Primate embryonic stem cells
First Claim
1. A purified preparation of primate embryonic stem cells which (i) is capable of proliferation in an in vitro culture for over one year, (ii) maintains a karyotype in which all the chromosomes characteristic of the primate species are present and not noticeably altered through prolonged culture, (iii) maintains the potential to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iv) will not differentiate when cultured on a fibroblast feeder layer.
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Reexamination
Accused Products
Abstract
A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (-); SSEA-3 (+); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation have normal karyotypes and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.
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Citations
11 Claims
- 1. A purified preparation of primate embryonic stem cells which (i) is capable of proliferation in an in vitro culture for over one year, (ii) maintains a karyotype in which all the chromosomes characteristic of the primate species are present and not noticeably altered through prolonged culture, (iii) maintains the potential to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iv) will not differentiate when cultured on a fibroblast feeder layer.
- 3. A purified preparation of primate embryonic stem cells wherein the cells are negative for the SSEA-1 marker, positive for the SSEA-3 marker, positive for the SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, and have karyotypes which includes the presence of all of the chromosomes characteristic of the primate species and in which none of the chromosomes are noticeably altered.
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9. A method of isolating a primate embryonic stem cell line, comprising the steps of:
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(a) isolating a primate blastocyst; (b) isolating cells from the inner cell mass of the blastocyst of (a); (c) plating the inner cell mass cells on embryonic fibroblasts, wherein inner cell mass-derived cells masses are formed; (d) dissociating the mass into dissociated cells; (e) replating the dissociated cells on embryonic feeder cells; (f) selecting colonies with compact morphologies and cells with high nucleus to cytoplasm ratios and prominent nucleoli; and (g) culturing the cells of the selected colonies. - View Dependent Claims (10)
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11. A cell line developed by the method of step 9.
Specification