Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

Abstract

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders

“…At present, human homologous feeder cells have been used widely in the culture of hES cells, but their use has not been described for hPGES cells. In previous studies, the derivation and culture of hPGES cells both have been performed on MEF feeder cells, which limit their potential application in clinical settings [3][4][5]. Here we used foreskin fibroblasts as feeder cells, and derived an hPGES cell line successfully.…”

“…There are two methods by which such ES cells can be derived: somatic nuclear cell transfer (SCNT) and parthogenesis. The derivation of hES cells by the SCNT method has yet to be reported, whereas the successful derivation of hES cells by parthenogenesis has been reported recently [3][4][5][6].…”

“…In general, parthenogenetic (PG) ES cells are derived from the inner cell mass (ICM) of blastocyst-stage embryos developed from metaphase II (MII) oocytes that have undergone spontaneous activation [4][5][6]. Lin et al suggested an alternative strategy in which human PGES cells were obtained from haploid oocytes after routine in vitro fertilization (IVF) treatment [3].…”

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

“…The immunosurgery method was applied for ICM isolation, as described in our previous work. 54 After 5-7 d, the human ICM formed a small colony, and the culture media were changed every 2 d. The small colony was allowed to grow for 7 more days and was mechanically dissociated into three to four small clumps using a micropipette. After five passages, the human ES cell colonies were propagated using 1 mg/ml of type IV collagenase every 4-7 d.…”

Pluripotent stem cells and their use in hearing loss

“…The pgES cell was first isolated from a mouse and later from a rabbit, a monkey, and a human by Kaufman et al (1983). The pgES cells show similarities with sperm-fertilized embryonic cells, express outside stem cell markers, form an embryoid body, and can differentiate into all cells across the three germ layers (Mai et al, 2007). Ethical issues, teratoma formation, and immune reactions in experiments with ES cells limit the clinical trials with those cells.…”