Prospectively Isolated NGN3-Expressing Progenitors From Human Embryonic Stem Cells Give Rise to Pancreatic Endocrine Cells

Abstract

Pancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3. We targeted the 3' end of NGN3 using zinc finger nuclease-mediated homologous recombination to allow selection of NGN3eGFP(+) cells without disrupting the coding sequence of the gene. Isolated NGN3eGFP(+) cells express PDX1, NKX6.1, and chromogranin A and differentiate in vivo toward insulin, glucagon, and somatostatin single hormone-expressing cells but not to ductal or exocrine pancreatic cells or other endodermal, mesodermal, or ectodermal lineages. This confirms that NGN3(+) cells represent pancreatic endocrine progenitors in humans. In addition, this hESC reporter line constitutes a unique tool that may aid in gaining insight into the developmental mechanisms underlying fate choices in human pancreas and in developing cell-based therapies.

Using stem cells to produce insulin

“…To address these limitations, homologous recombination to 'knock in' a fluorescent protein into a specific genetic locus can be used. Currently, hESC reporter lines exist that have been used to sort neuronal [145], cardiac [146] and b-cell precursors [147,148].…”

Pancreatic Stem Cells Remain Unresolved

“…Again, it remains to be clarified whether the ESC-derived Ngn3-expressing cells [54,57] were bona fide islet progenitors.…”

Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo

“…Ngn3 is a marker of endocrine progenitors [32, 33] and neogenesis [34, 35] in the adult pancreas. Additionally, Pdx1-expressing cells were confirmed in newly formed PIDs because Pdx1 is a critical transcription factor in the embryologic development [36, 37] and regeneration of the pancreas [38].…”