Reprogramming of Mouse Embryonic Fibroblast by External Stimuli and Vitamin C

Abstract

After estamblish reprogramming concept, many methods were developed such as using transgenes, synthesized small molecule, and recombinant protein. Induced pluripotent stem cells (iPSCs) can proliferate without limitation and differentiate into three germ lines. Reprogramming cells such as iPSC are good medicine resource because of differentiated to various tissues or cells. However, iPSCs which are generated by traditional methods that transgenes or virus vectors are utilized during generating iPSCs are not appropriate for clinic because of safety and risk of tumor. This research attempted to develop a new method for reprogramming using small molecule and external stimuli without transgene. First, 4 groups were selected

condition with both acid and shear stress, with acid, with shear stress , without acid and shear stress. In acid experiment, cells were soaked at pH 5.4 solution for 25min. In shear stress experiment, pipetting was carried out cells twice per day for 2 minutes under suspension culture and it continued for 7 days. 500ul of embryonic stem (ES) medium was added in 60mm the petri dish every day for 7 days. After 7 days, spheres were picked up and they were utilized in alkaline phosphate (ALP) staining immunocytochemistry to confirm pluripotency. Spheres of all group showed positive- ALP and pluripotent proteins but aspects of staining were not perfect. This experiment show that acid or shear stress assisted to generate spheres compared with group without any stimulation but acid induced cell death. For improving efficiency, stimulated period was modified much longer and vitamin C was selected instead of acid. Vitamin C had been reported that it promoted to accelerate reprograming and increased efficiency of generating iPSCs. 2 groups were established

shear stress under suspension culture and combination of shear stress and vitamin C under suspension culture. ES medium or ES medium with 10uM vitamin C was added into experimental group every day for 14 days according to experimental design. For 14 days, pipetting was carried out cells twice per day for 2 minutes. After 14 days, all spheres were picked up and were performed experiments for confirming pluripotency and capacity of differentiation such as ALP staining, immunocytochemistry with pluripotent markers and 3 germ layers marker, western blot, fluorescence activated cell sorter (FACS) and teratoma formation in vivo. Both groups were observed positive results in ALP staining, immunocytochemistry with pluripotent markers, western blot, FACS, differentiation in vitro although difference of degree existed. However, any group did not form teratoma as in vivo experiment. Although shear stress group and group of combination of shear stress and vitamin C were not significantly different in FACS data, most of data demonstrated the spheres under shear stress and 10uM vitamin C had better. In synthesizing all data, combination of 10uM vitamin C and shear stress under suspension culture was appropriate for generating spheres which had capacity of differentiation. This research showed possibility that methods using external stimuli and small molecule without transgenes can reprogram from somatic cells to differentiated cells. This method would be utilized in direct cell reprogramming field.