Studies On The Isolation, Culture, Evaluation And Differentiation Of Porcine Embryonic Germ Cells

The present research was carried out by use of different methods to study (1) the factors influencing the development of EG cell lines by use of Primordial germ cells, (2) the methods of identification and inducing differentiation of porcine EG cell, (3) establishment of WZSP chimera, and (4) the separation and cultivation of porcine spermatogonial stem cells. The results obtained are as follows. 1. By culturing porcine embryonic fibrolast obtained from piglet with the age of 26.5～29 days, it was found that the piglet could be used as the material of producing porcine embryonic fibrolast, and the porcine embryonic fibrolast as those of feeder cells and the donor nuclear of animal cloning.2. As the feeder cells of EG cells, the morphology of STO cells should proliferate quickly, maintain regular shapes and sizes, and have clear boundary between cells. No apoptotic phenomena of the STO cells were observed in 7～10 days after the STO cells were inoculated with PGC cells.3. The age of the embryo used to culture the EG cells should not be older than 28 days of age, otherwise, PGC would lose responsiveness to stimulating factors secreted by STO cells and showed the property of differentiation. No EG colonies were found in the culturing of embryo with the age of 29 days.4. As a serum used for cultivation of ES cells, we found that ES Cell Quality Fetal Bovine Serum (GIBCO) was the best one, the Fetal Bovine Serum (Sijiqing,Hangzhou) was the last one, and the Fetal Bovine Serum (Hyclon) was placed in the middle. There was a remarkably improvement in the speed of proliferation when the consistence of the same serum enhanced from 10% to 15%.5. Endogenous growth factors produced by either the STO feeder layer or the porcine PGC themselves were sufficient to support the survival and prolifieration of PGC in short-term chlture. The growth factors (SCF and bFGF) had been showed to be essential for stimulating proliferation and for inhibiting differentiation of porcine PGC in long-term culture.6. The passage time of the EG cells was closely associated with establishing porcine EG cell lines. It was very difficulty to form new EG colonies on new feeder when EG cells were passed 9～10 days after plating, 7. Six of 19 embryos differentiated distinctly when the embryos were incubated separately and no new EG cell colonies formed, while a lower degree of differentiation was observed when 2 ~4 embryos were cultured together. 8. It was found that the EG cell was positive to dying reaction of AKP, to immunofluorescence reaction for SSEA-1 antibodies, and to immunofluorescence reaction for Oct-4. This result indicated that SSEA-1 antigen and the expression of oct-4 gene existed in the surface of EG cells.9. EG cells would differentiate into fibroblasts-like cells, tyophoblast-like cells and neuron-like cells when porcine EG cells were passed onto a 0.1% gelatin-coated plate. 10. The EG cells would form embryoid bodies when they cultured in suspension. The embryoid bodies would differentiate into neuron-like cells, tyophoblast-like cells, morula-like colonied and ES-like colonies when they cultured and treated by using RA and DMSO.11. In the incubating of the testicle cells obtained from piglet with the age of 2-6 days, it was observed that the sertoli cells grown faster than spermatogonial stem cells (SSCs). Sertoli cells, formed the layer of feeder cells, nourished SSCs to grow and proliferate. The SSCs colonies were stained as positive to AKP activity. And its morphology was similar to that of EG cells. 12. Two chimeric piglets were obtained by using PGCs and EG cells of WZSP. Microsatellite technology was used to check the various tissues of the chimeric piglet, and the results indicated that the EG cells of WZSP contributed to the following tissues: skin, brain, pancreas, thyroid gland and blood.