Isolation And Characterization Of Human Umbilical Cord Mesenchymal Stem Cells And The Study Of Reprogramming With Defined Factors

?Background:The generation of induced pluripotent stem cell (iPSC) not only bypasses the ethic obstraction of embryonic stem cells for the emergence of tissue-engineered human, but also provides a new direction and wonderful materials for stem cell research. However, the iPSC technology is not yet mature, high costs and very low success ratio of induction has seriously hampered the development of iPSC, so looking for a highly efficient induction method is the urgent scientific problems. Mesenchymal stem cells (MSCs) is one of the low differentiation cells in somatic cells. For its horizontal differentiation ability, it plays a high-profile in adult stem cell research field. Compared with other adulult cells, MSCs have the greater plasticity, so it may be more easily induced with the reported retroviral vectors to become pluripotent stem cells. Adenovirus vector is a highly efficient gene transfection vector, its large capacity, high output, low toxic side effects, etc. make it widely used in gene therapy. This topic trying to preliminary explore using adenovirus vector carrying Oct4, Sox2, Klf4, Nanog four transcription factors to induce mesenchymal stem cells reprogramming, and hope to explore an efficient and economical iPSC induction method for clinical applications and lay the foundation for cell therapy.Methods: 1) Isolation mesenchymal stem cells from human umbilical cord, then culture and identified in vitro. 2) Induce the isolated cells with the reported retroviral vectors to iPSC, and identify the relevant indicators. 3) Construction of chimeric adenovirus Ad5/F11b, carrying Oct4, Sox2, Klf4, Nanog transcription factor genes. 4) Induce the isolated UCMSCs with the chimeric adenovirus Ad5/F11b to iPSC, and identify the relevant indicators.Resulults: 1) The mesenchymal-like cells isolated from human umbilical cord, through cell cycle, surface markers, differentiation and other indicators have proved their potential for mesenchymal stem cells. 2) Induction of isolated umbilical cord mesenchymal stem cells by reported retovirus formed the embryonic stem cells (hESCs)-like clone groups. The detection of the clone groups at the mRNA level shows four transcription factors were upregulated expression in different levels, and hESCs-related genes the expression level has improved markedly. Biochemical parameters could be further testing to determine whether its iPSC. 3) Construction of a carrying Oct4, Sox2, Klf4, Nanog transcription factor genes adenovirus backbone plasmid have been determined to be correct clone by enzyme digestion and packaging in HEK293 cells. 4) Induction of isolated umbilical cord mesenchymal stem cells by the chimeric Ad5/11b adenovirus formed the hESCs-like clone groups, and the relevant indicators need to be further identified.Conclusions: 1) Successfully isolated mesenchymal stem cells from human umbilical cord and formed hESCs-like clone group cells retrovirus inducing. The 4 transcription factors and hESCs related genes are upregulated expression, showing characteristics similar to hESCs. 2) Construction of a carrying Oct4, Sox2, Klf4, Nanog transcription factor genes adenovirus backbone plasmid and prodcted chimeric Ad5/11b virus. 3) Induction of isolated umbilical cord mesenchymal stem cells by the chimeric Ad5/11b adenovirus formed the embryonic stem cells (hESCs)-like clone groups, and the relevant indicators need to be further identified.