Pluripotential embryonic stem cells and methods of making same
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a technology embryos, applied in the field can solve the problems of inconvenient differentiation of embryonic stem cells, insufficient evidence presented in the evans application for the differentiation of cow and pig putative es cell lines into differentiated cell types in monolayer culture, in embryoid bodies and tumors,
Inactive Publication Date: 2007-08-02
HOGAN BRIGID L M
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Attempts at isolating ES cells from other animals apparently have failed.
In addition, the evidence presented in the Evans application for the differentiation of the cow and pig putative ES cell lines into differentiated cell types in monolayer culture, in embryoid bodies and in tumors, is not convincing.
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[0065] All the cell types and other materials listed below can be obtained through available sources and / or through routine methods.
Materials and Methods
Feeder Cells
[0066] The SI / SI4 cell line, derived from a homozygous null SI / SI mouse embryo, and its derivative, SI4-m220, which stably expresses only membrane bound murine SF lacking exon 6 encoding the proteolytic cleavage site, were obtained from Dr. David Williams (Howard Hughes Medical Institute, Indiana University Medical School). Other cell lines which produce adequate SF can be substituted for SI / SI4, for example mouse or human embryo fibroblasts or cell lines or somatic cell lines from gonads or genital ridges. Combinations of feeder cells can also be utilized. They were maintained in DMEM with 10% calf serum and 50 ug / ml gentamicin. For making feeder layers they were irradiated (500 rads) and plated at a density of 2×105 per well of 24-well plates (Falcon) in the same medium, 24 hrs before use. Wells were pre-treated w...
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Abstract
The present invention provides a non-mouse, including human, pluripotential embryonic stem cell which can: (a) be maintained on feeder layers for at least 20 passages; and (b) give rise to embryoid bodies and multiple differentiated cell phenotypes in monolayer culture. The invention further provides a method of making a pluripotential embryonic stem cell comprising culturing germs cells and germ cell progenitors in a composition comprising a growth enhancing amount of basic fibroblast growth factor, leukemia inhibitory factor, membrane associated steel factor, and soluble steel factor to primordial germ cells under cell growth conditions, thereby making a pluripotential embryonic stem cell. Also provided are composition useful to produce the pluripotent embryonic stem cells and methods of screening associated with the method of making the embryonic stem cell.
Description
[0001] This application is a continuation of, and claims the benefit of, application Ser. No. 08 / 813,829, filed Mar. 6, 1997, which status pending, which is a continuation of Ser. No. 08 / 217,921, filed Mar. 25, 1994, now U.S. Pat. No. 5,690,926 issued Nov. 25, 1997, which is a continuation-in-part of Ser. No. 07 / 958,562, filed Oct. 8, 1992, now U.S. Pat. No. 5,453,357, issued Sep. 26, 1995, which applications are hereby incorporated herein by this reference in their entireties. [0002] This invention was made with government support under grant number HD25580-04 from the National Institute of Health Child Health and Development. The United States government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] This invention relates to pluripotential embryonic stem cells and methods and compositions for making pluripotential embryonic stem cells. [0005] 2. Background Art [0006] Primordial germ cells (PGCS) in the mouse are thought to ...
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