Rodent spermatogonial stem cells and identification (excluding serum in vitro cell culture model)
Abstract
Spermatogonial stem cells with multi-potential stem group of the original cells can be formed under the appropriate regulation of mature sperm or embryo develop into a layer of cells and tissues needed. Therefore, to study the role of early spermatogenesis and the molecular mechanism related to the versatility of
spermatogonial stem cells in development potential, the establishment of in vitro serum free culture system for spermatogonial stem cells can make long-term culture and proliferation or induction of differentiation is the necessary, also help to further clarify the spermatogonial stem cells required for growth factor and its mechanism.
We successfully used in the ICR strain male mice testicular cells cultured in vitro with multi-functional characteristics of
spermatogonial stem cell community, our experimental results, the laminin (6.25ng/ml) of the coating material and EGF (10ng/ml ) + times Insulin / transferring - selenium in the culture, those cells that have specific and highly expressed alkaline phosphatase activity and Oct4 protein, revealing that these cells that have the potential of spermatogonial stem cells. We used reverse transcription
polymerase? Chain reaction and immunofluorescence staining to identify cells in spermatogonial stem cells to further specific
expression of genes and proteins, we found that mRNA level, cells that specific performance of spermatogonial stem cells to specific genes, including Oct4, Mvh, Fragilis, Stella, Dazl, Tex14, Piwil2 such as, but not the performance of c-kit and Gcnf (Oct4 the suppressor);
the protein level, the performance of Oct4, SSEA1, CD29, CD49f, etc., but do not show CD117 ( c-kit), CD30, CD34.
In addition, we used EGFP-lentivirus Our results confirm that the volume is not in the laboratory in vitro cell culture model of serum
can be successfully cultivated in the early spermatogonial stem cells (c-kit negative) of the cell community.
English Abstract
