No abstract

Islet transplantation is a promising approach to enable type 1 diabetic patients to attain glycemic control independent of insulin injections. However, up to 60% of islets are lost immediately following transplantation. To improve this outcome, islets can be transplanted within bioscaffolds, however, synthetic bioscaffolds induce an intense inflammatory reaction which can have detrimental effects on islet function and survival. In the present study, we first improved the biocompatibility of polydimethylsiloxane (PDMS) bioscaffolds by coating them with collagen. To reduce the inflammatory response to PDMS bioscaffolds, we then enriched the bioscaffolds with dexamethasone-loaded microplates (DEX-μScaffolds). These DEX-microplates have the ability to release DEX in a sustained manner over 7 weeks within a therapeutic range that does not affect the glucose responsiveness of the islets but which minimizes inflammation in the surrounding microenvironment. The bioscaffold showed excellent mechanical properties that enabled it to resist pore collapse thereby helping to facilitate islet seeding and its handling for implantation, and subsequent engraftment, within the epididymal fat pad (EFP). Following the transplantation of islets into the EFP of diabetic mice using DEX-μScaffolds there was a return in basal blood glucose to normal values by day 4, with normoglycemia maintained for 30 d. Furthermore, these animals demonstrated a normal dynamic response to glucose challenges with histological evidence showing reduced pro-inflammatory cytokines and fibrotic tissue surrounding DEX-μScaffolds at the transplantation site. In contrast, diabetic animals transplanted with either islets alone or islets in bioscaffolds without DEX microplates were not able to regain glycemic control during basal conditions with overall poor islet function. Taken together, our data show that coating PDMS bioscaffolds with collagen, and enriching them with DEX-microplates, significantly prolongs and enhances islet function and survival.

Misactivation of the Hedgehog pathway can cause cancers such as medulloblastomas, the most common malignant brain tumors in children. Hedgehog signals are transmitted through primary cilia, where Hedgehog ligands bind to Patched1 and activate Smoothened through interactions with cilia-associated sterol lipids. The gene expression programs driving cellular responses to ciliary Hedgehog signals are incompletely understood. Thus, to define Hedgehog target genes and elucidate mechanisms underlying Hedgehog-associated medulloblastomas, we performed RNA sequencing of cells after treatment with Hedgehog ligands (Shh, Dhh, Ihh), cilia-associated lipids (7b,27-dihydroxycholesterol, 24(S),25-epoxycholesterol), or synthetic lipids or small molecules that activate Smoothened (20(S)-hydroxycholesterol, SAG). Nonspecific gene expression changes were identified by performing RNA sequencing (1) after treatment of CRISPR mediated Smo-/- cells with the same Hedgehog pathway agonists, (2) after treatment with vehicle controls, or (3) after treatment with sterol lipids that are unable to activate Smoothened (7a,27-dihydroxycholesterol). Differentially expressed genes were integrated across RNA sequencing of human medulloblastomas (n=458) or the Math1-Cre SmoM2 mouse genetic model of Hedgehog-associated medulloblastoma. Mechanistic studies validating Hedgehog target genes were performed using CRISPR interference, genetic gain-of-function, molecular biology, quantitative immunofluorescence, or cell biology approaches. RNA sequencing after treatment with Hedgehog pathway agonists identified a core gene expression program comprised of 155 genes driving lipid synthesis, metabolism, signaling, adhesion, or angiogenesis. Integration of transcriptomic datasets revealed a conserved gene expression program driving cellular responses to ciliary Hedgehog signals in human or mouse medulloblastomas, including known target genes such as Gli1 or Ptch1, and novel target genes such as Hsd11b1 or Retnla. Retnla is a regulator of sterol synthase expression, and Hsd11b1 is a sterol synthase that opposes the action of Hsd11b2, a driver and druggable dependency underlying Hedgehog-associated medulloblastoma. In support of these findings, mechanistic studies demonstrated Retnla drives expression of Hsd11b2, and showed Hsd11b1 negatively regulates the Hedgehog pathway.