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The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.

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Although relatively resistant to oxidation, polycarbonate-based polyurethanes (PCNUs) are degraded by monocyte-derived macrophages (MDM) by a co-mediated mechanism involving both hydrolytic and oxidative pathways. Since a previous study showed that PCNU pretreatment with H(2)O(2) modulated degradation by esterases, human MDM were used to further elucidate this dual pathway mechanism of degradation for (14)C-radiolabeled PCNUs (synthesized with 1,6-hexane diisocyanate:polycarbonatediol: butanediol with different stoichiometry (HDI431 and HDI321) or another diisocyanate 4,4'-methylene bisphenyl diisocyanate (MDI321)). Scanning electron microscopy of PCNU slips pretreated with 20% H(2)O(2) showed that HDI431 had visible holes with more radiolabel release than from the other PCNUs. When MDM were seeded on H(2)O(2)-treated PCNUs, degradation of HDI321 and MDI321, but not HDI431 was decreased. Esterase activity was inhibited in MDM on all surfaces except MDI321, whereas inhibition of acid phosphatase occurred on all surfaces. The material surface itself, induced H(2)O(2) release from live MDM, with more H(2)O(2) elicited by phorbol myristate acetate treated MDM when cultured on HDI431 but not the other materials. H(2)O(2) pretreatment affected cell function by chemically altering the material surface and MDM-mediated degradation, known to be dependent on surface chemistry. The findings highlight that both oxidative and hydrolytic mechanisms need to be understood in order to tailor material chemistry to produce desired cell responses for in vivo applications.

Islet transplantation to treat type 1 diabetes (T1D) has shown varied long-term success, due in part to insufficient blood supply to maintain the islets. In the current study, collagen and collagen:chitosan (10:1) hydrogels, +/- circulating angiogenic cells (CACs), were compared for their ability to produce a pro-angiogenic environment in a streptozotocin-induced mouse model of T1D. Initial characterization showed that collagen-chitosan gels were mechanically stronger than the collagen gels (0.7kPa vs. 0.4kPa elastic modulus, respectively), had more cross-links (9.2 vs. 7.4/µm2), and were degraded more slowly by collagenase. After gelation with CACs, live/dead staining showed greater CAC viability in the collagen-chitosan gels after 18h compared to collagen (79% vs. 69%). In vivo, collagen-chitosan gels, subcutaneously implanted for up to 6 weeks in a T1D mouse, showed increased levels of pro-angiogenic cytokines over time. By 6 weeks, anti-islet cytokine levels were decreased in all matrix formulations ± CACs. The 6-week implants demonstrated increased expression of VCAM-1 in collagen-chitosan implants. Despite this, infiltrating vWF+ and CXCR4+ angiogenic cell numbers were not different between the implant types, which may be due to a delayed and reduced cytokine response in a T1D versus non-diabetic setting. The mechanical, degradation and cytokine data all suggest that the collagen-chitosan gel may be a suitable candidate for use as a pro-angiogenic ectopic islet transplant site.