Entrapped Collagen Type 1 Promotes Differentiation of Embryonic Pancreatic Precursor Cells into Glucose-Responsive β-Cells When Cultured in Three-Dimensional PEG Hydrogels

Abstract

Development of an alternative source of functional, transplantable beta-cells to replace or supplement cadaveric tissue is critical to the future success of islet cell transplantation therapy. Embryonic pancreatic precursor cells are desirable as a renewable source of beta-cells as they are both proliferative and inherently capable of pancreatic cell differentiation. We have previously shown that precursor cells undergo selective beta-cell differentiation when dissociated and photoencapsulated in a polyethylene glycol (PEG) hydrogel network; however, these cells remained immature and were not glucose responsive. Collagen type 1 supports mature cell viability and function in many cell types and we hypothesized that incorporating it within our gels may support differentiating beta-cells and facilitate beta-cell maturation. For these studies, collagen-1 was entrapped with dissociated pancreatic precursor cells in a PEG hydrogel matrix (PEGCol) with the following key findings: (1) mature, glucose-responsive, islet-like structures differentiated from spontaneously forming precursor cell clusters in PEGCol, but not unmodified PEG, hydrogels; (2) a balance existed between providing sufficient collagen-1 signaling to support precursor cell development and providing an overabundance of adhesive sites allowing contaminating mesenchymal cells to thrive' and (3) mechanical stability provided by the PEG hydrogel platform is important for successful precursor cell culture, as PEGCol hydrogels encourage glucose responsiveness and high-insulin gene expression, while pure collagen gel cultures, with the same collagen concentration, have negligible insulin gene expression. These results indicate that PEGCol hydrogels are a useful culture platform to promote differentiation of a glucose-responsive beta-cell population from dissociated precursor cells.

Inhibition of Gamma-Secretase Activity Promotes Differentiation of Embryonic Pancreatic Precursor Cells into Functional Islet-like Clusters in Poly(Ethylene Glycol) Hydrogel Culture

“…Consistent with our previously published work, vimentin gene expression was undetectable by day 7, indicating that contaminating mesenchymal cells present upon dissection/dissociation of the pancreatic bud were unable to survive in PEGCol hydrogel culture (data not shown). 22 Additionally, amylase gene expression, a marker for exocrine differentiation, did not change significantly from the low level of gene expression measured on day 0 for any culture time or condition tested (data not shown).…”

“…14 This initial cell population has been previously characterized and contains *65% Pdx1þ/insulinÀ pancreatic precursor cells, *25% vimentinþ mesenchymal cells, and <1% of differentiated insulin or glucagonþ cells. 22 For clustering experiments, cells were placed in nonadherent tissue culture wells on an orbital shaker (80 rpm) for 30, 60, or 120 min. Dissociated or clustered cells were photoencapsulated into 7.5wt% PEG hydrogels with 0.5 mg/mL entrapped collagen (PEGCol) at a cell concentration of 3Â10 6 cells/mL.…”

“…Dissociated or clustered cells were photoencapsulated into 7.5wt% PEG hydrogels with 0.5 mg/mL entrapped collagen (PEGCol) at a cell concentration of 3Â10 6 cells/mL. 22 Hydrogel discs (3.6 mm in diameter and 1.5 mm thick) were photopolymerized using 0.025wt% photoinitiator (Irgacure 2959) for 10 min at 365 nm ultraviolet light (*4 mW/cm 2 ).…”

“…[22][23][24] A 50:50 mixture of hydrolytically degradable and nondegradable PEG macromers were used to preserve stability over long-term in vitro culture, a modification that would also facilitate maintenance of hydrogel structural integrity for in vivo implantation. 25 PEGCol cultures were maintained at 378C/5% CO 2 in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.1% N2 (all vol./vol.…”

“…14,22,26 After incubation, supernatant C-peptide concentrations were measured using rat C-peptide ELISA kits (Mercodia).…”