Switching of mesodermal and endodermal properties in hTERT-modified and expanded fetal human pancreatic progenitor...

The ability to expand organ-specific stem/progenitor cells is critical for translational applications, although uncertainties often arise in identifying the lineage of expanded cells. Therefore, superior insights into lineage maintenance mechanisms will be helpful for cell/gene therapy.

We studied epithelial cells isolated from fetal human pancreas to assess their proliferation potential, changes in lineage markers during culture, and capacity for generating insulin-expressing beta cells. Cells were isolated by immunomagnetic sorting for epithelial cell adhesion molecule (EpCAM), and characterized for islet-associated transcription factors, hormones, and ductal markers. Further studies were performed after modification of cells with the catalytic subunit of human telomerase reverse transcriptase (hTERT).

Fetal pancreatic progenitor cells efficiently formed primary cultures, although their replication capacity was limited. This was overcome by introduction and expression of hTERT with a retroviral vector, which greatly enhanced cellular replication in vitro. However, we found that during culture hTERT-modified pancreatic progenitor cells switched their phenotype with gain of additional mesodermal properties. This phenotypic switching was inhibited when a pancreas-duodenal homeobox (Pdx)-1 transgene was expressed in hTERT-modified cells with a lentiviral vector, along with inductive signaling through activin A and serum deprivation. This restored endocrine properties of hTERT-modified cells in vitro. Moreover, transplantation studies in immunodeficient mice verified the capacity of these cells for expressing insulin in vivo.

Limited replication capacity of pancreatic endocrine progenitor cells was overcome by the hTERT mechanism, which should facilitate further studies of such cells, although mechanisms regulating switches between meso-endodermal fates of expanded cells will need to be controlled for developing specific applications. The availability of hTERT-expanded fetal pancreatic endocrine progenitor cells will be helpful for studying and recapitulating stage-specific beta lineage advancement in pluripotent stem cells.