Human pluripotent embryonic stem cells produced by nuclear transfer using a somatic cell nucleus treated with HVJ-E extract and an oocyte from a donor cycle that produced 15 or fewer oocytes

摘要

Human pluripotent embryonic stem cells produced by somatic cell nuclear transfer as well as methods of making and using said human pluripotent embryonic stem cells are disclosed.

主权项

1. A method of producing a human pluripotent embryonic stem cell, the method comprising:
enucleating a human oocyte from a donation cycle of 15 or fewer oocytes by removing the MII spindle in a manner that does not lower levels of maturation promoting factor, thereby producing a cytoplast; contacting a human donor nucleus with an HVJ-E extract; contacting the cytoplast with the human donor nucleus thereby producing an SCNT embryo; treating the human oocyte and/or the cytoplast and/or the donor nucleus and/or the SCNT embryo with a protein phosphatase inhibitor; applying a first electroporation pulse to the SCNT embryo, thereby producing an activated SCNT embryo; culturing the activated SCNT embryo in a first media comprising 6-DMAP; culturing the activated SCNT embryo in a second media comprising TSA; culturing the activated SCNT embryo in a third media thereby producing a blastocyst; culturing the blastocyst on a feeder layer; and selecting a cell with an embryonic stem cell-like morphology.