i.e., facilitating and/or promoting growth of the stem cell population without differentiation.
  • This factor is further characterized m that it is capable of being isolated from growth media or cell extract in which a population of cells of the invention is growing, or from supernate from a culture of the cells of the invention.
  • the factor thus isolated is preferably m pure or substantially pure form, e . g . at least 90%, preferably at least 95%, most preferably at least 99% pure form.
  • Immortalized cells of the invention are further useful as being cells which are responsive to a factor allowing for the regeneration of stem cells, e . g . in assays for survival, activation, or proliferation in the presence and absence of the putative stem cell growth factor.
  • parts are parts by weight, molecular weight is weight average molecular weight, temperature is m degrees centigrade; and pressure is at or near atmospheric .
  • Gestational age was estimated from developmental anatomic criteria. In all cases, informed consent to the use of the embryo in research was obtained from the patient, and embryos were collected according to the guidelines, and with the authorization, of the French Comite National D'Ethique. Gestational (i.e. post-conception) age is estimated from menstrual history and confirmed on development anatomic criteria. Embryos fixed overnight at 4'C m phosphate- buffered saline (PBS) 4% paraformaldehyde (v/v) are rinsed m PBS, dehydrated and embedded in paraffin.
  • PBS phosphate- buffered saline
  • v/v paraformaldehyde
  • Peroxidase activity is revealed with 0.025% (v/v) 3 , 3 ' -diammobenzidine (SIGMA) PBS containing 0.015% hydrogen peroxide.
  • Slides are countersta ed with Harris hematoxyl and mounted in aqueous medium (BioGenex Laboratories) for examination on an Opt ⁇ phot-2 microscope (NIKON) .
  • Endothelium-specific, biotm-labelled Ulex europaeus agglutimn I (commercially available from VECTOR) is incubated with transverse sections from an embryo as for the CD34 antibody preparation of example 1.
  • the lectin is seen to bind to aortic endothelial cells, but not to the CD34-cells clumped on the vessel floor.
  • Further immunohistology substantially as described above using antibodies to CD31 and CD45 show that CD31 is expressed on both endothelial cells and adherent intraaortic cells, but the latter also express the pan- leukocyte CD45 molecule which is absent from vascular endothelium.
  • Monoclonal antibodies to CD45 (HLE-1) and to CD31 (JC/70A) are available commercially from Becton- Dickmson and from DAKO, respectively.
  • Example 3 Growth of cluster-forming stem cells in culture
  • the trunk area containing the segment of aorta where the CD34+ cell clusters are detected is removed from human embryos of 30-40 days gestational age. Tissues are dissociated gently by pipetting. The tissue fragments are seeded in 96-well plates precoated with a confluent layer of MS-5 murine stromal cells using the techniques described in Isaad et al . Blood 81:2916 (1993) . Cultures are performed in long term culture medium [12.5% FCS (Techgen, Les Ulis, France) , 12.5% horse serum (Hyclone laboratories, Logan, UT) , 10 4 M 2-/3 mercaptoethanol in ⁇ MEM] at 37 * C.
  • the medium is changed twice weekly. No exogenous cytokines are added.
  • cell samples are harvested, pooled, counted, and processed for phenotypic analysis ( particular, CD34 and CD38 expression) , and optionally further purified, by flow cytometry using a FACSort (Becton-Dickenson) device equipped with Cellquest software.
  • FACSort Becton-Dickenson
  • Monoclonal antibodies to CD34 [HPCA-1 and phycoerythrin (PE) - HPCA-2] and to CD38 directly coupled to fluorescein isothiocyanate (FITC) suitable for use with a FACS, are commercially available from Becton-Dickonson (San Jose, CA) .
  • Tissue Exp. 1 Exp. 2 Exp. 3 preumbilical aortic 2000 460 280 region liver 350 ND 44 heart 0 ND 24 limbs 5 0 ND blood ND ND 4 umbilical cord 40 ND ND
  • the preumbilical aortic region generated a high number of progenitor cells giving rise to large colonies in methylcellulose assays.
  • a clearly higher number of clonogenic progenitors was recovered from the aortic region of the oldest embryo analyzed (35- 40 days) . At least 30-50% of the colonies included erythroid cells.
  • a high output of nonadherent, round, nucleated cells were observed in the wells, more than 10% of which expressed CD34, as detected by flow cytometry.
  • the production of clonogenic progenitors and nonadherent CD34+ cells was strikingly lower in the wells seeded with liver cells dissected from the same embryos.

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Abstract

L'invention concerne de nouvelles cellules souches formant des groupements, ainsi que le facteur de croissance isolé à partir desdites cellules.
PCT/US1996/020716 1995-12-19 1996-12-19 Cellules souches hematopoietiques embryonnaires de primate, formant des groupements WO1997022708A1 (fr)

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AU14309/97A AU1430997A (en) 1995-12-19 1996-12-19 Primate cluster-forming embryonic hematopoietic stem cells

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1192243A1 (fr) * 1999-06-23 2002-04-03 Oregon Health and Science University Procede destine a promouvoir l'hematopoiese
US6821513B1 (en) 1999-06-23 2004-11-23 Oregon Health & Science University Method for enhancing hematopoiesis

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US5061620A (en) * 1990-03-30 1991-10-29 Systemix, Inc. Human hematopoietic stem cell
US5199942A (en) * 1991-06-07 1993-04-06 Immunex Corporation Method for improving autologous transplantation

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US5061620A (en) * 1990-03-30 1991-10-29 Systemix, Inc. Human hematopoietic stem cell
US5199942A (en) * 1991-06-07 1993-04-06 Immunex Corporation Method for improving autologous transplantation

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Title
ANNUAL. REV. CELL. DEV. BIOL., 1995, Vol. 11, MORRISON S. et al., "The Biology of Hematopoietic Stem Cells", pages 35-71. *
BLOOD, 01 November 1993, Vol. 82, No. 9, WATT et al., "The Heparin Binding PECAM-1 Adhesion Molecule is Expressed by CD34+ Hematopoietic Precursor Cells with Early Myeloid and B-Lymphoid Cell Phenotypes", pages 2649-2663. *
COMPTES RENDUS DES SEANCES DEL LA SOCIETE DE BIOLOGIE ET DU SES FILIALES, 1995, Vol. 189, CHARBORD P. et al., "Early Ontogeny of the Human Hematopoietic System", pages 601-609. *
LABORATORY INVESTIGATION, 1982, Vol. 47, No. 1, HOLTHOFER H. et al., "Ulex Europaeus I Lectin as a Marker for Vascular Endothelium in Human Tissues", pages 60-66. *
NATURE, 01 July 1993, Vol. 364, GODIN I., "Para-Aortic Splanchnopleura from Early Mouse Embryos Contains B1a Cell Progenitors", pages 67-70. *
NATURE, 01 July 1993, Vol. 364, MEDVINSKY A. et al. "An Early Pre-Liver Intra-Embryonic Source of CFU-S in the Developing Mouse", pages 64-67. *
ROITT I, "Essential Immunology", LONDON: BLACKWELL SCIENTIFIC PUBLICATIONS, 1994, page 163. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1192243A1 (fr) * 1999-06-23 2002-04-03 Oregon Health and Science University Procede destine a promouvoir l'hematopoiese
EP1192243A4 (fr) * 1999-06-23 2003-05-28 Univ Oregon Health & Science Procede destine a promouvoir l'hematopoiese
US6821513B1 (en) 1999-06-23 2004-11-23 Oregon Health & Science University Method for enhancing hematopoiesis

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