WO2001000031A1 - Cellules souches derivees du muscle squelettique - Google Patents

Cellules souches derivees du muscle squelettique Download PDF

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Publication number
WO2001000031A1
WO2001000031A1 PCT/US2000/017064 US0017064W WO0100031A1 WO 2001000031 A1 WO2001000031 A1 WO 2001000031A1 US 0017064 W US0017064 W US 0017064W WO 0100031 A1 WO0100031 A1 WO 0100031A1
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Prior art keywords
stem cells
animal
cells
muscle stem
muscle
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PCT/US2000/017064
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English (en)
Inventor
Margaret A. Goodell
Kathyjo Ann Jackson
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Baylor College Of Medicine
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Priority to AU54988/00A priority Critical patent/AU5498800A/en
Publication of WO2001000031A1 publication Critical patent/WO2001000031A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C12N5/0659—Satellite 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the discovery of novel properties and uses for stem cells derived from skeletal muscle. More particularly, the present invention relates to materials and methods for regenerating various cell types in animals with the use of muscle stem cells.
  • the materials and methods are related to the novel discovery that muscle stem cells, also called satellite cells, when removed from their in situ sites can differentiate into other cell types, such as hematopoietic cells.
  • Bone marrow transplantation is increasingly used in, for example, the treatment of hematologic malignancy, breast cancer, and other metabolic disorders.
  • Bone marrow transplantation A key feature of bone marrow transplantation is the ability of transplanted hematopoietic stem cells to generate all the lineages of the blood of the transplant recipient over a period of time. Bone marrow cells are also presently a focus of interest for gene therapy, due to the possibility that genetic modification of the stem cell can be manifested in downstream progeny. However, while hematopoietic stem cells can be transduced, the efficiency of gene transfer is currently too low to allow realistic hope of therapeutic benefit from stem cell therapy using hematopoietic stem cells.
  • bone marrow transplantation Another serious limitation in the use of bone marrow transplantation is that, for example, in autologous bone marrow transplantation, a patient's bone marrow may be contaminated with transformed cells that are difficult to remove from the bone marrow cells. For example, many leukemia cells cannot be separated from the bone marrow stem cells.
  • a major requirement of hematopoietic stem cell biology is that true stem cells must be highly proliferative and able to generate progeny that can repopulate secondary recipients (Siminovitch, et al. , 1963 ; and Spangrude, etal. 1991), although this property has well established limits (Jones, et al.1989; and Mauch, et al. , 1989).
  • regenerative stem cells can be found in many adult tissues (Keller, et al, 1992; Gage, et al, 1995; Rudland, P. S., 1987; Sigal, et al, 1992; Watt, F. M., 1998; and Schultz, et al, 1994). Although possessing substantial proliferative and differentiative capacity, such cells are thought to be committed to differentiate exclusively into the tissue in which they reside. The myogenic potential of satellite cells is well established (Allbrook, D., 1981; Bischoff, R., 1986; Mauro, A., 1961; Snow, M. H., 1977).
  • U.S. Patent Number 5,750,376 discloses methods of producing genetically modified neuronal cells by transforming neuronal stem cells.
  • stem cells that are not bone marrow or embryo derived, are not likely to be contaminated with transformed cells, and have the ability to differentiate in the body into various cell types, including all blood cell types. Further, it is desired in the art that such cells be readily and stablely transformable allowing for their use in gene therapy.
  • An object of the invention is a method of replacing blood cells in an animal comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into blood cells.
  • An object of the invention additionally is a method of replacing bone marrow stem cells in an animal, comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells are grafted to the bone marrow and/or home to the bone marrow in the animal, and differentiate in the animal into blood cells.
  • An object of the invention additionally is a method of replacing a particular differentiated cell type in an animal, comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into a particular differentiated cell type.
  • An object of the invention additionally is a method of replacing muscle cells in an animal, comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into muscle cells.
  • An object of the invention additionally is a method of gene therapy, comprising the steps of transducing muscle stem cells with nucleic acid, thereby creating transduced muscle stem cells, and administering the transduced muscle stem cells to an animal.
  • a method of replacing blood cells in an animal comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into blood cells.
  • the method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or a different animal, as is administered the muscle stem cells.
  • the muscle stem cells administered to the animal may be transduced muscle stem cells, which may be transduced after being isolated from skeletal muscle but prior to being administered to the animal.
  • the animal may be a human.
  • a method of replacing bone marrow stem cells in an animal comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells are grafted to the bone marrow and/or home to the bone marrow in the animal, and differentiate in the animal into blood cells.
  • the method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or from a different animal, as is administered the muscle stem cells.
  • the muscle stem cells administered to the animal may also be transduced muscle stem cells, which may be transduced after being isolated from skeletal muscle and prior to being administered to the animal.
  • the animal may be a human.
  • a method of replacing a particular differentiated cell type in an animal comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into a particular differentiated cell type, or multiple cell types.
  • the method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or from a different animal, as is administered the muscle stem cells.
  • the muscle stem cells may be transduced muscle stem cells, and may be transduced after being isolated from skeletal muscle and prior to being administered to the animal.
  • the particular differentiated cell type may be a neuronal cell, an endothelial cell, or a muscle cell.
  • the animal may be a human.
  • a method of replacing muscle cells in an animal comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into muscle cells.
  • the method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or a different animal, as is administered the muscle stem cells.
  • the muscle stem cells administered to the animal can be transduced muscle stem cells, which may be transduced after being isolated from skeletal muscle and prior to being administered to the animal.
  • the animal may be a human.
  • a method of gene therapy comprising the steps of transducing muscle stem cells with nucleic acid, thereby creating a transduced muscle stem cells, followed by administering the transduced muscle stem cells to an animal.
  • the transduced muscle stem cells may differentiate into a differentiated cell type which can include a blood cell type, a neuronal cell, a muscle cell, or an endothelial cell.
  • the muscle stem cells can be transduced using retroviral-mediated gene transfer.
  • the animal may be a human.
  • Figures 1A, IB, and 1C show analysis of peripheral blood following transplantation with muscle stem cells.
  • Figure 2 shows the percentage of Ly-5.1+ cells in individual mice over time.
  • Figure 3 shows the percentage of Ly-5.1+ cells.
  • Figures 4A - 4D analyze the percentages of cells with stem cell phenotype, and Ly-5.1+ phenotype.
  • gene therapy refers to all methods of gene therapy whereby genetic information is utilized to change and/or supplement the genetic makeup of an animal, frequently for therapeutic purposes. This includes, but is not limited to, the use of recombinant DNA technology to deliver new and/or altered genetic sequences to an animal, including, for example, the use of a recombinant retrovirus to carry a particular genetic sequence.
  • muscle stem cell and “muscle satellite cell” are used synonymously herein to denote what is known and understood in the art as muscle stem cells, also called muscle satellite cells, briefly, cells found surrounding muscle fibers that have been, for example, shown to be involved in muscle regeneration after injury. Such cells can be obtained from skeletal muscle of any age, although their prevalence is higher in younger individuals (Snow, M. H, 1977; Campion, D. R.,
  • blood cell(s) denotes any cells known in the art to be blood cells. This includes, but may not be limited to, cells of lymphoid and myeloid lineages, B cells, T cells and granulocytes/macrophages.
  • isolated refers to isolation as known in the art.
  • isolating muscle stem cells from muscle it denotes any separation and/or segregation of muscle stem cells from muscle tissue regardless of the degree of separation or processes and/or methods used.
  • muscle satellite cells can be isolated to be free, or essentially free, or contaminating tumor cells, thereby providing a tumor-cell free (or lean) source of hematopoietic stem cells.
  • transduce(d) and “transform(ed)” as used herein are used as understood in the art, and are used broadly herein to cover any introduction of, and result of, genetic material (regardless of its nature, e.g., DNA, RNA, viral, etc.) by any means (e.g., mechanical, chemical, viral, electromagnetic, homologous recombination, gene replacement technology, and so forth) into a cell or tissue to create a cell or tissue having (permanently or transiently, expressed or non-expressed) introduced material capable of expressing or encoding a genetic sequence.
  • genetic material regardless of its nature, e.g., DNA, RNA, viral, etc.
  • any means e.g., mechanical, chemical, viral, electromagnetic, homologous recombination, gene replacement technology, and so forth
  • muscle stem cells when removed from muscle and administered intravenously, home to the bone marrow and give rise to all of the differentiated lineages of the adult blood system. This gives rise to materials and methods for use in regenerating cell lineages in animals, including uses in restorative therapy, treatment, and gene therapy.
  • muscle stem cells are more potent at generating blood cells than are transplanted bone marrow cells.
  • One aspect of the invention provides a method of replacing blood cells in an animal comprising the step of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into blood cells.
  • the method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or a different animal, as is administered the muscle stem cells.
  • the muscle stem cells that are administered to the animal may be transduced muscle stem cells. These cells may find use in methods of gene therapy as will be understood by one skilled in the art. This method is applicable to animals including humans.
  • Another example of the practice of an embodiment of the present invention relates to a method of replacing bone marrow stem cells in an animal, comprising the step of administering muscle stem cells to an animal, wherein the muscle stem cells home to the bone marrow in the animal and differentiate in the animal into blood cells.
  • the method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or a different animal, as is administered the muscle stem cells.
  • the stem cells may be transduced stem cells, thereby providing, for example, gene therapy use as is understood in the art. This method has application in animals including humans.
  • the practice of still another embodiment of the present invention provides a method of replacing a particular differentiated cell type in an animal, comprising the steps of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into a particular differentiated cell type.
  • the stem cells may be targeted to given locations in order for the cells to differentiate into particular cell types.
  • the muscle target cells may be targeted by implanting them into select tissue sites in the animal.
  • the muscle stem cells may be grafted directly into bone marrow in the animal.
  • intravenously administered muscle stem cells will home to the bone marrow to establish and produce differentiated blood cells.
  • the muscle stem cells can be targeted by injecting them and/or otherwise implanting them into particular tissues of interest, such as muscle and neuronal tissues.
  • Other means of targeting cells are also within the scope of the invention, such as the use of targeting antibodies, cell receptors, viral means, genetically engineered means and so forth.
  • the practice of this method may also comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same, or a different, animal as is administered the muscle stem cells.
  • the muscle stem cells administered to the animal may be transduced muscle stem cells which may be used in gene therapy methods as understood in the art.
  • the particular differentiated cell types may be, for example, blood cells, including cells of lymphoid lineages, cells of myeloid lineages, B cells, T cells, and granulocytes/macrophages, neuronal cells, endothelial cells, and for example, muscle cell.
  • the muscle stem cells may be caused to differentiate into particular progeny cell types by applying various chemical and/or physical signals known in the art to cause stem cells to differentiate into particular cell types. This can include targeting the stem cells to particular locations in the body, as discussed above, and/or applying various chemical and/or physicals signals both in the body and in vitro prior to administering the cells.
  • these methods are known to those skilled in the art and include, for example, the techniques set forth in U.S. Patent Numbers 5,750,376,
  • the animals used in this method may include humans.
  • various embodiments of the practice of the present invention result in the differentiation of muscle stem cells into a number of different types of differentiated cell types, including but not limited to skeletal muscle, cardiac muscle, hematopoietic cells, including red blood cells, platelets, T cells, B cells, granulocytes, macrophages, and other blood cells, neurons, glial cells, endothelial cells, smooth muscle cells, epithelial cells, including those of mammary gland, testes, gastrointestinal tract and liver, adipocytes, cartliage, bone, stromal cells of bone marrow, thymus, spleen, mesenchymal cells of other tissues, including liver, uterus, kidney, germ cells that may produce eggs or sperms, and pancreatic cells.
  • differentiated cell types including but not limited to skeletal muscle, cardiac muscle, hematopoietic cells, including red blood cells, platelets, T cells, B cells, granulocytes, macrophages, and other blood cells, neurons,
  • inventions of the practice of the present invention include methods of method of replacing muscle cells in an animal, comprising the step of administering muscle stem cells to an animal, wherein the muscle stem cells differentiate in the animal into muscle cells.
  • This method may further comprise the step of isolating muscle stem cells from skeletal muscle prior to administering the cells to the animal.
  • the muscle stem cells may be isolated from the same animal, or a different animal, as is administered the muscle stem cells.
  • the muscle stem cells can be transduced muscle stem cells.
  • the animal may be a human or other animal.
  • Another embodiment of the practice of the present invention involves a method of gene therapy, comprising the steps of transducing muscle stem cells with nucleic acid, thereby creating transduced muscle stem cells, and administering the transduced muscle stem cells to an animal.
  • This method may further include the transduced muscle stem cells differentiating in the animal into a differentiated cell type which may include a blood cell type, a neuronal cell, a muscle cell, an endothelial cell, or any number of different types of cells that differentiate from muscle stem cells.
  • the muscle stem cells may be transduced using retroviral-mediated gene transfer, or homologous recombination and/or gene replacement therapy and other techniques known in the art.
  • the animal may be a human.
  • the method of cell therapy may be employed by methods known in the art wherein a cultured cell containing a copy of a nucleic acid sequence or amino acid sequence for therapy of cancer is introduced.
  • the cells and methods of the present invention are utilized for gene therapy.
  • the cell contains a vector wherein the gene of interest is operatively limited to a promoter.
  • the antisense sequence of the gene of interest would be operatively linked to a promoter.
  • the promoter may be consitutive, inducible or tissue-specific.
  • sequences such as a 3' UTR regulatory sequence is useful in expressing the gene of interest.
  • Means known in the art can be utilized to prevent release and absorption of the composition until it reaches the target organ or to ensure timed-release of the composition.
  • a sufficient amount of vector containing the therapeutic nucleic acid sequence is administered to provide a pharmacologically effective dose of the gene product.
  • a vector into a cell of the present invention examples include: ( 1 ) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein said vector is complexed to another entity, such as a liposome or transporter molecule.
  • physical means such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure)
  • methods wherein said vector is complexed to another entity, such as a liposome or transporter molecule.
  • the present invention provides a method of transferring a therapeutic gene to a host, which comprises administering the vector inside a cell of the present invention.
  • Effective gene transfer of a vector to a host cell in accordance with the present invention can be monitored in terms of a therapeutic effect (e.g.
  • alleviation of some symptom associated with the particular medical condition being treated or, further, by evidence of the transferred gene or expression of the gene within the host (e.g., using the polymerase chain reaction in conjunction with sequencing, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer).
  • evidence of the transferred gene or expression of the gene within the host e.g., using the polymerase chain reaction in conjunction with sequencing, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer).
  • compositions can be further approximated through analogy to compounds known to exert the desired effect.
  • actual dose and schedule can vary depending on whether the cells are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism.
  • amounts can vary in in vitro applications depending on the particular cells utilized.
  • the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis).
  • One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.
  • cells containing the therapeutic gene may also contain a suicide gene (i.e., a gene which encodes a product that can be used to destroy the cell, such as herpes simplex virus thymidine kinase).
  • a suicide gene i.e., a gene which encodes a product that can be used to destroy the cell, such as herpes simplex virus thymidine kinase.
  • expression of the therapeutic gene in a host cell can be driven by a promoter although the product of said suicide gene remains harmless in the absence of a prodrug. Once the therapy is complete or no longer desired or needed, administration of a prodrug causes the suicide gene product to become lethal to the cell.
  • suicide gene/prodrug combinations which may be used are Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.
  • HSV-tk Herpes Simplex Virus-thymidine kinase
  • ganciclovir ganciclovir
  • acyclovir or FIAU oxidoreductase and cycloheximide
  • cytosine deaminase and 5-fluorocytosine thymidine kinase thymidilate kinase (Tdk:
  • the nucleic acid for therapy is a DNA or a RNA, and it is within the scope of the present invention to include any nucleic acid for a therapeutic purpose within the cells.
  • Specific examples include but are not limited to the dystrophin nucleic acid, such as for the treatment of muscular dystrophy, or the beta-globin gene, such as for the treatment of sickle cell anemia.
  • nucleic acids include ras, myc, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl abl, Rb, CFTR, pi 6, p21, p27, p53, p57, p73, C-CAM, APC, CTS-1, zacl, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF and thymidine kinase.
  • the nucleic acid for therapy is p53, which is often mutated in cancer.
  • a compound to stabilize the DNA binding domain of p53 in an active conformation is furthermore delivered via cells or methods of the present invention to enable a mutant p53 in a tumor cell to activate transcription and slow tumor growth.
  • the compound for stabilization comprises a hydrophobic group containing at least one cyclic group joined by a linker to an ionizable group, such as an amine.
  • Example 1 is an illustration of a preferred embodiments for practicing the present invention. However, they are not limiting examples. Other examples and methods are possible in practicing the present invention.
  • Example 1 is an illustration of a preferred embodiments for practicing the present invention. However, they are not limiting examples. Other examples and methods are possible in practicing the present invention.
  • Example 1 is an illustration of a preferred embodiments for practicing the present invention. However, they are not limiting examples. Other examples and methods are possible in practicing the present invention. Example 1
  • the gastrocneimius, soleus, andplantaris were dissected from three C57B1 6- Ly-5.1, 6-week old mice. Tendons, all bone, and fat were carefully discarded, the muscle tissue was thoroughly minced and then digested at 37° C with 0.2% collagenase (Worthington) for 45 minutes, followed by 0.1% trypsin (Gibco) for 45 minutes. The tissue was triturated vigorously, passed through a 70 ⁇ m filter, and the cells were collected by centrifugation. Cells were plated in Dulbecco's modified Eagle's medium (DME) containing 10% fetal calf serum (Hyclone), 5% chick embryo extract (Gibco), and antibiotics for 1 hour at 37° C.
  • DME Dulbecco's modified Eagle's medium
  • the non-adherent cells were then transferred to another plate and the adherent cells (primarily fibroblasts) were discarded. After 24 hours, the floating cells (blood cells) and debris were vigorously washed off the plate, and fresh medium was applied to the attached satellite cells.
  • Satellite cells were harvested by trypsinization after 5 days of culture, counted, and mixed with 200 x 10 5 nucleated whole bone marrow cells prepared from 6- to 12-week old C57Bl/6-Ly-5.2 mice. Recipients were also 6- to 12-week old C57Bl/6-Ly-5.2 mice that had been given 11 Gy in a split dose and maintained on acidified water and autoclaved food. Cell mixtures were injected retroorbitally in a volume of 300 ⁇ l under methoxyflurane anesthesia (Goodell, et al, 1996; Goodell, et al, 1997).
  • bone marrow was harvested from mouse 1, and 8 x 10 5 nucleated cells were injected into five C57Bl/6-Ly-5.2 recipients prepared as described herein. It is noted that in another embodiment, intravenous administration of muscle stem cells results in their homing to the bone marrow, providing, for example, another method for providing muscle stem cells in the bone marrow of an animal.
  • Example 3
  • Ly-5.1-biotin was detected by a subsequent staining with streptavidin-phycoerythrin (Molecular Probes). The stained blood samples were analyzed by flow cytometry on a FACSCalibur (Becton Dickinson). Background staining of Ly-5.2-positive peripheral blood by anti-Ly-5.1 antibody on was consistently less than 0.5%.
  • Hematopoietic stem cell enumeration Secondary transplant recipient mouse 5 was sacrificed and bone marrow was prepared and stained with Hoechst dye as known in the art (for example, Goodell, et al, 1996; and Goodell, et al, 1997) or downloaded, Goodell, M. A. http://www.bcm.tmc.edu/genetherapy/goodell/page2.htm.
  • bone marrow was suspended at 10 6 nucleated cells per ml in DME with 2% fetal calf serum (Hyclone), 10 mM HEPES buffer (Gibco) and 5 ⁇ g/ml Hoechst 33342 (Sigma) and incubated at 37°C for 90 minutes.
  • the bone marrow was pelleted by centrifugation and resuspended at 10 8 cells per ml in cold Hanks balanced salt solution containing 2% fetal calf serum and 10 mM HEPES (HBSS+) for staining with anti-Ly-5.1 -biotin and streptavidin-PE on ice.
  • the cells were resuspended in HBSS+ containing 2 ⁇ g per ml propidium iodide (Sigma).
  • Flow cytometric analysis was performed on a triple-laser instrument (MoFlow, Cytomation, Inc.). An argon laser tuned to 350 nm emission was used to excite the Hoechst dye.
  • Fluorescence emission was collected with a 405/30 BP filter (Hoechst blue) and 670/40 BP filter (Hoechst red).
  • a second 488 nm argon laser was used to excite phycoerythrin (emission was collected with a 575/40 BP filter).
  • a competitive bone marrow transplantation model was used in which the test cells are co-transplanted with whole bone marrow from a distinguishable strain of mice (Harrison, D. E., 1980). This material provides sufficient numbers of committed hematopoietic cells and stem cells to rescue the mice from lethal irradiation, allowing measurement of the stem cell activity of the test population against a known quantity of stem cells in the competitor population.
  • Satellite cells were prepared from C57Bl/6-Ly-5.1 mice, mixed with whole bone marrow from C57Bl/6-Ly-5.2 mice, and transplanted into lethally irradiated
  • mice C57Bl/6-Ly-5.2 mice.
  • peripheral blood was drawn from the recipients and analyzed by antibody staining and flow cytometry for the presence of B cells (B220), T Cells (Thy-1) and granulocytes/macrophages (Gr- 1+Mac-l) derived from the Ly-5.1+ satellite cells.
  • B220 B220
  • T Cells Thy-1
  • Gr- 1+Mac-l granulocytes/macrophages
  • Fig. 1 the total proportion of Ly-5.1+ cells in the peripheral blood was 56%. Such cells were present at high levels in each of the lymphoid and myeloid lineages (upper right quadrant of each panel).
  • the proportions of B cells, T cells, and granulocytes/macrophages derived from satellite cells were 42%, 76%, and 52%, respectively.
  • the overall distribution of B, T, and myeloid cells in this mouse was normal.
  • bone marrow was collected from mouse 1 (79% Ly-5.1+ cells) and tested it for regenerative capacity in lethally irradiated
  • mouse 5 was sacrificed (Fig. 3) and stained its bone marrow with the dye Hoechst 33342 and anti-Ly-5.1 antibody. Hematopoietic stem cells were identified on the basis of their high dye efflux activity, as determined by dual wavelength analysis of Hoechst dye fluorescence (Goodell, et al, 1996; Goodell, et al, 1997).
  • Fig. 4 A approximately 0.03% of the whole bone marrow had a stem cell phenotype and 63% of the whole bone marrow was positive for the Ly-5.1 marker (Fig. 4B).
  • Fig. 4C Closer examination of the hematopoietic stem cell population revealed that a majority of the cells (65%) were positive for the Ly-5.1 marker (Fig. 4D) and thus were derived from the satellite cells transplanted into the primary recipient, mouse 1.
  • the cells and methods of the present invention are used in gene therapy methods, for example by transplanting transduced murine satellite cells (containing a genetic sequence of choice for use in gene therapy).
  • One method of practicing this embodiment of the present invention is to utilize retroviral-mediated gene transfer for vectoring a genetic sequence of choice into muscle stem cells prior to transplantation.
  • Methods of gene therapy and well known in the art. In particular, but not in a limiting sense, it is noted that methods exist for the transduction of muscle stem cells.
  • transduced cells are then administered to an animal as directed herein and as understood in the art, providing transgenic differentiated cells within the animal for gene therapy uses.
  • these techniques are used in gene therapy of hematopoietic disease and gene therapy of neuro-muscular disease.
  • satellite cells adult muscle stem cells
  • lymphoid and myeloid progeny including B cells, T cells, granulocytes and macrophages for at least 3 months post-transplantation.
  • the cells retained their regenerative potential after they were transferred to secondary recipients, demonstrating their extremely primitive nature.
  • Such cells have numerous medical applications including, for example, the provision of hematopoietic potential to patients in whom autologous bone marrow is impaired in function or contaminated by tumor, and gene therapy use in treatment of a number of disorders including, for example, hematopoietic and neuro-muscular diseases.
  • All patents and publications mentioned in this specification are indicative of levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference. References
  • Snow, M. H. Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. I. A fine structural study. AnatRec 188, 181-199 (1977). Snow, M. H. The effects of aging on satellite cells in skeletal muscles of mice and rats. Cell Tissue Res 185, 399-408 (1977).

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Abstract

L'invention concerne des matériaux et des procédés se fondant sur la découverte selon laquelle les cellules souches musculaires (également appelées cellules satellites) peuvent générer au moins les principales lignées hématopoïétiques chez les souris avec le temps, comme le montre la figure. En outre, ces cellules conservent leur potentiel de regénérescence une fois transférés aux récepteurs secondaires.
PCT/US2000/017064 1999-06-25 2000-06-21 Cellules souches derivees du muscle squelettique WO2001000031A1 (fr)

Priority Applications (1)

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AU54988/00A AU5498800A (en) 1999-06-25 2000-06-21 Stem cells derived from skeletal muscle

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US14120699P 1999-06-25 1999-06-25
US60/141,206 1999-06-25

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001019966A2 (fr) * 1999-09-14 2001-03-22 Children's Medical Center Corporation, The Isolation de cellules souches derivees de muscle et utilisations associees
EP1495114A2 (fr) * 2002-04-10 2005-01-12 Medestea Internazionale S.r.l. Procede de preparation de cellules souches provenant du tissu musculaire humain et du tissu adipeux, et cellules souches pouvant etre obtenues par ledit procede
US7220582B2 (en) 2001-10-22 2007-05-22 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Stem cells that transform to beating cardiomyocytes
CN106916783A (zh) * 2013-05-29 2017-07-04 中国科学院上海生命科学研究院 肌肉干细胞体外培养方法及其应用

Citations (1)

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US5538722A (en) * 1989-06-13 1996-07-23 Stanford University Isolation, growth, differentiation and genetic engineering of human muscle cells

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US5538722A (en) * 1989-06-13 1996-07-23 Stanford University Isolation, growth, differentiation and genetic engineering of human muscle cells

Non-Patent Citations (5)

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Title
BJORNSON C.R.R. ET AL.: "Turning brain into blood: A hematopoietic fate adopted by adult neural stem cells in vivo", SCIENCE, vol. 283, 22 January 1999 (1999-01-22), pages 534 - 536, XP002930048 *
FERRARI G. ET AL.: "A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers", HUM. GENE. THER., vol. 6, June 1995 (1995-06-01), pages 733 - 742, XP002930047 *
FERRARI G. ET AL.: "Muscle regeneration by bone marrow-derived myogenic progenitors", SCIENCE, vol. 279, 6 March 1998 (1998-03-06), pages 1528 - 1530, XP002930046 *
GUSSONI E. ET AL.: "Dystrophin expression in the mdx mouse restored by stem cell transplantation", NATURE, vol. 401, 23 September 1999 (1999-09-23), pages 390 - 394, XP002930045 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001019966A2 (fr) * 1999-09-14 2001-03-22 Children's Medical Center Corporation, The Isolation de cellules souches derivees de muscle et utilisations associees
WO2001019966A3 (fr) * 1999-09-14 2001-08-09 Children S Medical Ct Corp The Isolation de cellules souches derivees de muscle et utilisations associees
US7220582B2 (en) 2001-10-22 2007-05-22 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Stem cells that transform to beating cardiomyocytes
EP1495114A2 (fr) * 2002-04-10 2005-01-12 Medestea Internazionale S.r.l. Procede de preparation de cellules souches provenant du tissu musculaire humain et du tissu adipeux, et cellules souches pouvant etre obtenues par ledit procede
CN106916783A (zh) * 2013-05-29 2017-07-04 中国科学院上海生命科学研究院 肌肉干细胞体外培养方法及其应用
CN106916783B (zh) * 2013-05-29 2020-07-17 中国科学院分子细胞科学卓越创新中心 肌肉干细胞体外培养方法及其应用

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