INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

Diabetologia. 2012 Mar;55(3):694-706. doi: 10.1007/s00125-011-2379-y. Epub 2011 Nov 26.

Abstract

Aims/hypothesis: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro.

Methods: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells.

Results: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells.

Conclusions/interpretation: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cell Line
  • Clone Cells
  • Diabetes Mellitus, Type 1 / therapy
  • Embryoid Bodies / metabolism
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / metabolism*
  • Embryonic Stem Cells / transplantation
  • Eye Proteins / genetics
  • Eye Proteins / metabolism
  • Gene Expression Profiling
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Insulin / genetics
  • Insulin / metabolism*
  • Insulin-Secreting Cells / cytology*
  • Insulin-Secreting Cells / metabolism*
  • Insulin-Secreting Cells / transplantation
  • LIM-Homeodomain Proteins / genetics
  • LIM-Homeodomain Proteins / metabolism
  • Oligonucleotide Array Sequence Analysis
  • PAX6 Transcription Factor
  • Paired Box Transcription Factors / genetics
  • Paired Box Transcription Factors / metabolism
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Recombination, Genetic
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Zebrafish Proteins

Substances

  • Eye Proteins
  • Homeodomain Proteins
  • Insulin
  • LIM-Homeodomain Proteins
  • NKX6-1 protein, human
  • Nkx2.2 protein
  • PAX6 Transcription Factor
  • PAX6 protein, human
  • Paired Box Transcription Factors
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Transcription Factors
  • Zebrafish Proteins
  • enhanced green fluorescent protein
  • insulin gene enhancer binding protein Isl-1
  • Green Fluorescent Proteins