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

Abstract

Introduction: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Figure 1

Fractionation of EpCAM-positive epithelial cells from fetal human pancreas. (a) shows intact 22-week fetal human pancreas with hematoxylin staining alone (extreme left) or histochemical staining for DPPIV, GGT and glycogen, as indicated, which were expressed in cells in ductal (arrows), periductal and acinar regions. (b) shows EpCAM-positive cells isolated by immunomagnetic cell sorting with EpCAM, DPPIV and GGT expression but absence of glycogen. (c) shows EpCAM-negative cell fraction with only occasional epithelial cells and more abundant glycogen-containing acinar cells (arrows, panel extreme right). Orig. mag., a, ×200; b and c, ×400.

Figure 2

Endocrine phenotype of EpCAM-positive fetal pancreatic cells. (a) shows immunostaining of 22-week fetal human pancreas to demonstrate cells in primitive pancreatic islets with coexpression of insulin and glucagon. Negative controls, where primary antibodies were omitted, are on extreme right. (b) shows isolated freshly EpCAM-positive cells with coexpression of insulin and glucagon in some cells. (c) shows EpCAM-negative fraction showing occasional cells with glucagon. Orig. mag., a, ×200; b and c, ×400.

Figure 3

Initial characterization of fetal pancreatic cells. (a) and (b) show morphology of cells in culture after 2 d and 7 d. Note epithelial morphology of EpCAM-positive cells. (c) shows RT-PCR for genes as indicated. Lanes 1 to 6 show results from mature human pancreatic islets, intact fetal pancreas, cells after early term culture (1 to 2 d) or longer culture (10 to 14 d). For comparisons, β-actin and glyceraldehyde phosphate dehydrogenase (GAPDH) genes were included.

Figure 4

Immortalization of EpCAM-positive fetal pancreatic cells by hTERT. (a) shows RT-PCR for hTERT expression after early and late passages of hTERT-FPC. Note the absence of hTERT expression in mature human islets and fetal pancreas, whereas hTERT-FH-B fetal human liver cells expressed hTERT (positive control). (b) shows kinetics of proliferation in primary EpCAM-positive fetal pancreatic cells and hTERT-FPC during culture over up to four weeks, which was 4-6-fold greater in the latter. Asterisks indicate P < 0.05. (c) shows RT-PCR for insulin, which was expressed in mature islets (lane 1), fetal pancreas (lane 2) and primary EpCAM-positive cells during early and late culture (lanes 3 and 4), as well as an early passage (P3) of hTERT-FPC (lane 5) but not in hTERT-FPC after further cell culture (lane 6).

Figure 5

Induction of insulin-expression in hTERT-FPC by Pdx1-LV. (a) shows schematic of LV with rat Pdx1 and GFP genes driven by hPGK promoter - IRES, intervening internal ribosomal entry site, cPPT, central polypurine tract, Wpre, posttranscriptional regulatory element of the woodchuck hepadnavirus. (b) shows Pdx1-LV-transduced hTERT-FPC under phase contrast (top) and under epifluorescence for GFP. (c) shows flow cytometric quantitation of GFP in nontransduced cells (top panel) and Pdx1-LV-transduced hTERT-FPC. MFI = mean fluorescence intensity. (d) shows RT-PCR for gene expression in control hTERT-FPC (lane 1), Pdx1-LV-transduced hTERT-FPC cultured without serum (lane 2) and without serum plus activin A (lane 3), and mature pancreatic islets (lane 4). (e) shows insulin and c-peptide expression in negative control hTERT-FPC-Pdx1 cells, where primary antibodies were omitted, and cells with expression of both insulin and c-peptide. Orig. Mag., × 200.

Figure 6

Phenotype alterations in fetal pancreatic cells. (a) shows RT-PCR for epithelial marker, CK-19, and mesenchymal marker, vimentin, along with TGF-β1, TGFβ2 and their receptors under various conditions indicated. (b) shows morphological changes in LV-Pdx1-transduced hTERT-FPC during culture with serum and in the absence of serum plus addition of Activin A (bottom panel). These data indicated that cells became more rounded and less flattened in the absence of serum and presence of Activin A. (c) shows changes in vimentin expression by immunostaining in LV-Pdx1-transduced hTERT-FPC cultured with serum (top left), and with Activin A and no serum (bottom left). No immunostaining was detected when vimentin antibody was omitted (top right). The panel at bottom right in c shows quantitation of vimentin immunofluorescence signals by image analysis to indicate that culture without serum and with activin A perturbed cell phenotype, which was in agreement with morphological changes in LV-Pdx1-transduced hTERT-FPC.

Figure 7

Transplantation studies with hTERT-FPC. (a) shows DNA PCR for human sequences to identify hTERT-FPC in the liver of NOD/SCID mice 24 hours, 1 week, 2 weeks and 1 month after intrasplenic transplantation. (b-f) show in situ hybridization for alphoid satellite sequences in human centromeres to verify that transplanted cells were present in tissues. (b), fetal human liver as positive control to show hybridization signals in cell nuclei (arrow); (c), mouse liver showing absence of hybridization signals; (d), (e) and (f) show tissues from animals 24 hours, 1 week and 1 month after cell transplantation, respectively, with transplanted cells localized by nuclear in situ signals (arrows). (g-i) show sequential immunostaining for GFP (g) and insulin (h) with merged image of these two panels (i) two weeks after transplantation in the liver to verify β cell phenotype in transplanted hTERT-FPC.