Successful Generation of Donor Specific Hematopoietic Stem Cell Lines From Co-Cultured Bone Marrow With Human Embryonic Stem Cell Line: A New Methodology

Abstract

We report the generation of 30 healthy human embryonic stem cell (h-ESC) lines from 33 voluntary oocyte donors using a donor somatic cell nuclear transfer (SCNT) technique on 190 oocytes. Our aim was to coculture them with their own bone marrow (BM) to generate hematopoietic progenitor cells for therapeutic purposes. Pluripotency and undifferentiated stage were confirmed using molecular cell surface markers. Normal karyotype of these cell lines was confirmed. Here we demonstrate that SCNT-h-ESCs differentiate to hematopoietic precursors when cocultured with unmodified, nonirradiated donor BM. We did not use any xenogeneic material for this hematopoietic differentiation. Hematopoietic precursors derived from them expressed cell surface antigens CD45/34. When further cultured with hematopoietic growth factors these hematopoietic precursors formed characteristic myeloid, erythroid, and megakaryocyte lineages. Phenotypic CD34+ cells derived from NT-h-ESCs were functionally similar to their counterparts in primary hematopoietic tissues like BM, umbilical cord, and blood. More terminally differentiated hematopoietic cells derived from h-ESCs under these culture conditions also expressed normal surface antigens like glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. We report generation of hematopoietic progenitor cells from h-ESC lines by a SCNT technique, with differentiation into further lineages with structural and functional similarities to their adult counterparts in vivo. This novel alternative source of CD34+ stem cells from h-ESC lines generated without any xenogeneic material might be used to create transplantation tolerance, to implement regenerative medicine, and to treat autoimmune disorders.