PDX1+/NKX6-1+ pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of β cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2+ cells generate β-like cells (C-PEPTIDE+/NKX6-1+) more efficiently compared to GP2− and unsorted populations, underlining the potential therapeutic applications of GP2.

SummaryPrimary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

The development and maintenance of the zones of the adrenal cortex and their steroidal output are extremely important in the control of gluconeogenesis, the stress response, and blood volume. Sonic Hedgehog (Shh) is expressed in the adrenal cortex and signals to capsular cells, which can respond by migrating into the cortex and converting into a steroidogenic phenotype. Delta-like homologue 1 (Dlk1), a member of the Notch/Delta/Serrate family of epidermal growth factor-like repeat-containing proteins, has a well-established role in inhibiting adipocyte differentiation. We demonstrate that Shh and Dlk1 are coexpressed in the outer undifferentiated zone of the male rat adrenal and that Dlk1 signals to the adrenal capsule, activating glioma-associated oncogene homolog 1 transcription in a β1 integrin- and Erk1/2-dependent fashion. Moreover, Shh and Dlk1 expression inversely correlates with the size of the zona glomerulosa in rats after manipulation of the renin-angiotensin system, suggesting a role in the homeostatic maintenance of the gland.

Exogenous insulin administration is currently the only treatment available to all type 1 diabetes patients, but it is not a cure. By helping to regulate circulating blood glucose levels, it has significantly improved life expectancy, but there are still long-term complications associated with the disease that may be preventable with a treatment strategy that can provide better glycemic control. Isolated islet (or whole pancreas) transplantation, xenotransplantation, and the transplant of human pluripotent stem cell-derived β-cells provide the potential to restore endogenous insulin production and reestablish normoglycemia. Here, we discuss the latest advances in these fields, including the use of encapsulation technology, as well as some of the hurdles that still need to be overcome for these alternative therapies to become mainstream and/or progress to clinical development.

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. In this study we investigate the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells cilia loss correlated with a reduction in ligand dependent hedgehog signalling. Heat shock dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by HDAC6 which localises to ciliary axonemes. The rate of cilia resorption was reduced in thermotolerant cells. This implies a role for molecular chaperones in primary cilia maintenance. The cytosolic chaperone Hsp90 localised to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells Hsp90 is known to exist in a complex with HDAC6. Immediately after heat shock Hsp90 levels were reduced in remaining ciliary axonemes. We hypothesise that cilia resorption in response to heat shock is regulated by the disassembly of an HDAC6/Hsp90 complex and would serve to attenuate cilia mediated signalling pathways and reduce the translational load on the cell in times of stress.

Primary cilia play key roles in development, cell signalling and cancer, and are involved in signal transduction pathways such as Hh and Wnt signalling. The adrenal cortex produces steroid hormones essential for controlling homeostasis and mediating the stress response. Signalling pathways involved in the process of its development and differentiation are still being identified but include Hh and Wnt, and adrenal development is thus likely to require cilia. I have demonstrated that inhibiting cilia formation, using siRNA targeted to different ciliary components, results in reduced differentiation of the human adrenal carcinoma cell line H295R towards a zona glomerulosa (zG)-like phenotype. These data suggest that primary cilia play a key role in adrenal differentiation, but which signalling pathways are involved still remains unclear. I have also discovered that adrenals from Bardet-Biedl syndrome (BBS) mice, the most prominently studied ciliopathy, have thin capsules, the proposed adrenal stem cell niche, and abnormal histology, while zebrafish embryos injected with morpholinos targeting BBS genes show delayed and reduced expression of ff1b, a marker of interrenal tissue. These data further suggest a role for primary cilia in adrenal development and maintenance. These studies are the foundation for elucidating the role of primary cilia in the development and function of the adrenal gland, and furthering our understanding of adrenocortical development. This promises to lead to improved management of adrenal dysfunction, and demonstrating that adrenal defects are a characteristic of ciliopathies will potentially inform new strategies for patient care.