β-Cell Differentiation of Human Pancreatic Duct–Derived Cells AfterIn VitroExpansion

Abstract

β-Cell replacement therapy is a promising field of research that is currently evaluating new sources of cells for clinical use. Pancreatic epithelial cells are potent candidates for β-cell engineering, but their large-scale expansion has not been evidenced yet. Here we describe the efficient expansion and β-cell differentiation of purified human pancreatic duct cells (DCs). When cultured in endothelial growth-promoting media, purified CA19-9(+) cells proliferated extensively and achieved up to 22 population doublings over nine passages. While proliferating, human pancreatic duct-derived cells (HDDCs) downregulated most DC markers, but they retained low CK19 and SOX9 gene expression. HDDCs acquired mesenchymal features but differed from fibroblasts or pancreatic stromal cells. Coexpression of duct and mesenchymal markers suggested that HDDCs were derived from DCs via a partial epithelial-to-mesenchymal transition (EMT). This was supported by the blockade of HDDC appearance in CA19-9(+) cell cultures after incubation with the EMT inhibitor A83-01. After a differentiation protocol mimicking pancreatic development, HDDC populations contained about 2% of immature insulin-producing cells and showed glucose-unresponsive insulin secretion. Downregulation of the mesenchymal phenotype improved β-cell gene expression profile of differentiated HDDCs without affecting insulin protein expression and secretion. We show that pancreatic ducts represent a new source for engineering large amounts of β-like-cells with potential for treating diabetes.

V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog A Synthetic Modified mRNA Drives Reprogramming of Human Pancreatic Duct-Derived Cells Into Insulin-Secreting Cells

“…Although our initial HDDC populations were devoid of insulin or amylase expression from passage (P) 1 onward (data not shown), transfection experiments were carried out on HDDCs from P 2 to 5 to avoid the possibility of contamination with β or acinar cells. As described elsewhere [15], HDDCs are a uniform lineage of cells arising from purified CA19‐9 + human pancreatic ductal cells and presenting partial EMT features with coexpression of mesenchymal and ductal epithelium markers. HDDCs have advantageous characteristics in terms of proliferation [15] and resistance to cryopreservation, as we calculated a recovery rate of 73.2% ± 9.9% ( n = 5 ) after one cycle of cryopreservation at −196°C.…”

“…The exocrine tissue was obtained through the collaboration with the Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy, within a human islet distribution program for basic research supported by the Juvenile Diabetes Research Foundation [30]. DCs were isolated within 48 hours using MACS Separation columns to purify CA19‐9 + DCs as previously described [15]. CA19‐9 + DCs were initially plated at 3 × 10 5 cells per cm 2 in EGM‐2‐MV medium (Lonza, Allendale, NJ, http://www.lonza.com) without hydrocortisone.…”

“…Recent data on α‐to‐β transdifferentiation revealed a crucial role of the pancreatic duct lining in the recruitment of newly formed β cells [14] and the involvement of a preliminary duct‐to‐α switch occurring after a transitory state of epithelial‐mesenchymal transition (EMT). Because human DCs—like all epithelial lineages—undergo early loss of proliferation and of epithelial phenotype in vitro, we previously developed a new strategy to culture and expand human DCs via the forcing of a partial EMT [15]. The resulting cells, called human pancreatic duct‐derived cells (HDDCs), had potential for β‐cell differentiation, with glucose‐unresponsive insulin secretion detected in populations containing 2%–3% insulin‐positive (insulin + ) cells.…”

La thérapie cellulaire du diabète

“…En partenariat avec l'équipe de Susan Bonner-Weir, nous avons développé un système d'expansion in vitro de cellules ductales CA19-9 + isolées de pancréas humains. Après isolement, les cellules sont incubées avec un milieu de croissance endothéliale qui a la particularité d'induire une TEM des cellules CA19-9 + qui entrent en phase de prolifération et réalisent jusqu'à 22 doublements de population [58]. Après expansion, environ 3 à 5 % de ces cellules dérivées des canaux humains (en anglais human duct-derived cells ou HDDC) acquièrent des capacités de sécrétion d'insuline après 14 jours de contacts successifs avec des molécules et facteurs de croissance, selon un schéma reproduisant l'ontogenèse des cellules β. Les HDDC ont l'avantage de leur nombre (il est possible d'en collecter 10 11 en 1 mois de culture) et de la résistance des cellules différenciées au stockage à -196 °C.…”

Distribution of Islet Hormones in Human Adult Pancreatic Ducts

“…Adult pancreatic ducts seem to be highly differentiated, but the capillary contacted ductal endothelial cell retains its proliferative capability [5]. During the process of transdifferentiation, pancreatic ducts reprogram islet ontogenesis that renders new beta-cells [6]. Duct-derived beta-cell supplement may result from raised metabolic requirement or pancreatic impairment [7].…”