VERY SMALL EMBRYONIC-LIKE (VSEL) STEM CELLS AND METHODS OF ISOLATING AND USING THE SAME
First Claim
Patent Images
1. A method of forming an embryoid body-like sphere from a population of very small embryonic-like (VSEL) stem cells or derivatives thereof, the method comprising:
- (a) providing a population of CD45−
cells comprising VSEL stem cells or derivatives thereof; and
(b) culturing the VSEL stem cells or derivatives thereof in a medium comprising one or more factors that induce embryoid body-like sphere formation of the VSEL stem cells or derivatives thereof for a time sufficient for an embryoid body-like sphere to form.
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Abstract
The presently disclosed subject matter provides populations of stem cells that are purified from bone marrow, peripheral blood, and/or other sources. Also provided are methods of using the stem cells for treating tissue and/or organ damage in a subject.
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Citations
73 Claims
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1. A method of forming an embryoid body-like sphere from a population of very small embryonic-like (VSEL) stem cells or derivatives thereof, the method comprising:
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(a) providing a population of CD45−
cells comprising VSEL stem cells or derivatives thereof; and(b) culturing the VSEL stem cells or derivatives thereof in a medium comprising one or more factors that induce embryoid body-like sphere formation of the VSEL stem cells or derivatives thereof for a time sufficient for an embryoid body-like sphere to form. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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- 22. An embryoid body-like sphere comprising a plurality of very small embryonic-like (VSEL) stem cells.
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24. A method of differentiating a very small embryonic-like (VSEL) stem cell into a cell type of interest, the method comprising:
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(a) providing an embryoid body-like sphere comprising VSEL stem cells or derivatives thereof; and (b) culturing the embryoid body-like sphere in a culture medium comprising a differentiation-inducing amount of one or more factors that induce differentiation of the VSEL stem cells or derivatives thereof into the cell type of interest until the cell type of interest appears in the culture. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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43. A method of treating an injury to a tissue in a subject, the method comprising administering to the subject a composition comprising a plurality of isolated CD45−
- stem cells comprising VSEL stem cells in a pharmaceutically acceptable carrier, in an amount and via a route sufficient to allow at least a fraction of the population of CD45−
stem cells to engraft the tissue and differentiate therein, whereby the injury is treated. - View Dependent Claims (44, 45, 46, 47, 48, 49)
- stem cells comprising VSEL stem cells in a pharmaceutically acceptable carrier, in an amount and via a route sufficient to allow at least a fraction of the population of CD45−
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50. A method of producing a chimeric animal, the method comprising adding one or more of a population of CD45−
- stem cells comprising VSEL stem cells to an embryo such that the one or more of the CD45−
stem cells develop into one or more cell types of the embryo. - View Dependent Claims (51, 52, 53)
- stem cells comprising VSEL stem cells to an embryo such that the one or more of the CD45−
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54. A method of purifying a very small embryonic-like (VSEL) stem cell for a cell type of interest from a population of CD45−
- stem cells, the method comprising;
(a) providing a population of CD45−
stem cells comprising VSEL stem cells;(b) identifying a subpopulation of the CD45−
stem cells that express a marker of VSEL stem cells; and(c) purifying the subpopulation.
- stem cells, the method comprising;
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55. The method of claim 55, wherein the population and the subpopulation are both CD34+/CXCR4+/lin−
- or Sca-1+/lin−
in addition to being CD45−
. - View Dependent Claims (56, 57, 59, 60, 61, 62, 63, 64, 65)
- or Sca-1+/lin−
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58. The method of claim 58, wherein the marker is selected from the group consisting of GFAP, Nestin, β
- III tubulin, Olig1, Olig2, Myf5, MyoD, Myogenin, Nsx2.5/Csx, GATA-4, α
-Fetoprotein, CK19, Nkx 2-3, Tcf4, Nkx 6.1, Pdx 1, VE-cadherin, Krt 2-5, Krt 2-6a, BNC, DCT, TYR, and TRP.
- III tubulin, Olig1, Olig2, Myf5, MyoD, Myogenin, Nsx2.5/Csx, GATA-4, α
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66. A method of identifying an inducer of embryoid body-like sphere formation, the method comprising:
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(a) preparing a cDNA library comprising a plurality of cDNA clones from a cell known to comprise the inducer; (b) transforming a plurality of cells that do not comprise the inducer with the cDNA library; (c) culturing a plurality VSEL stem cells or derivatives thereof in the presence of the transformed plurality of cells under conditions sufficient to cause the VSEL stem cells or derivatives thereof to form an embryoid body-like sphere; (d) isolating the transformed cell comprising the inducer; (e) recovering a cDNA clone from the transformed cell; and (f) identifying a polypeptide encoded by the cDNA clone recovered, whereby an inducer of embryoid body-like sphere formation is identified. - View Dependent Claims (67, 68, 69, 70)
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71. A method of isolating a subpopulation of CD45−
- stem cells comprising VSEL stem cells from umbilical cord blood or a fraction thereof, the method comprising;
(a) contacting the umbilical cord blood or the fraction thereof with a first antibody that is specific for CD45 and a second antibody that is specific for CD34 or Sca-1 under conditions sufficient to allow binding of each antibody to its target, if present, on each cell of the population of cells; (b) selecting a first subpopulation of cells that are CD34+ or Sca-1+, and are also CD45−
;(c) contacting the first subpopulation of cells with one or more antibodies that are specific for one or more cell surface markers selected from the group consisting of CD45R/B220, Gr-1, TCRα
β
, TCRγ
δ
, CD11b, and Ter-119 under conditions sufficient to allow binding of each antibody to its target, if present, on each cell of the population of cells;(d) removing from the first subpopulation of cells those cells that bind to at least one of the antibodies of step (d); and (e) collecting a second subpopulation of cells that are either CD34+/lin−
/CD45−
or Sca-1+/lin−
/CD45−
, whereby a subpopulation of CD45−
stem cells comprising VSEL stem cells is isolated. - View Dependent Claims (72, 73)
- stem cells comprising VSEL stem cells from umbilical cord blood or a fraction thereof, the method comprising;
Specification