title: Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid-derived stem cells to insulin-producing clusters.
abstract: This study investigated the differentiation of human amniotic fluid-derived stem cells (hAFSCs) into insulin-producing clusters in vitro. Adenovirally-delivered mouse Pdx1 (Ad-Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream β-cell markers. When Ad-Pdx1-transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly-l-ornithine, the expression of islet-associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C-peptide ELISA confirmed that Ad-Pdx1-transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic β-cells. To sustain the β-cell-like phenotype and investigate the effect of three-dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1-transduced cells were encapsulated in alginate and cultured long-term under serum-free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a β-cell-like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets.
authors.affiliation: Joint Institute for Regenerative Medicine, Kyungpook National University Hospital, Daegu, Korea.
authors.forename: So Young
authors.initials: SY
authors.lastname: Chun
authors.forename: David L
authors.initials: DL
authors.lastname: Mack
authors.forename: Emily
authors.initials: E
authors.lastname: Moorefield
authors.forename: Se Heang
authors.initials: SH
authors.lastname: Oh
authors.forename: Tae Gyun
authors.initials: TG
authors.lastname: Kwon
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Pettenati
authors.forename: James J
authors.initials: JJ
authors.lastname: Yoo
authors.forename: Paolo De
authors.initials: PD
authors.lastname: Coppi
authors.forename: Anthony
authors.initials: A
authors.lastname: Atala
authors.forename: Shay
authors.initials: S
authors.lastname: Soker
mesh_terms: D000256:Adenoviridae / Q000378:metabolism; D000653:Amniotic Fluid; D000818:Animals; D002096:C-Peptide / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D003470:Culture Media; D016895:Culture Media, Serum-Free / Q000737:chemistry; D003920:Diabetes Mellitus / Q000628:therapy; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology; D051379:Mice; D013234:Stem Cells / Q000166:cytology*; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D002096:C-Peptide; D003470:Culture Media; D016895:Culture Media, Serum-Free; D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein
keywords: Pdx1; amniotic fluid-derived stem cells; cell therapy; diabetes; differentiation; extracellular matrix components; growth factors