title: Osteo-/odontogenic differentiation of induced mesenchymal stem cells generated through epithelial-mesenchyme transition of cultured human keratinocytes.
abstract: BACKGROUND
  Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT).
  
  
  METHODS
  We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities.
  
  
  RESULTS
  Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation.
  
  
  CONCLUSIONS
  The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp.
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California; Kyung Hee University School of Dentistry, Seoul, South Korea.
authors.forename: Jin-Kyu
authors.initials: JK
authors.lastname: Yi
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California.
authors.forename: Shebli
authors.initials: S
authors.lastname: Mehrazarin
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California.
authors.forename: Ju-Eun
authors.initials: JE
authors.lastname: Oh
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California.
authors.forename: Anu
authors.initials: A
authors.lastname: Bhalla
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California.
authors.forename: Jenessa
authors.initials: J
authors.lastname: Oo
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California.
authors.forename: Min
authors.initials: M
authors.lastname: Lee
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California; Jonsson Comprehensive Cancer Center at University of California, Los Angeles, Los Angeles, California.
authors.forename: Reuben H
authors.initials: RH
authors.lastname: Kim
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California; Jonsson Comprehensive Cancer Center at University of California, Los Angeles, Los Angeles, California.
authors.forename: Ki-Hyuk
authors.initials: KH
authors.lastname: Shin
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California; Kyung Hee University School of Dentistry, Seoul, South Korea; Jonsson Comprehensive Cancer Center at University of California, Los Angeles, Los Angeles, California; David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California.
authors.forename: No-Hee
authors.initials: NH
authors.lastname: Park
authors.affiliation: University of California, Los Angeles School of Dentistry, Los Angeles, California; Jonsson Comprehensive Cancer Center at University of California, Los Angeles, Los Angeles, California. Electronic address: mkang@dentistry.ucla.edu.
authors.forename: Mo K
authors.initials: MK
authors.lastname: Kang
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D015820:Cadherins / Q000032:analysis; D002113:Calcification, Physiologic / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D005353:Fibronectins / Q000032:analysis; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D009805:Odontogenesis / Q000502:physiology*; D015675:Osteocalcin / Q000032:analysis; D010012:Osteogenesis / Q000502:physiology*; D015676:Osteonectin / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis; D034741:RNA, Small Interfering / Q000235:genetics; D016615:Telomere / Q000648:ultrastructure; D054457:Tissue Scaffolds / Q000737:chemistry; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics; D014746:Vimentin / Q000032:analysis
chemical_list: D015820:Cadherins; D005353:Fibronectins; D015676:Osteonectin; D011498:Protein Precursors; D034741:RNA, Small Interfering; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014746:Vimentin; D015675:Osteocalcin; C032253:involucrin; D003094:Collagen; D000469:Alkaline Phosphatase
keywords: Epithelial–mesenchymal transition; keratinocytes; mesenchymal stem cells; p63; pulp regeneration