Human pancreatic ductal cells: Large-scale isolation and expansion

The in vitro differentiation of pancreatic stem cells has recently been sho wn to represent a new source of beta cells for cell therapy in diabetes. Hu man ductal cell differentiation, in vitro, has been documented in three-dim ensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex v ivo are mandatory for clinical relevance. We compared three sources of huma n ductal cells (hDC) (method A1-2, B, and C). The classical main duct isola tion of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fract ion in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal c omponent in the form of ductal cysts, as we described previously: monolayer s derived from digested cysts were 80% ductal (CK19). Method C: initially a dherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7 ) ductal cells. One-week exocrine cultures were amylase negative and 46% (C K19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81 <plus/minus> 12% (B), 91 +/- 2% (C). Extrapolating total yields we obtained (+/- SEM): 10.5 +/- 4.6 x 10(3) (Al), 36 +/- 18 x 10(3) (A2), 292 +/- 50 x 10(6) (B), 1696 +/- 526 x 10(6) (C) viable hDC per pancreas. A secondary monolayer exp ansion of cyst-derived hDC (method B) was achieved with NuSerum (R) (4.2-fo ld on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plast ic). First passage exocrine-derived ductal cells also responded to matrix a nd to growth factors, albeit not significantly. In conclusion, this study d emonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. if their d ifferentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic an d autologous diabetes cell therapy.