Induction Of Meiosis In Primordial Germ Cells And Renal Mesenchymal Stem Cells

In this work, primordial germ cells(PGCs) were isolated from the 5.5 d of chicken embryos genital ridge. We established an in vitro culture system for chicken PGCs. To investigate in vitro development potential of chick PGCs, the biological characteristics and induction of spermatogenesis of PGCs were been studied. And the renal MSCs were isolated from the kidney tissue of 6-week-old sheep fetus. The biological characteristics of MSCs were studied, and we induced it into spermatogenesis in vitro. Provided a theoretical base for induction of stem cell spermatogenesis in vitro. Results:1. Gonads of the chicken embryos were isolated and dissociated into single cells with 0.125 % trypsin. Then cultured PGCs in PGC medium which consisted of H-DMEM medium replenished with 10 % FBS, 2 % chicken serum and supplemented with 1 mM sodium pyruvate, 2 mM L-Glutamine, 55 μM β-mercaptoethanol, 10 ng/mL of hSCF, 10 units/mL LIF, 20 ng/mL of bFGF and 10 ng/mL IGF. The single PGCs were attached to surface of gonadal stroma cells and grown up into small cell colonies after seeding for 24 h. After culture 48 h, small cell PGCs colonies turned to bigger cell clusters and the colonies numbers increased. After 3 days later, PGCs were seeded onto feeder cells of CEFs which treated with 10 μg/mL Mitomycin-C. Compare different inoculation method of PGCs plated together with their own gonadal stroma cells and CEFs for PGCs culture, we found that the primary cultivation of the chicken PGCs plated together with their own gonadal stroma cells could form higher efficiency of PGCs colonies.2. The PGCs were cultured for 6 passages in this culture system. This study showed that PGCs were stained mauve with AKP staining and red by PAS staining. The size of PGCs, large amounts of glycogen granules in the cytoplasm and large nuclei, are bigger than somatic cells. RT-PCR identification shown the cells colonies expressed PGCs specific gene CVH, BLIMP1, POUV and NANOG. Immunofluorescence identification shown the cells colonies expressed SSEA-1, SSEA-3, BLIMP1, OCT4 and SOX2 antigen.3. PGCs could been induced into stages of meiosis and formed haploid sperm cells by 1 nmol/mL RA after 24 h. RT-PCR identification shown the cells expressed SYCP1, BOULE, DAZL、STRA8, DMC1 and ACR gene for meiotic and haploid cells after induction. RA and SCF improve SYCP1, BOULE and DMC1 expression, rise the differentiating efficiency of PGCs into haploid spermatids.4. The kidney tissues were isolated and dissociated into single cells with 0.25 % trypsin. Then isolated cells were cultured in medium which consisted of DMEM/F12 medium replenished with 10 % FBS for primary culture. Cells culture medium were changed to remove non-adherent cells after 24 h later. When cultures reached 80 % confluence, cells were digested and then subcultured onto new plates. After 3–4 passages cells were purified. The flow analysis shown the cell-purity was over 95 %. The results of RT-PCR and immunofluorescence shown that the MSCs expressed OCT4, CD44, VIM, PAX2 and FN1. The growth curve of MSCs from three different passages was appeared as typically sigmoidal, which consisted of a latent phase, a logarithmic phase, a plateau phase and a degenerating stage. The passage 4, 10 and 16 MMSCs colony-forming efficiency rates were respectively 56.33 % ± 2.52 %, 34.33 % ± 3.06 % and 19.67 % ± 2.08 %. The MSCs were diploid(2n=54), and diploid cells accounted for 95 %, implying that the cultured cells possessed of genetic stability.5. We successfully induced the MSCs to differentiate into adipocytes, hepatocyte and chondrocyte cells under the corresponding induced condition. The purpose of cells were identified by RT-PCR and specific staining.6. RA and testicular extracts can stimulate MSCs to differentiate into meiotic stage in vitro. The results of RT-PCR shown that the cells induced by RA and testicular extracts expressed DAZL, PRM1, TNP1, TNP2, DDX4, MLH1 and SCYP3. Immunofluorescence shown that the cells induced by RA and testicular extracts expressed markers of meiotic cells and haploid spermatids of DAZL, C-KIT, CTDSPL and ACR. Haploid spermatids were remarkably increased with testicular extracts and RA treatment.