Establishment and identification of congenital clubfoot induced pluripotent stem cells

Objective To establish specific induced pluripotent stem cells (iPSCs)fromthe patient with congenitalclubfoot by taking exfoliated cells in urine as source. Methods 180 ml of urine from clubfoot patient were collected, and the exfoliated cells in urine were isolated and cultured by centrifugation. Then, the cells were reprogrammed with virus system and identified by pluripotency gene detection, alkaline phosphatase (ALP) staining, karyotype identification, Fluorescein activated cell sorter (FACS) surface markers detection, embryoid body (EB) suspension ball assay and methylation assay. Results The expression of pluripotentgenes between obtained cellsand human embryonic stem cells H9 was similar, ALP staining showed strong positive. The cells possessed normal karyotype and 84.1% positive rate of pluripotency marker Tra-1-60, they could form a large number of EB suspension balls and almost lost methylation of the promoter regionsin Nanog homeobox (NANOG) and organic cation/carnitine transporter4 (Oct4) genes. Therefore, werevealed that the obtained cells owned all of the characteristics of pluripotent cells, and wecan identified them as iPSCs. Conclusion iPSCs were successfully constructed in this study, which provided cell materials for the clarification of evolution characteristics of congenital clubfootdisease. Key words: Congenital clubfoot; Induced pluripotent stem cells; Urine-derivedcells; Reprogramming