Key Biological Characteristics Research Of Marrow Mesenchymal Stem Cell And Embryonic Germ Cell From Beijing Fatty Chicken

To clarify the key biological characteristics of bone marrow mesenchymal stem cell and embryonic germ cell from Beijing fatty chicken, the study researched on isolation, multiplication, identification, transfection and differentiation of two kinds of stem cells. The results are as follows:1. The MSCs can be derived from 30～60d Beijing fatty chicken bone marrow by density gradient centrifuge separation or cell attachment to tissue culture plates. MSCs can be expanded stably in vitro, and sub-cultured to 7th passage. In morphology, the goat MSCs are fusiform, polygonal, and round.8 samples, 25 freezing tubes have been cryopreserved. The survival rate before and after cryopreservation were 98％,87.5％.2. The chicken MSCs have been identified by the following method: Microbial detection indicate no bacteria, fungi, virus and mycomplasma contamination in cell culture.Karyotype analysis showed the cells kept diploid condition and the hereditary feature is stable. PAS resulted positive, Akp negative, and cellular markers detection positive in CD44, CD54, SSEA-4, and negative in CD34, CD45, SSEA-1, c-kit.3. MSCs were transferred by lipofectamine-mediated plasmid expressing pEGFP, The expression of pEGFP reached optimal state in 48h～96h, with the transfection efficiency about 40％, remained strong expression within 1 week.4. Under induction of medium with dexamethasone, ascorbate-phosphate,β-glycerophosphate, and medium with fresh bone particles, MSCs differentiated into osteoblast, which expressed high-level alkaline phosphatase, alizarin red stain positive and tetracycline stain positive.5. Primordial germ cells were got by isolating embryonic gonad and surrounding tissue from chicken embryo (5.5 days of incubation). PGCs were co-cultured with their gonadal somatic cells were well grown in primary culture. When subculture, using primary mice embryonic fibroblast (PMEF) as feeder cells, EG were sub-cultured to the 7th passage. The PGCs had a high nucleus to cytoplasm ratio, prominent nucleoli, and clonies were uniformly round, multi-layered and well delineated. In the research, 8 samples, 25 freezing tubes have been cryopreserved.6. The chicken EG have been identified by the following method: Microbial detection indicate no bacteria, fungi, virus and mycomplasma contamination in cell culture. Karyotype analysis showed the cells kept diploid condition and the hereditary feature is stable. The putative EG cells continued to be PAS positive and expressed alkaline phosphatase, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81.7. EG were transferred by lipofectamine-mediated plasmid expressing pEGFP. The expression of pEGFP reached optimal state in 24h, with the transfection efficiency about 18.4％.8. Under suspension culture without cytokines, the putative EG clonies could be induced to form simple embryoid bodies.Under induction of dexamethasone, ascorbate-phosphate, andβ-glycerophosphate, PGCs and MSCs differentiated into osteoblast, which can be stained by alizarin red.