Selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

Urszula Polak, Calley Hirsch, Sherman Ku, Joel Gottesfeld, Sharon Y.R. Dent, Marek Napierala

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals, human newborn fibroblasts, as well as human keratinocytes.

Original languageEnglish (US)
Article numbere3416
JournalJournal of Visualized Experiments
Issue number60
DOIs
StatePublished - Feb 20 2012
Externally publishedYes

Keywords

  • Developmental Biology
  • Induced pluripotent stem cells
  • iPSC
  • Issue 60
  • Pluripotency
  • Retroviral transduction
  • Somatic cell reprogramming
  • Stem cells

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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