Method Improvement Of Generating IPSCs By Four Factors And The Generationof Rat Kidney Inherent Cells-derived IPSCs

Background:Recently, the morbidity chronic kidney disease (CKD) has been increasingly grown all over the world. For the patients with end-stage renal disease, the main replacement therapies are dialysis and kidney transplant. However, neither of them can maintain the stability of the internal environment in the body quite well let alone replacement of the kidney’s secretion function. Moreover the donors are quite limited. The rapid development of tissue engineering and stem cell bring hopes for the organ regeneration. Studies show that embryonic stem cell (ESc) can settle inside the decellulrized kidney scaffold and differentiate into kidney inherent cells. However, there are lots of ethical issues of ESc. In 2006, induced pluripotent stem cells(iPSCs) was found by Janpanese scientist Yamanaka, the cell has the similar pluripotency as ESc. Therefore iPSCs can be a perfect replacement of ESc for the ethical issues. Previously, our group has succeeded in constructing of rat decellulrized kidney scaffold. Here we isolated and cultured main rat kidney inherent cells and generated different derived iPSCs by the improved method. Because of the memory of iPSCs, we can infuse them into the rat decellulrized kidney scaffold to observe their differentiation.Objective:We synthesized several methods to improve the efficiency of generation iPSCs and obtaind rat kidney inherent cells-derived iPSCs by that. At last we infusd the iPSCs into rat decellulrized kidney scaffold to observe their survival condition.Methods:(1) The efficiency of generating iPSCs was improved by inhibiting the expression of Mbd3 gene and add Vc and KOSR to the induced medium combined with the classical four factors method, as well as increasing the density of seeding. (2) The main rat kidney inherent cells were isolated and cultured to obtain the different derived iPSCs, these iPSCs were also marked with GFP or RFP. (3) The marked iPSCs were infused into the rat decellulrized kidney scaffold to observe their survival condition.Results:(1) We succeeded in impoving the efficiency of generating iPSCs, from 7 days to 5 days. (2) We succeeded in isolating main rat kidney inherent cells and obtain the different derived iPSCs by the improved method. (3) The RFP marked iPSCs were infuesd into the rat decellulrized kidney scaffold, the survival condition was quite well after 48 hours.Conclusions:(1) The improved method can increase the efficiency of generating iPSCs. (2) The main rat kidney inherent cells can be isolated by double screen and adherent process, and the different derived iPSCs can be obtained by the improved method. (3) The iPSCs can survive in the rat kidney scaffold.