Pluripotent stem cells derived without the use of embryos or fetal tissue
First Claim
1. A purified preparation of pluripotent non-embryonic stem cells, which (i) is capable of proliferating in an in vitro culture for more than one year;
- (ii) maintains a karyotype in which the cells are euploid and are not altered through culture;
(iii) maintains the potential to differentiate into cell types derived from the endoderm, mesoderm and ectoderm lineages throughout the culture, and (iv) is inhibited from differentiation when cultured on fibroblast feeder layers.
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Abstract
This invention provides a method for deriving precursors to pluripotent non-embryonic stem (P-PNES) and pluripotent non-embryonic stem (PNES) cell lines. The present invention involves nuclear transfer of genetic material from a somatic cell into an enucleated, zona pellucida free human ooplastoid having a reduced amount of total cytoplasm. The present invention provides a new source for obtaining human and other animal pluripotent stem cells. The source utilizes as starting materials an oocyte and a somatic cell as the starting materials but does not require the use, creation and/or destruction of embryos or fetal tissue and does not in any way involve creating a cloned being. The oocyte never becomes fertilized and never develops into an embryo. Rather, portions of the oocyte cytoplasm are extracted and combined with the nuclear material of individual mature somatic cells in a manner that precludes embryo formation. Murine, bovine, and human examples of the procedure are demonstrated. Subsequently, the newly constructed P-PNES cells are cultured in vitro and give rise to PNES cells and cell colonies. Methods are described for culturing the P-PNES cells to yield purified PNES cells which have the ability to differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers. Methods are described for maintaining and proliferating PNES cells in culture in an undifferentiated state. Methods and results are described for analysis and validation of pluripotency of PNES cells including cell morphology, cell surface markers, pluripotent tumor development in SCID mouse, karyotyping, immortality in in vitro culture.
140 Citations
76 Claims
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1. A purified preparation of pluripotent non-embryonic stem cells, which
(i) is capable of proliferating in an in vitro culture for more than one year; -
(ii) maintains a karyotype in which the cells are euploid and are not altered through culture;
(iii) maintains the potential to differentiate into cell types derived from the endoderm, mesoderm and ectoderm lineages throughout the culture, and (iv) is inhibited from differentiation when cultured on fibroblast feeder layers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A purified preparation of pluripotent non-embryonic stem cells, which
(i) is capable of proliferating in an in vitro culture for an indefinite period; -
(ii) maintains a karyotype in which the cells are euploid and are not altered through culture; and
(iii) maintains the potential to differentiate into cells types derived from the endoderm, mesoderm and ectoderm lineages throughout the culture. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18)
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- 19. A stem cell which does not originate from a fertilized egg, but which originates from the combination of a somatic cell nucleus and an enucleated ooplastoid.
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30. A stem cell which is produced by the method of (i) contacting a desired somatic cell or somatic cell nucleus with an ooplastoid, wherein said ooplastoid is derived from an enucleated oocyte;
- (ii) combining said somatic cell or somatic cell nucleus with said ooplastoid to create a nascent cell, and (iii) culturing said nascent cell to obtain pluripotent non-embryonic stem cells.
- View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
- 41. A nascent cell produced from the combination of a somatic cell nucleus and an enucleated zona pellucida free ooplastoid.
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47. A method of producing pluripotent, non-embryonic stem cells comprising the following steps:
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(i) contacting a desired somatic cell or somatic cell nucleus with an ooplastoid, wherein said ooplastoid is derived from an enucleated oocyte;
(ii) combining said somatic cell or somatic cell nucleus with said ooplastoid to create a nascent cell;
(iii) activating said nascent cell; and
(iv) culturing said nascent cell to obtain pluripotent non-embryonic stem cells. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. A method of producing pluripotent non-embryonic stem cells comprising the following steps:
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(i) contacting one or more desired somatic cells or somatic cell nuclei with a super-ooplast derived from one or more enucleated zona pellucida free oocytes;
(ii) dividing said super-ooplast into single nucleus containing nascent cells;
(iii) activating said nascent cells; and
(iv) culturing said nascent cells to obtain pluripotent non-embryonic stem cells. - View Dependent Claims (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)
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69. A method of producing an ooplastoid comprising the following steps:
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(i) harvesting an oocyte from a female;
(ii) maturing said oocyte to metaphase II;
(iii) breaching or removing the zona pelucida of said metaphase II oocyte;
(iv) enucleating said oocyte by removing the polar body and nuclear DNA of said oocyte through the breach of the zona pelucida or by oocyte partitioning; and
(v) aspirating and pinching off an ooplastoid from said enucleated oocyte. - View Dependent Claims (70, 71, 72, 73, 74, 75, 76)
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Specification