Study Of Isolation, Culture, And Study Of The Proliferation And Differentiation Of Meishan Swine Bone Mesenchymal Stem Cells

Mesenchymal stem cells derived from bone marrow (BMSC) are a population of self-renewing multipotent cells. These stem cells are capable of differentiating into various cellular lineages, including the osteogenic, chondrogenic, adipogenic, and myogenic lineages, as well as are widely used in clinical investigations. The expansion and differentiation potential of these BMSC in vitro is essential for use in various stem cell therapies. However, the cells lose their proliferation and differentiation capacity after several passages. The key transcription factors Oct4 and Sox2 have been extensively studied in embryonic stem (ES) cells. Oct4 is a critical transcription factor for regulating self-renewal and differentiation of ES cells. It is expressed in ES cells and germ cells. Overexpression of Oct4 results in ES cell differentiation into primitive endoderm and mesoderm. Repression of Oct4 expression induces loss of ES cell pluripotency and dedifferentiation to trophectoderm. The transcription factor Sox2 is expressed in pluripotent cells and embryonic and extraembryonic cells, and plays an important role in embryonic development and in maintaining the pluripotency and self-renewal of ES cells. Oct4 and Sox2 have been shown to be critical factors for reprogramming somatic cells into induced pluripotent stem cells (iPS).In the current study, three experiments were conducted to isolate and culture BMSC in Meishan swine. The characteristics of proliferation and growth, as well as the differentiation of cultured BMSC in vitro were investigated.The effects of Oct4 or Sox2 genes overexpression on proliferation and differentiation of Meishan swine BMSC were also studied. The founds of the study will lay theoretical basis for BMSC’s application in clinical practice.The main content of the three experiments are as follows,1. BMSC were isolated from the whole bone marrow and cultured in vitro. The morphology of cultured BMSC was observed by an invert microscope. Chromosome analysis was checked by a method of pressing root tip cells. The cell cycle, surface markers and pluripotency genes of BMSC in third passage (P3) were analyzed. The results showed that, the swine BMSC grew well in vitro. The adherent cells proliferated obviously, and most of which were presented in spindle shape. BMSC expressed CD44, CD29, CD90, Fou5Fl, Sox2 and Nanog, and did not expressed CD45 and Lin28. In conclusion, Meishan BMSC can be isolated, purified and well cultured in vitro by whole bone marrow method, which can be served as optimal cell source for further research.2. To compare the differences of cell morphology, proliferation and multiple differentiation potential. Cultured BMSCs between 3th and 12th passage were comparedby counting method and multiplelineage induction. The results showed that cells showed long fusiform, high proliferation rate, and the short passage cycle at the beginning. After the 7th or 8th passage, cells extended to become magnanimous, flat and thin, and proliferated slow. BMSCs were induced into adipogenic for 21 days, Oil red O staining results showed that the lipid drops of 3th passage BMSC was more than 3th passage BMSC. The mRNA levels of PPARy and CEBPa in 3th passage BMSC were higher than that of 12 th passage BMSC by real-time PCR (P< 0.05). BMSC were induced into osteoblasts for 2 weeks, the mineralization nodules of 3th passage BMSC were more than 3th passage BMSC by alkaline phosphatase (ALP) staining. The mRNA levels of Osteocalcin, Osteonectin and Type Ial in 3th passage BMSC were higher than that of 12th passage BMSC by real-time PCR (P<0.05).3. To study that the effects of Oct4 or Sox2 genes overexpression on proliferation and differentiation of Meishan swine BMSC. Counting method, Fibroblast-colony-forming unit (CFU-F) assay and multiplelineage induction were used. The results showed that, Oct4 or Sox2 genes overexpression of BMSC had higher proliferation rate and colony formation rate compared with BMSC from control treatment. BMSC were induced into adipogenic for 21 days, the lipid drops of Oct4 or Sox2 genes overexpression of BMSC were more than control BMSC by Oil red O staining. The mRNA levels of PPARy and CEBPa in Oct4 or Sox2 genes overexpression of BMSC were higher than that of control BMSC by real-time PCR (P< 0.05). BMSC were induced into osteoblasts for 2 weeks, the mineralization nodules of Oct4 or Sox2 genes overexpression of BMSC were more than that of control BMSC by ALP staining. The mRNA levels of Osteocalcin, Osteonectin and Type Ial in Oct4 or Sox2 genes overexpression of BMSC were higher than that of control BMSC by real-time PCR (P<0.05), In conclusion, Oct4 or Sox2 genes overexpression of BMSC improve colony formation rate, proliferation and differentiation potential of Meishan swine BMSC, respectively. Consequently, these results will provide theoretical basis for BMSC application in the clinical practice.