Epigenomic Regulations of Patient-Specific and Primate-Specific Embryonic Stem Cells Derived after Nuclear Transfer: Differences between ESC from Fertilized Blastocysts versus NT-ESC

Gerald Schatten
Departments of Obstetrics ; University of Pittsburgh School of Medicine

Patient-specific, immune-matched human embryonic stem cells (hESC) are anticipated to be of great biomedical importance for studies of disease and development, and to advance clinical deliberations for stem cell transplantation. Embryonic stem cells (ESCs) have unique properties of unlimited self-renewal and pluripotent differentiation, but have only been established robustly and investigated intensively in mice (mESCs) and humans (hESCs) from fertilized-blastocysts and recently after nuclear transfer (NT; NT-mESCs, NT-hESCs). Eleven hESC lines were established by nuclear transfer (SCNT; NT) of skin cells from patients with disease or injury into donated oocytes. These lines (NT-hESCs), grown on human feeders from the same NT-donor or genetically-unrelated individuals, were established at high rates, regardless of NT-donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and match NT-patient's DNA. Major Histocompatibility Complex (MHC) identity of each NT-hESC with the patient's showed immunological compatibility, important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made.

Pluripotentiality is unequivocally shown with mESCs by germ-line transmission in chimeric offspring as well as differentiation into tissue types in teratomas: Overwhelming ethical constraints limit hESC assays only to teratoma analysis. Nonhuman primate ESCs (nhp-ESCs) could bridge the research gaps between mESCs and NT-mESCs with hESCs and NT-hESCs, and nhp-ESCs had previously been derived after fertilization and parthenogenesis (°_-nhp-ESCs). Here nhpESCs from IVF-blastocysts and NT-nhp-ESCs from somatic cell NT-blastocysts are demonstrated to be ESCs by pluripotency marker expressions (Oct-4; SSEA-3 and 4, but not the control SSEA-1; Alkaline Phosphate [AP]; Tra-1-61 and Tra-1-80), stable karyotypes, and differentiation into tissues from all three germ layers in vitro and in teratomas. Nhp-ESCs integrate into NHP embryos to form chimerae, but they do not form chimeric embryos when reaggregated with mouse embryos. NhpESCs and NT-nhpESCs are established at 3.7 times and 29.5 times lower rates compared with hESCs and NT-hESCs, respectively. Furthermore, the only successfully derived NT-nhpESC line is shown by DNA fingerprinting, cytogenetics and HLA analysis as a stable tetraploid (NT-4N-nhpESC) with complete sets of both the male somatic cell genome, as well as the maternal oocyte chromosomes (84, XXXY). Tetraploids reprogramming models made by fusing hESCs with somatic cells have been proposed, so these NT-4N-nhpESCs address questions regarding 4N ESC reliability. Nonhuman primate ESCs and NT-ESCs can ethically address questions regarding immune-matching, chimeric pluripotency, tumorigenesis and differentiation stability for clinical evaluations of stem cell transplantation. (Nonhuman Primate research is sponsored by the NIH whereas hESC research is performed and supported by the Government of Korea.)


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