Abstract

Although human undifferentiated keratinocytes (HUKs) can be reprogrammed to become induced pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the endogenous expression of reprogramming factors in cultured HUKs is not clear at different stages. In this study, keratinocytes were isolated from foreskin of adult subject (18 years old) and cultured on collagen type IV-coated culture dishes in a low-calcium, serum-free medium (Epilife, Invitrogen). In order to clarify the expression patterns of RFs and other stem cell markers in cultured human keratinocytes, total RNA was extracted using Trizol reagent, and polymerase chain reaction (PCR) was performed using established GenBank sequences to design primers. The subsequent PCR analysis was carried out by agarose gel electrophoresis. The expression levels of RFs and other stem cell markers in human HUKs clearly fluctuated during culturing, which supports the hypothesis that HUKs might be reprogrammed into a pluripotent state when the maximum levels of RFs expression are maintained by appropriate culture conditions.

Key words: Human undifferentiated keratinocytes, reprogramming factors, expression fluctuation.

Abbreviation

HUKs, Human undifferentiated keratinocytes; iPSCs, induced pluripotent stem cells; RFs, reprogramming factors; PCR, polymerase chain reaction;ESCs, embryonic stem cells; Oct4, octamer-binding transcription factor 4; Sox2,SRY (sex determining region Y)-box 2; Klf4, Krüeppel-like factor 4; c-Myc, v-myc myelocytomatosis viral oncogene homolog; CRIPTO, teratocarcinoma-derived growth factor 1; REX1, RNA exonuclease 1; KRT, keratin; RT-PCR, reverse transcription polymerase chain reaction.