Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells

J Dent Res. 2016 Jan;95(1):110-8. doi: 10.1177/0022034515611602. Epub 2015 Oct 14.

Abstract

Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs.

Keywords: culture media; feeder cells; gingiva; insulin-like growth factor II; regenerative medicine; serum-free.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Autografts / cytology
  • Cell Adhesion Molecules / analysis
  • Cell Culture Techniques
  • Cell Differentiation / physiology
  • Cell Movement / physiology
  • Cell Proliferation
  • Cells, Cultured
  • CpG Islands / genetics
  • Cross-Linking Reagents / pharmacology
  • Culture Media
  • Culture Media, Serum-Free
  • Fibroblasts / drug effects
  • Fibroblasts / physiology*
  • Gingiva / cytology*
  • Homeodomain Proteins / analysis
  • Humans
  • Induced Pluripotent Stem Cells / physiology*
  • Insulin-Like Growth Factor II / analysis
  • Karyotype
  • Laminin / analysis
  • Mice
  • Middle Aged
  • Mitomycin / pharmacology
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / analysis
  • Promoter Regions, Genetic / genetics
  • Skin / cytology

Substances

  • Cell Adhesion Molecules
  • Cross-Linking Reagents
  • Culture Media
  • Culture Media, Serum-Free
  • Homeodomain Proteins
  • Laminin
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • kalinin
  • laminin alpha5
  • Mitomycin
  • Insulin-Like Growth Factor II