Earticle

Recent technological progress in producing germline chimeras has provided an important means of studying genetic resource conservation, and transgenics production. Primordial germ cells (PGCs) are called gonadal germ cells (GGCs), after migration to the gonadal ridge. GGCs ultimately differentiate into spermatogonia in the testes or oogonia in the ovaries. Germline chimeras can be produced in domestic chicken by transferring GGCs collected from the embryonic gonad into the bloodstream of 2-dayold recipient embryos. However, it is diffcult to purify GGCs from the majority number of somatic cells after digesting embryonic gonads using proteinases such as trypsin and EDTA. The aim of the present study was to develop a simple method for isolating GGCs from the gonads of 7-day-old chick embryos. A simple method for isolating viable GGCs was developed in the domestic chicken. Developing gonads were isolated from 7-day-old chick embryos and cultured for up to 24 hours at 38.5℃ in phosphate buffered saline without Ca+ and Mg+ (PBS [-]). A discharge of GGCs from the gonad was observed within 40 minutes after introducing the embryonic gonad into PBS [-], and the number of discharged GGCs increased until 12 hours of incubation. The purity of the GGCs (number of discharged GGCs/total number of discharged cells) was approximately 48% for the initial 1 hours of incubation and decreased thereafter. These results indicate that high-purity, viable GGCs can be collected by simply introducing isolated developing gonads into PBS [-]. The results provide an alternative means for producing germ line chimeras to conserve genetic resources and to produce transgenic avian species.

Human pluripotent stem cells (hPSCs) self-renew indefinitely as highly organized pluripotent colonies; this self-renewal is evident for both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Unlike mouse pluripotent stem cell colonies, human colonies form a uniform, flat epithelium-like monolayer. Interestingly, it has been reported that colony morphology is closely correlated with the maintenance of pluripotency. However, the molecular mechanisms that underlie human pluripotent colony formation and organization are poorly understood. In this study, using real-time imaging tools, we examine in vitro colony formation by enzymatically dissociated single hPSCs (specifically CHA15 cells, H9 hESCs and hiPSCs) under feeder-free conditions. We showed that colony formation consists of 4 stages: attachment, migration, colony formation (aggregation), and colony organization, which are facilitated in an intracellular calcium-dependent manner. Moreover, we found that blocking Gi-coupled GPCR signaling results in enhanced cell-cell interactions and plays an integral role in promoting the survival of hESCs in culture. Using various visualization methods, we identified conditions required for colony formation, and we suggested a promising new target for modulating hPSC colony formation and organization. These results are likely to be useful for engineering hPSCs to further study the mechanisms involved in pluripotency.

Porcine pluripotent stem cells have important in modeling embryonic development and disease processes in biomedical research, especially to transplantation medicine, immunology and circulatory system. However, despite years of effort, the pluripotency machinery of porcine PSCs has not been demonstrated clearly. Porcine induced plu-ripotent stem cells (piPSCs) were generated using combination of six human tran-scriptional factors under stem cell culture condition. To confirm dependency of these piPSCs, they were separately cultured in stem cell media supplemented with i) 20 ng/ml LIF, ii) 20 ng/ml bFGF, iii) 20 ng/ml BMP4, iv) 20 ng/ml LIF and Su5402, or v) 20 ng/ml bFGF and Jak inhibitor I, for 48 h, respectively. Upon bFGF treated groups, colonies show signs of differentiation with dispersing colony morphology and losing alkaline phosphatase activity. In contrast, colonies with tightly expanded and strong AP activity are shown in LIF or BMP4 treated groups. Western blotting shows phospho- Stat3 and phospho-Smad1/5 are significantly suppressed by Jak inhibitor I. We con-clude that piPSCs generated by six human pluripotent factors, are dependent on LIF or BMP4 to sustain self-renewal and pluripotency.