JP2006254872A - Method for promoting stem cell differentiation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for promoting stem cell differentiation because transplantation of differentiated embryonic stem cell to specific internal organs is expected to obtain important curability. <P>SOLUTION: The method comprises introducing a nucleic acid encoding Tall/Scl transcription factor in a tissue embryoid formed from undifferentiated embryonic stem cell by a vector-intervening gene transfer method, and cultivating the transduced cell in a differentiation medium. The method is useful for differentiation of ES cells of primates and can certify those long-term hematogenous reconstruction and safety of ES cells introduced with Tall/Scl cells by heterotransplantation of ES cells containing Tall/Scl into an immunodeficient mouse. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for differentiating stem cells. In particular, the present invention promotes differentiation of stem cells such as embryonic stem cells by introducing a nucleic acid encoding a Tall / Scl transcription factor into the cells and culturing the transduced cells in a differentiation medium. Regarding the method.

  Embryonic stem cells (ES cells) arise from pluripotent cells derived from mammalian early embryos and are capable of infinitely undifferentiated proliferation in vitro (Evans and Kaufman, Nature 292: 154 (1981); Martin , G., Proc. Natl. Acad. Sci. USA 78: 7634 (1981)). ES cells differentiate into cells of all lineages in vivo (Evans et al., Nature 292: 154-156 (1981); Bradley et al., Nature 309: 255-256 (1984)), and diverse cells in vitro (Doetschman et al., J. Embryol. Exp. Morph., 87: 27-45 (1985); Wobus et al., Biomed. Biochim. Acta, 47: 965-973 (1988); Robbins et al. , J. Biol. Chem., 265: 11905-11909 (1990)). Non-human primate and human embryonic stem cell lines have been established (eg Thomson and Marshall, Curr Top Dev Biol 38: 133-65 (1998); Thomson et al., Proc Natl Acad Sci USA 92: 7844-7848 ( 1995); and Thomson et al. Science 282: 1145-1147 (1998)). Primate embryonic stem cells are described, for example, in US Pat. No. 5,843,780 issued Dec. 1, 1998. Embryonic stem cells originating from monkeys are disclosed in US Patent Application Publication No. 2003/0157710 A1, published on August 21, 2003.

  Mature stem cells (also referred to as tissue stem cells, somatic stem cells or postnatal stem cells) are also isolated from a large number of mature tissues, umbilical cords, and other non-embryonic sources, and are of different tissues and cell types It has been shown that it can differentiate into

  In vitro, various cell types such as cardiac myocytes, hematopoietic progenitor cells, yolk sac, skeletal myocytes, smooth muscle cells, adipocytes, chondrocytes, endothelial cells, melanocytes, neurons, glia, islet cells, and early in Various culture conditions have been evaluated in order to induce differentiation of stem cells, particularly ES cells, into germ layers. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and morphogenic protein 4 (BMP4) have been reported to promote early or eventual hematopoiesis in mouse ES cells ( Johansson and Wiles, Mol. Cell. Biol. 15: 141-151 (1995); Faloon et al., Development 127: 1931-1941 (2000); and Nakayama et al., Blood 95: 2275-2283 (2000)). Granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), interleukin 3 (IL-3), stem cell factor (SCF), thrombopoietin (TPO), vascular endothelial growth factor (VEGF), and bone morphogenetic protein (BMP) Hematopoietic differentiation from non-human primate ES cells in the presence of cytokines and / or growth factors has been tested by Umeda et al. (Development 131: 1869-1879 (2004)). In particular, exogenous VEGF, bFGF and EPO have proven effective in this study.

  Since the successful establishment of the human embryonic stem (ES) cell line in 1998 (Thomson et al., Science 282: 1145-1147 (1998)), transplantation of differentiated embryonic stem cells to specific organs has been important Expected to have therapeutic potential.

  The present invention, at least in part, is a basic helix-loop-helix transcription factor, Tall / Scl (acute T cell lymphoblastic leukemia / stem cell leukemia transcription factor). This is based on experimental data demonstrating that introduction of a nucleic acid coding for ES into ES cells facilitates differentiation of ES cells into hematopoietic cells.

  In one aspect, the present invention provides a method for promoting differentiation of embryonic stem cells, wherein a nucleic acid encoding a Tall / Scl transcription factor is introduced into a tissue embryoid body (EB) formed from undifferentiated embryonic stem cells. And culturing an EB carrying a nucleic acid encoding TL1 / SCl in a differentiation medium until differentiation is confirmed.

  In one embodiment, the differentiation is hematopoietic differentiation.

  In another embodiment, the nucleic acid is introduced into the EB by vector-mediated gene transfer.

  In yet another embodiment, the vector is a viral vector, in particular a retroviral vector, preferably a lentiviral vector such as, for example, a VSV-G pseudotype lentiviral vector. A lentiviral vector can carry a Tall / Scl gene under the control of various promoters known in the art, including, but not limited to, EF-1α, CAG, PGK and CMV promoters are included.

  Hematopoietic differentiation is the confirmation of the presence of multiple blood cells, immunochemical testing, morphological analysis, detection of local expression of at least one embryonic marker specific to the cell type, or Any combination of such detection methods can be confirmed by methods known in the art.

  In certain embodiments, the embryonic marker is selected from the group consisting of ζ (zeta) globin, neurofilament 68 kD, and α-fetoprotein. ES cell differentiation typically involves one or more of cytokines and / or growth factors such as IL-3, IL-6, GM-CSF, G-CSF, SCF, and / or erythropoietin (EPO). It can be carried out according to any technique in any medium known in the industry.

Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd edition, J. Wiley & Sons (New York, NY 1994), provides those skilled in the art with many terms used in this application along with general criteria. .

  Those skilled in the art will recognize many methods or materials similar or equivalent to those described herein that can be used in the practice of the present invention. Indeed, the present invention is not limited in any way to the methods and materials described.

  The term “embryonic stem cells” is used herein to refer to primitive (undifferentiated) cells derived from embryos that have the potential to differentiate into a wide variety of differentiated cell types.

  The term “mature stem cell” is an undifferentiated cell found in a differentiated tissue that can regenerate itself and (with certain restrictions) any tissue that originates the cell. Used herein to refer to cells capable of differentiating into differentiated cell types.

  The term “hematopoietic stem cell” is used to refer to the stem cell from which all red blood cells and white blood cells originate.

DETAILED DESCRIPTION The practice of the present invention, unless otherwise indicated, includes conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, and biochemistry that are within the skill of the art. adopt. Such techniques are described in “Molecular Cloning: A Laboratory Manual” 2nd edition (Sambrook et al., 1989); “Animal Cell Culture” (RI Freshney, 1987); “Gene Transfer Vectors for Mammalian Cells” (JM Miller & MP Calos 1987); “Current Protocols in Molecular Biology” (FM Ausubel et al., 1987); and “Embryonic Stem Cells: Methods and Protocols” (Kursad Turksen, Humana Press, Totowa NJ, 2001) Explained.

  The present invention provides improved methods for stem cell differentiation, particularly for ES cell differentiation. Protocols for maintaining and differentiating stem cells in cell culture are known in the art. Such techniques include, but are not limited to, ES cell isolation, media preparation, ES cell passage technology, ES cell cryopreservation technology, ES cell transfection technology, ES cell and Protocols for techniques for the analysis of these differentiation derivatives, as well as techniques for the differentiation of ES cells into various cell types, including, for example, hematopoietic, neural, or cardiac cells, are included.

  Human and non-human primate ES cells are typically isolated by transferring the inner cell mass to the culture medium. Conventionally, the surface of a cell culture dish is covered with mouse fetal skin cells and treated to prevent the differentiation of ES cells (usually referred to as the “supporting cell layer”). The feeder cell layer releases nutrients during cell culture. However, ES cells can be cultured in the absence of such a feeder cell layer. After the passage of permissive time for growth, ES cells are passaged multiple times, resulting in a multipotent ES cell line that can be frozen and maintained without differentiation.

  Differentiation of ES cells into different cell types can be induced and regulated in a variety of ways, such as changing the composition of cell culture media, culturing on specific stromal cells, or modifying ES cells by inserting various genes Can do. Basic protocols for differentiation of ES cells, including differentiation media, are described in, for example, chapters 5-9 of the NIH report and Annexes B and C (Stem Cells: Scientific Progress and Future Research Directions, June 2001, http: //stemcells.nih.gov/info/scireport), the entire contents of which are expressly incorporated herein by reference.

  Briefly, ES cells, such as primate ES cells such as humans, begin to differentiate after being removed from the feeder cell layer and grown without attaching to the surface in suspension culture. The result is the formation of tissue embryoid bodies (EB) that can be separated and replate in monolayer culture and exposed to specific growth factors that affect further differentiation.

  Some growth factors induce cell types that normally arise from the embryonic ectoderm. These growth factors include, for example, retinoic acid, epidermal cell growth factor (EGF), bone morphogenetic protein 4 (BMP4), and basic fibroblast growth factor (bFGF). Other growth factors such as activin A and transforming growth factor β1 (TGF-β1) induce differentiation of cells derived from mesoderm. Hepatocyte growth factor (HGF) and nerve growth factor (NGF) promote differentiation into all three germ layers, including the endoderm. Differentiated human EB-derived cell types can be determined by their morphology, growth characteristics and expression of messenger RNA (mRNA) for specific markers (eg, Shamblott et al., Proc. Natl. Acad. Sci. USA). 95: 13726-13731 (2001)).

  Differentiation of human ES cells into hematopoietic progenitor cells includes, for example, human ES cells and mouse bone marrow stromal cells (irradiated to prevent replication) containing fetal bovine serum but without the addition of growth factors This can be achieved by co-culturing in a growth medium. Partially differentiated cells express CD34, a marker for blood cell precursors. When these partially differentiated human ES cells are replanted under conditions that allow them to form hematopoietic colonies, they differentiate into erythroblasts, macrophages, granulocytes, and megakaryocytes ( Odorico et al., Stem Cells 19: 193-204 (2001)).

  Media for differentiation into hematopoietic cells that form colonies of human ES cells have been described in several publications such as, for example, Kaufman et al. Proc. Natl. Acad. Sci. USA 98: 10716-10721 (2001). It is disclosed. Specific media for differentiation into hematopoietic cells that form colonies of primate ES cells are described in the Examples below.

Suitable cell markers for identification of primate hematopoietic stem cells, including humans, include CD34 ⁺ , CD59 ⁺ , Thy1 ⁺ , CD38 ^{low / −} , C-kit ^{− / low} , and lin ⁻ . These markers can be used to confirm hematopoietic differentiation, for example by using antibody-based detection techniques.

  According to the present invention, ES cell differentiation involves transducing a viral vector carrying the Tall / Scl gene, preferably a lentiviral vector, into an ES cell, particularly a tissue embryoid body (EB) formed from the ES cell. Promoted by.

  Vectors derived from lentiviruses are well known in the art and are considered efficient gene delivery systems. Lentiviral vectors derived from human immunodeficiency virus (HIV) (Kafri et al., Nat. Genet. 17: 314-317 (1997); Naldini et al., Science 272: 163-167 (1996); Peng et al., Gene Ther. 8 : 1456-1463 (2001)), lentiviral vectors derived from feline immunodeficiency virus (FIV) (Johnston et al., J. Viol. 73: 4991-5000 (1999); Poeschla et al., Nat. Med. 4: 354- 357 (1998); Wang et al., Am. J. Respir. Cell. Mol. Biol. 22: 129-138 (1999)) and other lentiviral vectors transfer genes to somatic cells in vitro and in vivo. It is used for. One of the attractions of lentivirus-based vectors is that they can transduce both dividing and non-dividing cells, resulting in stable integration and long-term transgene expression. For the purposes of the present invention, lentiviral vectors pseudotyped with vesicular stomatitis virus G (VSV-G) are particularly useful, although other lentiviral vectors are also included.

  Further details of the invention will be apparent from the following non-limiting examples.

Promotion of stem cell differentiation
ES cells are multipotent cells that are expected to be one of the most important transplantable cell sources for future medicine. However, preclinical studies using animal models including non-human primates are essential to assess the safety and efficacy of ES cell therapy in vivo. It has already been proved that non-human primates such as common marmoset (CM) are suitable as experimental animal models. This example focuses on the induction of hematopoietic stem cells using gene transfer in this model system.

  In order to introduce foreign DNA into CMES, VSV-G pseudotype human immunodeficiency virus (lentiviral) vector containing EF1a promoter and several hematopoietic genes such as tal1 / scl, gata1, gata2, lh2 and hoxB4 genes And was introduced into CM ES cells using the following protocol.

Common marmoset ES cells were collected from the fetal fibroblast layer irradiated with radiation (40 Gy) using 0.25% trypsin / 1 mM EDTA, and 1-2 × 10 ⁴ cells were re-inoculated in a petri dish having a diameter of 9 cm. 15 Tissue embryos in IMDM containing 1% fetal bovine serum, 200 μg / ml transferrin, 50 μg / ml ascorbic acid, 10 μg / ml insulin, 0.45 mM monothioglycol, 100 μg / ml streptomycin and 100 U / ml penicillin Acquired a body (EB). In the obtained EB, a VSV-G pseudotype lentiviral vector (Bai et al., Gene Ther) capable of expressing cDNA encoding the hematopoietic genes listed above including the cDNA of tall / scl under the promoter EF-1α. 10: 1446-1457 (2003)) was transduced in 1-2 days.

Cells transduced with cDNA were cultured for an additional 10-14 days in the absence of cytokines. The medium was replaced with fresh one every 3-4 days. Subsequently, for the CFU (colony forming unit) assay, the cells were treated with 1% methylcellulose, 30% fetal calf serum, 1% bovine serum albumin, 3 U / ml erythropoietin, 10 ⁻⁴ M 2-mercapto. Ethanol, 2 mM L-glutamine, 50 ng / ml SCF (stem cell factor), 20 ng / ml GM-CSF (granulocyte macrophage colony stimulating factor), 20 ng / ml IL (interleukin) -3, 20 ng / ml IL-6, 20 ng / ml G-CSF (granulocyte colony stimulating factor) (StemCell Technologies) was inoculated into a semi-solid medium consisting of IMDM. Cells obtained from each colony were collected, washed with IMDM medium, and cytospun onto glass slides. Cells were stained with Mei Giemsa staining solution, esterase staining solution and antibody according to standard methods (Umeda et al., Development 131 (8): 1869079 (2004)).

In the absence of cDNA transfer, no clear CFU cells were observed. Very rarely, few CFU-M (monocyte colony forming units) were observed in each dish. CFU was observed in cells into which cDNA was introduced, but hematopoietic induction from CM ES cells was dramatically increased only when an HIV vector containing tall / scl cDNA was introduced. Thus, a significant number of CFU was observed in each petri dish in which EB cells into which tal / scl had been introduced were planted. More than 40 CFU colonies (CFU: 20-30%, CFU-GM: 5-10%, CFU-M: 60-75%) were observed from 10 ⁵ EB cells. These colonies were microscopically confirmed to contain red blood cells, myeloid cells, monocyte macrophage cells, and megakaryocytes after cytospun cells were stained with May Giemsa, double esterase and anti-hemoglobin antibody. (Figs. 1-3). This data was reproducible 3 times. This result shows that introduction of tal1 / scl cDNA effectively promotes hematopoietic differentiation of marmoset ES cells, and is also useful for differentiating other primate ES cells including humans. It proves what is expected. By xenotransplanting ES cells containing tall / scl into immunodeficient mice, it is possible to confirm their long-term hematopoietic reconstitution ability and the safety of ES cells into which tall / scl cells have been introduced.

  All references cited throughout this specification are expressly incorporated herein by reference in their entirety.

FIG. 1 shows hematopoietic cells obtained from marmoset EB cells into which the Tall / Scl gene has been introduced. FIG. 2 shows identification of erythroid cells generated from marmoset EB cells into which the Tall / Scl gene has been introduced, using an anti-human hemoglobin antibody. FIG. 3 shows the identification of granulocyte macrophage cells generated from marmoset EB cells introduced with the Tall / Scl gene using the double esterase staining method.

Claims (20)

  A method for promoting the differentiation of embryonic stem cells by introducing a nucleic acid encoding Tall / Scl transcription factor into a tissue embryoid body formed from undifferentiated embryonic stem cells, and encoding Tall / Scl transcription factor The method as described above, comprising culturing a tissue embryoid body carrying a nucleic acid to be cultured in a differentiation medium until differentiation is confirmed.   2. The method of claim 1, wherein the differentiation is hematopoietic differentiation.   3. The method according to claim 2, wherein the nucleic acid is introduced into the tissue embryoid body by vector-mediated gene transfer.   4. The method of claim 3, wherein the vector is a viral vector.   5. The method of claim 4, wherein the vector is a lentiviral vector.   6. The method according to claim 5, wherein the lentiviral vector is a VSV-G pseudotype.   7. The method of claim 6, wherein the lentiviral vector comprises a Tall / Scl transcription factor gene under the control of a promoter selected from the group consisting of EF-1a, CAG, PGK and CMV promoters.   8. The method of claim 7, wherein the promoter is an EF-1a promoter.   3. The method according to claim 2, wherein differentiation is confirmed by confirming the presence of a plurality of types of blood cells.   10. The method according to claim 9, wherein the presence of multiple types of blood cells is confirmed by an immunochemical test.   10. The method according to claim 9, wherein the presence of multiple types of blood cells is confirmed by morphological analysis.   3. The method of claim 2, wherein differentiation is confirmed by detecting local expression of at least one embryonic marker specific for the cell type.   13. The method of claim 12, wherein the embryonic marker is selected from the group consisting of ζ (zeta) globin, neurofilament 68 kD, and α-fetoprotein.   The method of claim 2, wherein the differentiation medium comprises one or more cytokines and / or growth factors.   3. The method of claim 2, wherein the differentiation medium comprises one or more components selected from the group consisting of IL-3, IL-6, GM-CSF, G-CSF, SCF, and erythropoietin.   16. The method of claim 15, wherein the differentiation medium contains all of the components.   2. The method of claim 1, wherein the embryonic stem cell is a primate cell.   18. The method of claim 17, wherein the primate is a non-human primate.   19. The method of claim 18, wherein the non-human primate is a marmoset.   18. The method of claim 17, wherein the primate is a human.

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