Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid-derived stem cells to insulin-producing clusters

J Tissue Eng Regen Med. 2015 May;9(5):540-9. doi: 10.1002/term.1631. Epub 2012 Nov 13.

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.

Keywords: Pdx1; amniotic fluid-derived stem cells; cell therapy; diabetes; differentiation; extracellular matrix components; growth factors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / metabolism
  • Amniotic Fluid
  • Animals
  • C-Peptide / metabolism
  • Cell Culture Techniques / methods*
  • Cell Differentiation
  • Cell- and Tissue-Based Therapy / methods
  • Culture Media
  • Culture Media, Serum-Free / chemistry
  • Diabetes Mellitus / therapy
  • Enzyme-Linked Immunosorbent Assay
  • Extracellular Matrix / metabolism
  • Homeodomain Proteins / metabolism*
  • Humans
  • Insulin / metabolism*
  • Insulin-Secreting Cells / cytology
  • Mice
  • Stem Cells / cytology*
  • Trans-Activators / metabolism*

Substances

  • C-Peptide
  • Culture Media
  • Culture Media, Serum-Free
  • Homeodomain Proteins
  • Insulin
  • Trans-Activators
  • pancreatic and duodenal homeobox 1 protein