ESCs can differentiate into PGCs known to appear in the proximal epiblast of the egg cylinder by 6.5 dpc as soon as the BMP is expressed. Specific growth factors are essential in enhancing the appearance and survival of germ cells increasing their chances of steering them into meiosis. Here, by adding a cocktail of growth factors, we designed a favorable in vitro environment aimed at increasing PGC appearance among ESCs through embryoid body (EBs) development and attempted to correlate PGCs so derived with those observed in in vivo-derived mouse embryos. Mouse ESCs were maintained in a standard medium supplemented with LIF. EBs were generated in standard DMEM+serum, BMP4, or a combination of BMP4, 7, and 8b. At regular time intervals, cells were assessed for early germ cell specific markers, namely VASA, Fragilis, and c-Kit. Pre- (E6.5) and post- (E12.5) migratory PGCs were identified in embryos sectioned following vascular perfusion. Embryo slices were either 5 or 8 μm, depending on the age. Dissociation of EBs were sorted to enrich putative PGCs prior to culture into an in vitro maturation medium. The size of the embryos at E6.5 was about 15 × 22 μm. Approximately 3 sections (5 μm each) were obtained per embryo. A total of 14.6% (12/82) of the cells per embryo slice expressed fragilis – confirming premigratory PGCs. For E12.5 embryos, with a developed gonadal ridge estimated to be 25 × 87.5 μm, germ cell component of early post-migratory PGCs was depicted as 25.9% (45/174) by VASA expression. EB development was comparable in all media and after cell dissociation the cell number ranged from 550 to 3000. Spontaneous germ cell appearance on day 3 of culture was detected in standard medium at ∼1% (1/98). The presence of BMP4, increased PGC appearance to 36.2% (58/160) topping with the combination of the BMP family to 44.9% (92/205). Although there was an early and consistent appearance of PGC since day 3, they only self-renewed for up to 8 days and progressively declined thereafter. Allocation to the in vitro maturation medium failed to drive PGCs into meiosis. We confirm that germ cell differentiation from ESCs can be greatly enhanced by the action of specific growth factors. Morphology and marker presence mimicked their counterpart of epiblastic origin. Although we were able to enrich and stimulate self-renewal and proliferation of PGCs in early EBs, their spontaneous apoptosis impeded their further passage to later maturational stages.

Aiming to ease the ethical debates surrounding the ESC techniques, we have shown that microsurgical isolation of inner cell mass (ICM) cells is a feasible method to harvest embryonic stem cells (ESCs) maintaining embryo viability. Mouse embryonic fibroblasts (MEF) as a feeder cell layer have a paramount role for establishing ESCs, however, represent a source of xenogenic contamination, are labor intensive, and at times produce inconsistent results. Single cells were isolated from the ICM of mouse blastocysts. The efficiency of ESC derivation was compared between two different culture conditions according to presence or absence of adjuvant cell support. Pluripotency characteristics of the derived ESC lines were confirmed as well as pre- and post-implantation development of the biopsied embryos. Blastocysts were obtained by culturing zygotes, from mated mice after ovarian stimulation. Individual cells (3–5) were dislodged from the ICM and aspirated into a micropipette loaded with trypsin, and plated individually either on adjuvant cell system with conventional DMEM (MEF-D) or directly on a gelatin coated dish with ESGRO CompleteTM (ESGRO-C). Once ICM cells developed into epiblast-like aggregates, first dissociation was carried out by enzymatic digestion. Coincidentally, the biopsied embryos were transferred to pseudopregnant females. Intact blastocysts were used as controls. The ESC lines so derived were tested for pluripotency. Of a total of 119 blastocysts 46 were biopsied and 43 yielded isolated ICM cells that were allocated to either MEF-D (n = 30), or ESGRO-C (n = 16) system. The remaining 73 intact blastocysts were assigned to the same culture conditions serving as controls. Biopsied ICM cells formed 12 cell plaque aggregates and yielded 8 ESC colonies (23.5%) in MEF-D while no cell aggregates developed in ESGRO-C. ESC harvesting from intact control embryos in MEF-D was 17.6 and 9.1% in ESGRO-C, respectively. All the putative ESC lines showed pluripotent characteristics. Live birth rate of manipulated blastocysts was 43.3%, not different from the control (55.2%). The isolation of individual epiblast cells from blastocysts allows ESC harvesting of comparable quality to those derived from methodology sacrificing the entire embryo. Moreover, ICM biopsy did not impair embryo developmental competence. This study confirmed that individually isolated ICM cells necessitated adjuvant cells for stem cell generation.