title: Directed differentiation of human embryonic stem cells into keratinocyte progenitors in vitro: an attempt with promise of clinical use.
abstract: Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus, efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA), bone morphogenetic protein-4 (BMP4), and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA, AA, and BMP4; the third was commercial-defined keratinocyte serum-free medium, which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR, immunofluorescence, and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology, while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12, all P < 0.05) After hESC induction by AA+BMP4+RA, CK14 mRNA expression was upregulated after day 21, peaking by 35 d of differentiation. Combined RA, BMP4, and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering.
authors: Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, 200011, China.
authors: Hanqing
authors: H
authors: Li
authors: Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, 200011, China. haiwen39@126.com.
authors: Haiwen
authors: H
authors: Zhou
authors: Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100144, China.
authors: Xin
authors: X
authors: Fu
authors: Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100144, China.
authors: Ran
authors: R
authors: Xiao
mesh_terms: D001205:Ascorbic Acid / Q000008:administration & dosage; D055415:Bone Morphogenetic Protein 4 / Q000008:administration & dosage; D002454:Cell Differentiation / Q000187:drug effects*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000637:transplantation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects; D009061:Mouth Mucosa / Q000254:growth & development; D012333:RNA, Messenger / Q000096:biosynthesis; D012867:Skin / Q000166:cytology / Q000254:growth & development; D023822:Tissue Engineering; D014212:Tretinoin / Q000008:administration & dosage
chemical_list: C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D012333:RNA, Messenger; D014212:Tretinoin; D001205:Ascorbic Acid
keywords: Ascorbic acid; Bone morphogenetic protein-4; Differentiation; Human embryonic stem cells; Keratinocyte progenitors; Retinoic acid