Human embryonic germ cells isolation from early stages of post-implantation embryos

Abstract

Human embryonic germ (hEG) cell is a very important alternative pluripotent stem cell resource. We describe the derivation of hEG cells from human embryonic fetal gonads over 6-8 weeks postconception. A large number of EG-like cell clumps were obtained at passage 1 and thus facilitated the following routine culture when the donor tissues were trypsinized with gentle pipetting and plated on feeder layer cells in the initial culture. Eight diploid hEG cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent stem cells. Human EG cells expressed transcription factor Oct4, a marker of pluripotency in mouse EG cells, at a high and steady level. Expression of markers indicative of differentiation along the three germ lineages was also observed in EBs. High level of alkaline phosphatase activity was shown in EG cells. These encouraging findings provide a starting point for potential applicability of hEG cells.

Stem cell factor affects fate determination of human gonocytes in vitro

“…As shown in Fig. 3, the gonocyte colonies were stained positive for Oct-4, SSEA-4, and c-Kit, suggesting that these colonies share similar pluripotent markers as well as embryonic germ cells (EGCs) that were cultured from PGCs (Shamblott et al 1998, Turnpenny et al 2003, Liu et al 2004, Park et al 2004, Pan et al 2005. The result also showed that the cultured gonocyte colonies were only dotted stained with c-Kit, suggesting that the c-Kit might be down-regulated in the present culture system.…”

Techniques and Conditions for Embryonic Germ Cell Derivation and Culture

“…To date, six laboratories have reported successful derivation of human EGC lines (Shamblott et al, 1998;Turnpenny et al, 2003;Liu et al, 2004;Park et al, 2004;He et al, 2007;Hua et al, 2009). This very limited number of groups is due to the difficulties of acquiring human PGCs (therapeutic termination of pregnancy and ethical approval is needed), and the complexity of the derivation and culture of EGCs (Turnpenny et al, 2006;Kerr et al, 2006;Perret et al, 2008).…”

“…Although it is a clonal cell line, there are several phenotypic variations in STO cells from different isolates or even depending on the time in culture that affects the human EGC derivation (Kerr et al, 2006). Some groups have also successfully utilized primary MEFs (CF1) as feeder layers for EGC derivation (Liu et al, 2004;Hua et al, 2009) and even human embryonic fibroblast-like cells derived from gonadal ridges and dorsal mesenteries (see below, He et al, 2007). The feeder layer can be mitotically inactivated with mitomycin C or by irradiation, either before or after plating.…”

“…Finally, cells are cultured into gelatinized culture plates at ~1.5 x 10 5 cells/cm 2 and allowed to adhere overnight. Alternatively, feeder layers can be mitotically inactivated with 10 μg/ml mitomycin C (Sigma) (Park et al, 2004;Liu et al, 2004;Hua et al, 2009). When MEFs are used instead of STO cells, these have to be seeded at 7.5 ×10 4 cells/cm 2 , half the density of STO cells (Pan et al, 2005;Hua et al, 2009).…”

“…Growth medium is added to the tube to stop the digestion and finally the cell suspension is pipetted 30-50 strokes with a 200 μl Pipetman (Gilson) (Kerr et al, 2006). Slight variations in these protocols are used by other groups: A previous 0.01% EDTA treatment for 10 minutes (Turnpenny et al, 2003), different enzymatic dissociation treatments between using only trypsin (without EDTA) (Liu et al, 2004), collagenase IV + DNase I (Turnpenny et al, 2003;Park et al, 2004), and even without mechanically dissociation and just incubation with 0.125% trypsin and 0.02% EDTA for 10-20 min or with 0.125% collagenase for 20-40 min at 37°C (Hua et al, 2009). No comparisons between different PGC isolation protocols have been reported, so it is unknown which one is the more efficient.…”