Growth characteristics of the nonhuman primate embryonic stem cell line cjes001 depending on feeder cell treatment.

Embryonic stem cells (ESC) hold tremendous potential for therapeutic applications, including regenerative medicine, as well as for understanding basic mechanisms in stem cell biology. Since numerous experiments cannot be conducted in human ESC because of ethical or practical limitations, nonhuman primate ESC serve as invaluable clinically relevant models. The novel marmoset (Callithrix jacchus) ESC line cjes001 was characterized using different stem cell markers. The cells were stained positively with Oct4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and Sox-2 underscoring their status as undifferentiated ESC. ESC are typically grown on mouse embryonic fibroblasts (MEF) as feeder cells whose proliferation is arrested either by treatment with Mitomycin C or by gamma-irradiation. To assess the impact of these treatments on the ability of MEF to support the growth of undifferentiated ESC, we used an MTT assay to evaluate the cellular metabolic activity of growth arrested feeder cells. There was a significant (p < 0.02) difference in gamma-irradiated cells displaying a higher metabolic activity compared to Mitomycin C inactivation. Also we quantified 69 soluble factors in the supernatant of both Mitomycin-treated and gamma-irradiated MEF by bead-based multiplex analysis, and thus established a profile of MEF-secreted factors. The time course of secretion was analyzed by monitoring the supernatant at 0, 6, 12, and 24 h after changing the medium. Comparing gamma-irradiated and Mitomycin-treated MEF suggested higher amounts of some cytokines including FGF or SCF by the former. We also assessed whether the method of inactivation had an effect on growth kinetics and differentiation of primate ESC. There appeared to be a trend to a lower number of differentiated ESC colonies on the gamma-irradiated feeder cells, suggesting that this may be the preferable method of growth arrest.

A novel embryonic stem cell line derived from the common marmoset monkey (Callithrix jacchus) exhibiting germ cell-like characteristics.

BACKGROUND: Embryonic stem cells (ESC) hold great promise for the treatment of degenerative diseases. However, before clinical application of ESC in cell replacement therapy can be achieved, the safety and feasibility must be extensively tested in animal models. The common marmoset monkey (Callithrix jacchus) is a useful preclinical non-human primate model due to its physiological similarities to human. Yet, few marmoset ESC lines exist and differences in their developmental potential remain unclear. METHODS: Blastocysts were collected and immunosurgery was performed. cjes001 cells were tested for euploidy by karyotyping. The presence of markers for pluripotency was confirmed by immunofluorescence staining and RT-PCR. Histology of teratoma, in vitro differentiation and embryoid body formation revealed the differentiation potential. RESULTS: cjes001 cells displayed a normal 46,XX karyotype. Alkaline phosphatase activity, expression of telomerase and the transcription factors OCT4, NANOG and SOX2 as well as the presence of stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection antigens (TRA)-1-60, and TRA-1-81 indicated pluripotency. Teratoma formation assay displayed derivatives of all three embryonic germ layers. Upon non-directed differentiation, the cells expressed the germ cell markers VASA, BOULE, germ cell nuclear factor and synaptonemal complex protein 3 and showed co-localization of VASA protein within individual cells with the germ line stem cell markers CD9, CD49f, SSEA-4 and protein gene product 9.5, respectively. CONCLUSIONS: The cjes001 cells represent a new pluripotent ESC line with evidence for enhanced spontaneous differentiation potential into germ cells. This cjes001 line will be very valuable for comparative studies on primate ESC biology.