EP1370642A2 - Pluripotente erwachsene stammzellen aus regenerationgeweben - Google Patents

Pluripotente erwachsene stammzellen aus regenerationgeweben

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Publication number
EP1370642A2
EP1370642A2 EP02712824A EP02712824A EP1370642A2 EP 1370642 A2 EP1370642 A2 EP 1370642A2 EP 02712824 A EP02712824 A EP 02712824A EP 02712824 A EP02712824 A EP 02712824A EP 1370642 A2 EP1370642 A2 EP 1370642A2
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Prior art keywords
cells
adult stem
stem cell
composition
pluripotent
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English (en)
French (fr)
Inventor
Bernat Soria Escoms
Genoveva Berna Amoros
Juan Antonio Reig Macia
Franz Martin Bermudo
Roberto Ensenat Wasser
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SORIA ESCOMS, BERNAT
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Cardion AG
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    • 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
    • C12N5/0679—Cells of the gastro-intestinal tract
    • C12N5/068—Stem cells; Progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
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    • 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
    • C12N5/0676—Pancreatic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • 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"
    • C12N2502/1323—Adult fibroblasts
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/23—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Definitions

  • Pluripotent adult stem cells derived from regenerative tissue Pluripotent adult stem cells derived from regenerative tissue
  • the present invention relates generally to isolated, enriched, mammalian, preferably human, adult stem cell and progenitor cell populations derived from regenerative tissue, having pluripotent stem cell-like characteristics, their differentiation into any cell type, and therapeutic use thereof.
  • Regenerative stem cell populations constitute only a small percentage of the total number of cells, but are of immense interest because of their ability to repopulate the body. There is significant commercial interest in these methods because stem cells have a number of clinical uses. Using stem cells for generation of organs and tissues for transplantation provides a promising alternative therapy for diabetes, neuronal disease, liver disease, heart disease and autoimmune disorders, to name a few. Diabetes, for instance is an excellent candidate for stem cell treatment (Soria et al. (2001) Diabetalogia 44: 407-415; Soria et al. (2000) Eur. J. Physiol. 440: 1-18; and Berna et al. (2001) Biomed. Pharmacother. 55: 206-212).
  • Regenerative stem cells can be found in many adult tissues, participating in normal replacement due to physiological turnover or in response to injury.
  • a mouse model of intestinal stem cells has been developed (Roth et al. (1991) Cell Biol. 88: 9407-9411; and Slorach et al. (1999) J. Cell Sci. 11 : 3029-3038).
  • neural stem cells from the adult mouse brain produced hematopoietic progeny and incorporated into an embryo, contributing to all the tissues of the resulting chimeric mouse (Bjomson et al. (1999) Science 283: 534; and Clarke et al. (2000) Science 288: 1666).
  • clonally derived neural stem cells from mice and humans can produce skeletal myotubes in vitro and in vivo (Galli et al. (2000) Nat. Neurosci. 3: 986).
  • bone marrow cells can differentiate into myogenic progenitors, myocytes, vascular structures, neurons, or liver cells (Ferrari et al. (1998) Science 279: 1528; Orlic et al. (2001) Proc. Natl. Acad. Sci. USA 98: 10344; Woodbury et al. (2000) Neurosci. Res. 61 : 364; and Alison et al. (2000) Nature 404: 257).
  • stem cells isolated from skeletal muscle have shown hematopoietic potential (Jackson et al. (1999) Proc. Natl. Acad. Sci. USA 96: 14482).
  • An object of the present invention is to provide isolated pluripotent adult stem cell and progenitor cell populations, derived from regenerative tissue, which can differentiate into any cell type, and methods for isolating and enriching pluripotent adult stem cell and progenitor cell populations.
  • the object is achieved by means of providing an isolated pluripotent adult stem cell or stem cell population and a composition comprising said pluripotent adult stem cell or stem cell population, derived from regenerative tissue, and having alkaline phosphatase activity, high levels of telomerase activity and the ability to form derivatives of all three embryonic germ layers and/or the ability to form an embryoid body.
  • the object is further achieved by a method for isolating and enriching the pluripotent adult stem cell population, comprising the steps of: a) isolating a population derived from regenerative tissue containing somatic adult stem cells; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer; c) selecting and replating colonies which have a pluripotent morphology; wherein the cells of the selected colonies are preferably expanded over 2-100 passages, very particularly over 2-15 passages.
  • Another object of the present invention is to provide a differentiated cell population and methods for selecting and differentiating the pluripotent adult stem cell population.
  • the object is achieved by a method for selection and differentiation of a pluripotent adult stem cell population to a desired differentiated cell population, comprising the steps of: a) isolating a population derived from regenerative tissue containing somatic adult stem cells; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer; c) selecting and replating colonies which have a pluripotent morphology; d) causing differentiation by deleting the fibroblast feeder layer; and e) selecting the cells of said desired cell lineage.
  • the object is further achieved by a method for selection and differentiation of a pluripotent adult stem cell population to a desired differentiated cell population, comprising the steps of: a) isolating a population derived from regenerative tissue containing somatic adult stem cells; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer; c) selecting and replating colonies which have a pluripotent morphology; d) transfecting cells in selected colonies to introduce a marker gene that enables the selection of one desired cell lineage from the other cell lineages that result from differentiation of the stem cells; e) causing differentiation by deleting the fibroblast feeder layer and culturing at high density in suspension; and f) selecting the cells of said desired cell lineage based on the marker gene.
  • the object is further achieved by a method for selection and differentiation of a pluripotent adult stem cell population to a desired differentiated cell population, comprising the steps of: a) isolating a population derived from regenerative tissue containing somatic adult stem cells; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer; c) selecting and replating colonies which have a pluripotent morphology; d) transfecting cells in selected colonies to introduce a lineage- specific gene for the purpose of driving differentiation toward a chosen cell lineage, and a marker gene for the purpose of selection; e) causing differentiation by deleting the fibroblast feeder layer and culturing at high density in suspension; and f) selecting the enriched chosen cell lineage based on the marker gene.
  • Another objects of the invention are to provide a method of treating a disease, comprising administering said pluripotent adult stem cell population or said differentiated cell population to a patient in need thereof, and to provide a therapeutic and/or diagnostic composition comprising said pluripotent or said differentiated cell population.
  • the present invention relates to the isolation of pluripotent adult stem cell and progenitor cell populations, derived from regenerative tissue, which can differentiate into any cell type, and methods for the isolation and enrichment of pluripotent adult stem cell and progenitor cell populations.
  • the isolated pluripotent adult stem cell or stem cell population is derived from regenerative tissue, and has alkaline phosphatase activity, high levels of telomerase activity and the ability to form derivatives of all three embryonic germ layers.
  • the isolated pluripotent adult stem cell population also has the ability to form an embryoid body.
  • the method for isolating and enriching the pluripotent adult stem cell population comprises the steps of: a) isolating a population derived from regenerative tissue containing somatic adult stem cells, preferably human; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer, preferably human; c) selecting and replating colonies which have a pluripotent morphology; wherein the cells of the selected colonies are preferably expanded over 2-100 passages, more preferably over 2-15 passages and most preferably over 10-15 passages. Specific methods for pursuing each step are provided for instance, in the Examples herein.
  • the undifferentiated cells are mammalian cells, and even more preferably, human cells, which have the potential to differentiate into any cell lineage when subjected to differentiating conditions.
  • the undifferentiated cells preferably can proliferate and propagate in vitro, and can maintain an undifferentiated state when cultured up to 15 passages on a fibroblast feeder layer.
  • the undifferentiated cells preferably have a total expansion capacity of more than 1x10 10 , more preferably of more than 1x10 11 .
  • the undifferentiated cells express SSEA-3, and preferably can express any other characteristic stem cell markers known in the art.
  • the regenerative tissue can be selected from the group consisting of peripheral blood, cord blood, bone marrow, brain, skin, retina, hair papilla, muscle, liver, pancreas, epithelium.
  • the regenerative tissue are epithelia, preferably mammalian intestinal epithelia, most preferably human intestinal epithelia derived from the small intestine, in particular, derived from crypts.
  • HISC adult human intestinal stem cells
  • Epithelia are usually composed of several distinct cell types, and the ability to form all of them is an aspect of stem cell behavior.
  • the mammalian intestinal epithelium is a rapidly proliferating tissue, with stem cells residing at the crypt base and giving rise to all cell types found within the crypt (Bach et al. (2000) Carcinogenesis 21 : 469).
  • stem cell is meant, in accordance with the present invention, a cell that can undergo self-renewing cell division, for an indefinite time, to give rise to phenotypically and genotypically identical daughter cells, and can ultimately differentiate into at least one final cell type.
  • the quintessential stem cell is the embryonic stem (ES) cell, as it has unlimited self- renewal and multipotent and/or pluripotent differentiation potential, thus possessing the capability of developing into any organ, tissue, or cell type.
  • ES embryonic stem
  • EG embryonic germ
  • ES and EG cells have been derived from mice and, more recently, from non-human primates and humans (Evans et al. (1981) Nature 292: 154-6; Matsui et al. (1991) Nature 353: 750-2; Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 7844-8; Thomson et al. (1998) Science 282: 1145-7; and Shamblott et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13726-31).
  • Stem cells have been identified in most organs and tissues. The best-characterized is the hematopoietic stem cell, which can give rise to any of the different types of terminally differentiated blood cells. This is a mesoderm-derived cell, purified based on cell surface markers and functional characteristics (Hill et al. (1996) Exp. Hematol. 24: 936-43). Also well-characterized are the neural stem cell and a number of mesenchymal stem cells derived from multiple sources (Flax et al. (1998) Nature Biotechnol. 16: 1033-1039; Clarke et al. (2000) Science 288: 1666; Bruder et al. (1997) J. Cell. Biochem.
  • adult stem cells and progenitor cell populations are to be understood, in accordance with the present invention, as meaning cells that can be derived from any source of adult tissue or organ, can replicate as undifferentiated cells, and have the potential to differentiate into at least one, preferably multiple, cell lineages.
  • “In vitro” means, for the purposes of the present invention, a conversion, i.e. a reaction, which proceeds outside a live organism.
  • Regenerative tissue is a mammalian, preferably human, fluid, tissue or organ, which is preferably selected from the group consisting of peripheral blood, cord blood, bone marrow, brain, skin, retina, hair papilla, muscle, liver, pancreas and intestine.
  • human intestinal epithelia derived from the small intestine are most preferred. Such intestinal stem cells reside just above the crypt base in the small intestine and/or at the crypt base in the colon.
  • HISC can overcome their ectodermal commitment and show a very broad plasticity that is not restricted to ectodermal derivatives.
  • the preferred HISC in spite of being adult stem cells, have an enormous capacity to proliferate. These cells preferably can divide at least 50-80 times, more preferably 60- 70 times, without losing their ability to proliferate and differentiate. After about 15 passages, the cells may become senescent and telomerase activity may decrease. Changes in the environment could have an influence on loss of potency; it has been calculated that during a human life-time, approximately 5,000 intestinal stem-cell divisions occur (Booth and Potten (2000) J. Clin. Invest. 105: 1493). Furthermore, the invention relates to a pluripotent adult stem cell or pluripotent adult stem cell population, wherein the cell can be induced to differentiate to form at least one differentiated cell type of mesodermal, ectodermal and endodermal origin.
  • the pluripotent adult stem cell or stem cell population can be used to generate differentiated cell lineages.
  • the differentiated cell types can be identified or selected by any method known in the art.
  • lineage specific markers can be used for sorting methods such as fluorescent activated cell sorting (FACS) or other surface antigen based enrichment.
  • FACS fluorescent activated cell sorting
  • Other examples include DNA chip technology, which can be used to detect lineage-specific genes, or reverse transcription polymerase chain reaction (RT-PCR), which can be used to analyze lineage-specific gene expression.
  • RT-PCR reverse transcription polymerase chain reaction
  • the object is preferably achieved by a method for selection and differentiation of a pluripotent adult stem cell population to a desired differentiated cell population, comprising the steps of: a) isolating a population derived from regenerative tissue containing somatic adult stem cells; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer; c) selecting and replating colonies which have a pluripotent morphology; d) causing differentiation by deleting the fibroblast feeder layer; and e) selecting the cells of said desired cell lineage.
  • such a differentiated cell lineage can be obtained by selecting and differentiating a pluripotent adult stem cell population to a desired differentiated cell population, by: a) isolating a population derived from regenerative tissue containing somatic adult stem cells, preferably human; b) plating and culturing the cells of a) on mitotically inactivated fibroblast feeder layer, preferably human; c) selecting and replating colonies which have a pluripotent morphology; d) transfecting cells in selected colonies to introduce a marker gene that enables the selection of one desired cell lineage from the other cell lineages that result from differentiation of the stem cells; e) causing differentiation by deleting the fibroblast feeder layer and culturing at high density in suspension; and f) selecting the cells of said desired cell lineage based on the marker gene.
  • the desired cell lineage for example can be neuronal, myocardial, hepatic, pancreatic, pancreatic islet, renal, or dermal.
  • the marker gene is under the control of a promoter that causes expression of the marker gene to enable selection of one desired cell lineage from other cell lineages. This is achieved by using a promoter which is activated only in the desired cell lineage. Furthermore, the marker gene can confer antibiotic resistance, wherein selection includes contacting the mixed differentiated cellular population with an antibiotic to which cells of the desired cell lineage have been conferred resistance by the expression of the first marker gene, thereby killing cells of other cell lineages, but not cells of the desired one cell lineage that express the marker gene.
  • WO 95/14079 The cell preparation and process for the differentiation and selection thereof is disclosed in WO 95/14079, which is encompassed in the disclosure of the present invention. Therefore a further aspect of the invention relates to a method for the selection and differentiation of pluripotent adult stem cells to a desired differentiated cell lineage comprising the steps a) - f), by using the indicated promoter (as in Table 1) for obtaining the desired differentiated cell lineage.
  • a differentiated cell lineage can be obtained by selecting and differentiating a pluripotent adult stem cell population to a desired differentiated cell population by performing steps a) - c) as above, and then, as step (d), transfecting cells in selected colonies to introduce a lineage-specific gene for the purpose of driving differentiation toward a chosen cell lineage, and a marker gene for the purpose of selection.
  • the cells or cell population can then be differentiated and selected according to steps e) and f).
  • the marker gene can confer antibiotic resistance, wherein selection includes contacting the mixed differentiated cellular population with an antibiotic to which cells of the desired cell lineage have been conferred resistance by the expression of said lineage-specific gene, thereby killing cells of the other cell lineages, but not cells of the desired, one cell lineage that express the lineage-specific gene.
  • the following constructs can be used to drive undifferentiated stem cells toward a pancreatic, insulin-secreting fate: CMV-pdx, L32-pdx, ⁇ -actin-pdx, CMV-cdx, hlns-pGK-hygro, where CMV (cytomegalovirus), L32 and ⁇ -actin are constitutive promoters, and pdx (pancreas/duodenum homeobox) and hlns (human insulin) are pancreatic cell-specific genes. Suitable genes for these constructs are described for instance in the literature and are known to those of skill in the art. See e.g., Sakakibara et al. (1996) Dev. Biol.
  • Pluripotent adult stem cells can also be differentiated by any method known in the art. For instance, co-culture with differentiated cells, gene knockout, and the addition of growth factors or other modification of culture conditions can be used in the differentiation of stem cells.
  • Another subject matter of the invention is a drug product which comprises pluripotent adult stem cells as mentioned before and/or differentiated cells as mentioned before wherein the differentiated cells are selected from the group consisting of neuronal, myocardial, hepatic, pancreatic (including islet cells), renal and/or dermal cells.
  • the drug product preferably further comprises suitable additives, adjuvants and/or a pharmaceutically acceptable vehicle.
  • the drug product can be used for treatment of diseases such as diabetes, neuronal diseases, liver diseases, heart diseases and/or autoimmune disorders.
  • Subject matter of the invention is further a process for the preparation of such a drug product.
  • Another subject matter of the present invention is a method of treating a disease, wherein pluripotent cells as mentioned before or differentiated cells as mentioned before or a composition containing these cells are administered to a patient in need thereof.
  • the differentiated cell or cell population is here preferably a neuronal, myocardial, hepatic, pancreatic (including islet cells), renal, or dermal differentiated cell or cell population.
  • the disease can be diabetes, neuronal disease, liver disease, heart disease or autoimmune disorders.
  • the therapy can be human cell or gene therapy, preferably in transplantation therapy.
  • the invention additionally relates to providing a diagnostic composition comprising the pluripotent cell or cell population or the differentiated cell or cell population or a composition comprising at least one of these cells or cell populations.
  • the composition may contain a pharmaceutically acceptable vehicle.
  • vehicle is meant a carrier for the administration of differentiated cells, preferably sterile isotonic saline.
  • the composition may also contain reagents for determining the physiological or morphological response of the differentiated cells or cell line to mutagenic, toxic or beneficial agents or treatments.
  • A Phase contrast image of human intestinal epithelial cells isolated from fresh small intestine specimens obtained from cadaveric donors.
  • B Phase contrast image of an HISC-2 colony showing pluripotent-like morphology after 23 days of culture.
  • C HISC-2 colony (indicated by arrow) showing histochemical staining for alkaline phosphatase. The fibroblast feeder layer (indicated by triangle) was always negative for alkaline phosphatase staining.
  • Figure 2 shows some adult human stem cells stained with an antibody recognizing the SSEA-4 epitope (A) and a phase contrast view of the same field of cells (B).
  • Figure 3 shows alkaline phosphatase staining of a human intestinal stem cell EB (A) and a negative control (B).
  • Adult human stem cells after 6 days formed EBs and were subsequently differentiated by plating at high density, in the absence of fibroblasts, on gelatin-treated tissue culture plates. After 1-3 weeks of culture in these conditions a variety of differentiated cells appeared.
  • the structures identified were (A) hyaline-like structures (indicated by arrow), (B) neural ganglionic-like structures with elongated processes that extended out from their cell bodies forming networks (indicated by arrow), and (C) secretory glandular-like structures.
  • HISC-2 cells strongly reacted in immunocytochemistry assays with monoclonal antibodies (mAb) against ectoderm proteins, such as neurofilament-200 (A), syntaxin (B), and glial fibrillary acidic protein (C), and against mesoderm proteins, such as vimentin (C), actin (D), and desmin (E).
  • mAb monoclonal antibodies
  • ectoderm proteins such as neurofilament-200 (A), syntaxin (B), and glial fibrillary acidic protein (C)
  • mesoderm proteins such as vimentin (C), actin (D), and desmin (E).
  • F -fetoprotein
  • Figure 6 shows G418 selection of human intestinal stem cells using a human atrial natriuretic factor (hANF) promoter (A) and a human insulin (hlns) promoter (B).
  • hANF human atrial natriuretic factor
  • hlns human insulin
  • the mucosa was dissected from the submucosa, cut into strips that were rinsed again in 100 ml of sterilized PBS, supplemented with 250 ng/ml amphotericin B (Sigma), at 37 9 C, and then incubated in 20 ml of PBS, supplemented with 250 ng/ml amphotericin B and containing 10 mM dithiothreitol (Sigma), for 30 minutes at room temperature.
  • the strips were rinsed again in 100 ml of sterilized PBS supplemented with 250 ng/ml amphotericin B, at 37 e C and then incubated for 45 minutes in 1 mM EDTA (Sigma) plus 0.25% trypsin (Gibco-BRL) in Dulbecco's modified essential medium (DMEM) (Gibco-BRL) at 37 9 C.
  • DMEM Dulbecco's modified essential medium
  • a line of human fibroblasts was isolated from the piece of duodenum of a donor. For isolating this cell line, 10 7 cells obtained from the previous protocol, were plated onto 100-mm tissue culture dishes (25020-100, Corning) and cultured in DMEM supplemented with 10% fetal bovine serum (Gibco-BRL), 2 mM glutamine (Gibco-BRL), 100 lU/ml penicillin (Gibco-BRL), 0.1 mg/ml streptomycin (Gibco-BRL) and 250 ng/ml of amphotericin B (Sigma). Every 48 hours, the culture medium was changed. After ten days of culture, fibroblast growth was evident.
  • human fibroblasts were selected, transferred to new dishes and expanded by culturing in the fibroblast culture medium.
  • 5 X 10 6 fibroblasts were plated onto 100-mm tissue culture dishes, cultured in the same fibroblast culture medium, and grown to confluency (3-5 days). The medium was changed every 24 hours. When confluent, the fibroblasts were trypsinized and split.
  • isolated intestinal epithelial cells died rapidly, but after 3 days of culture, 27% of the cells remained as monolayers. To assess the purity of these cells, they were stained for the epithelial cell marker, cytokeratin-18, with specific primary mAb (Sigma) and localized with fluorescent (TRITC) secondary antibody (Sigma) by confocal microscopy. The percentage of cells positive for cytokeratin-18 was determined by immunocytochemistry to be 95%.
  • epithelial cells continued proliferating. After this time, small colonies with a morphology resembling pluripotent stem cells were occasionally observed growing from the intestinal epithelial cell cultures. These cells were selected, transferred to new dishes and expanded by culturing in the same culture medium. For each passage, 2 x 10 6 cells were cultured in tissue culture flasks (Falcon 75 cm 2 , 353084, Becton Dickinson), on new inactivated human fibroblast feeder layer (75.000 cells/cm 2 ), in the same culture medium, and grown to confluence (5-7 days). The medium was changed every 48 hours. When confluent, the cells were trypsinized and split. Using this methodology, four clones were established (clones: HISC 1 to 4; Table 3). Although the four clones showed similar characteristics, only data from clone HISC-2 are shown.
  • Alkaline phosphatase was detected with Vector Blue substrate (Vector Labs) and SSEA-3 was detected by immunocytochemistry with specific primary mAb (Hybridoma Bank) and localized with fluorescent (TRITC) secondary antibody (Sigma) by confocal microscopy.
  • SSEA-1 , SSEA-2, GCTM-2, TRA-1-60 and TRA-1-81 can also be used to characterize pluripotent stem cells. Shamblott et al. (1998) Proc. Natl. Acad. Sci. USA 95:13726-31.
  • telomerase activity was measured with the TRAP assay.
  • Cells were lysed as previously described.
  • TRAP analysis the reaction mixture was performed in 0.2 ml thin-walled tubes, in a 10 ⁇ l volume containing 50 ⁇ M dNTPs, TRAP buffer (final concentrations of 20 mM Tris-HCI, pH 8.3, 1.5 mM MgCls, 68 mM KCl, 0.05% Tween ® , 20.1 mM EGTA), 100 ng TS primer (AATCCGTCGAGCAGAGTT) (SEQ ID NO: 1), 100 ng CX primer (CCCTTACCCTTACCCTTACCCTAA) (SEQ ID NO: 2) labeled with HEX phosphoramidites (Perkin-Elmer Applied Biosystems), 2.5 U AmpliTaq Golg (Perkin
  • the tubes were incubated at room temperature for 20 min, then heated to 95 C for 10 min, followed by 35 cycles at 94 Q C for 30 sec, 50 9 C for 30 sec and 72 Q C for 45 sec, with a final extension step at 72 9 C for 1 min in a Techne Thermal Cycler.
  • an electrophoresis was carried out in an ABI PRISM 377 DNA sequencer (Perkin-Elmer). The samples were run in the GS 36C- 2400 module for 3 h. DNA fragment analysis was performed using GeneScan 2.1 software (Applied Biosystems). Other methods for characterization of isolated HISC can also be employed. Telomere length can be determined by Southern blotting for telomere restriction fragment.
  • Quantitative RT-PCR can be used to confirm the origin and character of isolated stem cells by detecting expression of embryonic transcription factors such as TCF4, Sox2 and Oct-4.
  • embryonic transcription factors such as TCF4, Sox2 and Oct-4.
  • teratoma studies can be performed to demonstrate the pluripotency of cells. This data can be used to distinguish HISC from intestinal epithelial non-stem cells
  • HISC-2 cells When allowed to differentiate in vitro by culturing at high density, in plastic Petri dishes, in the absence of fibroblasts and rhLIF and in the same medium as that used for initial culture, HISC-2 cells (passages 2-12) formed EBs ( Figure 1 D). Subsequent in vitro differentiation, by plating trypsinized EBs at high density, in the absence of fibroblasts, on gelatin-treated tissue culture plates, in the last mentioned medium, for three to six days, revealed a variety of differentiated cells. Thus, in vitro differentiation occurred when HISC-2 cells were cultured in the absence of embryonic fibroblasts and at a high density, both in the presence and absence of rhLIF.
  • Hyaline structures (Figure 4A), neural ganglionic-like structures with elongated processes that extended out from cell bodies forming networks (Figure 4B), secretory glandular structures (Figure 4C) and contracting muscle, pacing at frequencies of 30-70 pulses/min., were observed.
  • the approach relies on the use of two transcriptional units, introduced on a common vector backbone into undifferentiated intestinally derived pluripotent cells.
  • the first transcriptional unit comprises a promoter expressed in undifferentiated stem cells, linked to a marker gene suitable for enrichment of cells carrying the DNA.
  • a marker gene suitable for enrichment of cells carrying the DNA In this example, the phosphoglycerate kinase promoter and a cDNA encoding resistance to hygromycin were used; the transcriptional unit is designated pGK-hygror.
  • the second transcriptional unit comprises a cell lineage-restricted promoter, linked to a marker gene suitable for enrichment of the desired cells.
  • the cardiomyocyte- restricted ⁇ -cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase were used; the transcriptional unit is designated MHC-neor.
  • Undifferentiated, intestinally derived pluripotent cells are transfected with the MHC- neor/pGK-hygror construct. Cells incorporating the DNA are enriched based on their resistance to hygromycin. Differentiation is then induced, and once evidence of cardiomyogenesis is observed (i.e. spontaneous contractile activity), the cultures are treated with G418. Since the ⁇ -MHC promoter is only active in cardiomyocytes, only these cells express aminoglycoside phosphotransferase and survive G418 treatment.
  • the genetic enrichment approach has the advantage that very long-term cultures of terminally differentiated cardiomyocytes can be generated, since all non- cardiomyocytes are eliminated from the culture. Moreover, the approach is easily amenable to gene transfer, either prior to differentiation or after the generation of terminally differentiated cells. In addition, the genetic enrichment approach is applicable to all pluripotent stem cell systems.
  • the Methods are divided into sub-sections describing: (1) the transfection and selection of undifferentiated intestinally derived pluripotent cells; (2) the "en mass” differentiation of the selected cells; and (3) the generation of highly enriched cardiomyocyte cultures.
  • the selection cassette Prior to transfecting intestinally derived pluripotent cells, the selection cassette (pMHC-neor/PGK-hygror) was digested with Xho I/Hind III and the fragment containing the entire MHC-neor/PGK-hygror sequence was isolated using a gene clean kit.
  • Intestinally derived pluripotent cells are maintained in an undifferentiated state by culturing on mitotically inactivated human fibroblast feeder layer in Growth Medium: DMEM (Gibco-BRL) supplemented with 15% fetal bovine serum (Gibco-BRL), 0.1 mM nonessential amino acids (Gibco-BRL), 0.1 mM 2-mercaptoethanol (Gibco-BRL), 2 mM glutamine (Gibco-BRL), 500 ng/ml human recombinant fibroblast growth factor-2 (rhFGF-2) (Upstate Biotech) and 1 ,000 units/ml of human recombinant leukemia inhibitory factor (rhLIF) (Sigma).
  • DMEM Gibco-BRL
  • fetal bovine serum Gibco-BRL
  • 0.1 mM nonessential amino acids Gibco-BRL
  • 0.1 mM 2-mercaptoethanol Gibco-BRL
  • MHC-neor/PGK-hygror DNA (1 ⁇ g) were mixed with salmon testes DNA (25 ⁇ g) in a total volume of 70 ⁇ l and sonicated. This mixture was added to the cells and left in the electroporation chamber on ice for 15 minutes.
  • DMEM fetal bovine serum
  • 0.1 mM nonessential amino acids Gibco-BRL
  • 0.1 mM 2-mercaptoethanol Gibco-BRL
  • 2 mM glutamine Gibco-BRL
  • 500 ng/ml human recombinant fibroblast growth factor-2 rhFGF-2 (Upstate Biotech). Cells grow in suspension under these conditions.
  • the cells were supplemented with 5 ml of Differentiation Medium on the next day to facilitate EB formation.
  • EBs were collected on the fifth day by gravity and resuspended in 10 ml of Differentiation Medium. EBs were plated in 100 mm Corning cell culture dishes at different dilutions (1 :2, 1 :5, 1 :10 etc).
  • pancreatic ⁇ cells from differentiating intestinally derived pluripotent cells.
  • Example 4 is modified such that a second expression cassette is introduced to produce a gene product coding for a transcription /growth factor involved in beta-cell differentation (for example pdx-1).
  • the first transcriptional unit comprises hlNSp-neo-pGK-hygro where hlNSp stands for the human insulin promoter and the second transcriptional unit comprises pGK-pdx-1 where pdx-1 represents the human pdx-1 gene.
  • Undifferentiated, intestinally derived pluripotent cells are transfected with the hlNSp-neo-pGK-hygro / pGK-pdx-1 constructs as described in Example 4. Cells which have incorporated the DNA are enriched based on their resistance to hygromycin, and are directed toward an insulin-secreting cell fate by their expression of the pdx-1 gene.
  • hygromycin-resistant HISC cells obtained by Method 1 of Example 4, are plated onto a 100 mm bacterial Petri dish and cultured in DMEM lacking supplemental LIF. After 8-10 days in suspension culture, the resulting EBs are plated onto plastic 100 mm cell culture dishes and allowed to attach for 5-8 days.
  • the differentiated cultures are grown in the same medium in the presence of 200 ⁇ g/ml G418 (GIBCO/BRL). Single G418 EB clones are selected, isolated into 24 well plates, and then expanded. This approach avoids the dilution of robust insulin-secreting clones by clones that are weaker insulin producers.
  • the resulting clones are trypsinized and plated in a 100 mm bacterial Petri dish and gown for 14 days in DMEM supplemented with 200 ⁇ g/ml G418 and 10 mM nicotinamide (Sigma, Madrid). Finally, the resulting clusters (pseudo-islet structures) are cultured for 1-5 days in RPMI 1640 supplemented with 10% fetal bovine serum, 10 mM nicotinamide, 200 ⁇ g/ml G418, 100 lU/ml penicillin, 0.1 mg/ml streptomycin, and 5.6 mM glucose.
  • Pancreatic ⁇ cells produced by the methods of Example 5 can be screened for insulin secretion. For static incubations, cells are plated in 12-well dishes (Corning Glass
  • the buffer is removed form the vials. Each condition is assayed in triplicate.
  • Perfusion studies are performed as described in Perrusa et al. (1999) J. Physiol. 520:470-80. Briefly, 1 x 10 5 HISC-derived insulin-secreting cells (as free floating clusters) are packed into a 1 cm diameter column and sandwiched between two layers of swollen Sephadex G-200 microcarrier beads (Sigma, St. Louis, MO). The column is perfused at a flow rate of 1 ml/min at 37 ° C with fresh modified Krebs buffer with 1% BSA plus glucose (3 or 22 mM).
  • the HISC-derived insulin-secreting cells are first perfused in 3 mM glucose for 30 min to reach a state of stable insulin release.
  • the solutions are prewarmed at 37"C and continuously gassed.
  • Insulin is assayed by radio- immunoassay (RIA) using the Coat-a-Count kit (DPC, Los Angeles, CA) that detects both rat and human insulin.
  • RIA radio- immunoassay
  • cell pellets are sonicated in 1 mM acetic acid containing 1% BSA and cellular extract is also determined by RIA. Secretion is normalized for cell number by measuring total protein in each experiment by the method of Bradford. Bradford (1976) Anal. Biochem. 72:248-54.
  • CD34 + bone marrow cells highly enriched for pluripotent stem cells Exp. Hematol.
  • Sakakibara et al. (1996) “Mouse-Musashi-1 , a neural RNA-binding protein highly enriched in the mammalian CNS stem cell” Dev. Biol. 176:230-242. Sakakibara et al. (1997) "Expression of neural RNA-binding proteins in the postnatal CNS: Implications of their roles in neuronal and glial cell development” J. Neurosci.

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