Optimized surface markers for the prospective isolation of high-quality hiPSCs using flow cytometry selection

Sci Rep. 2013:3:1179. doi: 10.1038/srep01179. Epub 2013 Jan 31.

Abstract

hiPSC derivation and selection remains inefficient; with selection of high quality clones dependent on extensive characterization which is not amenable to high-throughput (HTP) approaches. We recently described the use of a cocktail of small molecules to enhance hiPSC survival and stability in single cell culture and the use of flow cytometry cell sorting in the HTP-derivation of hiPSCs. Here we report an enhanced protocol for the isolation of bona fide hiPSCs in FACS-based selection using an optimized combination of cell surface markers including CD30. Depletion of CD30(+) cells from reprogramming cultures almost completely abolished the NANOG and OCT4 positive sub-population, suggesting it is a pivotal marker of pluripotent cells. Combining CD30 to SSEA4 and TRA-1-81 in FACS greatly enhanced specificity and efficiency of hiPSC selection and derivation. The current method allows for the efficient and automated, prospective isolation of high-quality hiPSC from the reprogramming cell milieu.

MeSH terms

  • Animals
  • Antigens, Surface / metabolism
  • Cell Differentiation
  • Cell Line
  • Cell Separation*
  • Cellular Reprogramming
  • Flow Cytometry*
  • Homeodomain Proteins / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Karyotyping
  • Ki-1 Antigen / genetics
  • Ki-1 Antigen / metabolism
  • Mice
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / metabolism
  • Stage-Specific Embryonic Antigens / metabolism
  • Teratoma / pathology

Substances

  • Antigens, Surface
  • Homeodomain Proteins
  • Ki-1 Antigen
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • Stage-Specific Embryonic Antigens
  • TRA-1-81 antigen, human
  • stage-specific embryonic antigen-4