Promotion Of Bone Marrow Mesenchymal Stem Cells Differentiation By Co-culture With Sertoli Cells

Testis Sertoli cells (SCs) can not only secrete multitudinous trophic factors, growth factors and physiologically regulatory proteins to nourish and promote other cells, but also have function of local immune privilege. Bone marrow mesenchymal stem cells (bmMSCs) have unlimited self-renewal capacity and the remarkable ability to produce mature cells with specialized functions. So bmMSCs have recently gained attention as a useful resource in the fields of regenerative medicine and tissue engineering. In this thesis, the identification and seperation of SCs and bmMSCs were carried out, the differentiation of bone marrow mesenchymal stem cells co-cultured with SCs were succesfully achieved, and the large-scale of bmMSCs cultivation were established in a stirred biotector. The results are as follows:(1) SCs were successfully separated from testis of10-day-old mouse and SCs was identified with Hematoxylin-Eosine staining and scan electronic microscope scanning. In addition, the culture process of SCs was optimized by microcarrier technology in spinner flask culture. SCs were expanded in vitro as well. The maximum cell density of2.37×106cells/ml was obtained from initial cell density of2.25x105cells/ml.(2) bmMSCs were separated from bone marrow by density gradient centrifugation and presented a unique whirl-like arrangement. Flow cytometry detection showed positive rates of surface markers of bmMSCs were98.56%for CD29,83.18%for CD90and0.15%for CD34to identify bmMSCs. The different serum concentrations culture indicated that low-serum cell culture was beneficial for cells in maintaining their morphological characteristics.(3) When co-cultured with SCs and CM induction, most of bmMSCs were induced into chondrogenic and osteogenic lineages were remarkably promoted by cytochemistry, immunocytochemistry, RT-PCR and Western-blot analysis.(4) In this work we established a culture process for the in vitro expansion of bmMSCs in a1.5L stirred bioreactor. Under an inoculum of2.5×105cells/ml, microcarrier at4mg/ml and real-time control stirring regime condition, bmMSCs were successfully expanded in the1.5L bioreactor on Cytodex3microcarriers in combination with a low-serum (1%) medium. The highest number of bmMSCs population doublings (10.4doublings) and the maximum cell density (2.6×106cells/ml) were obtained by replacing50%of the medium each day of cultivation. After enzymatic harvest from microcarriers, the bmMSCs retained their phenotypic properties and potentials to differentiate into chondrogenic and osteogenic lineages.