Hypoxia-inducible factor-1alpha (HIF1alpha) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. Here, we demonstrate that the abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2, under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression was completely inhibited in Siah1a/2 null cells, yet could be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia displayed an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Decreased Fas expression during tumor progression often results in a loss of Fas-ligand (FasL)-mediated apoptosis. Human and mouse melanoma exhibit an inverse correlation between the degree of Fas cell surface expression, tumorigenicity, and metastatic capacity. The expression of dominant negative Stat3 or c-Jun in melanoma cells efficiently increased Fas expression and sensitized cells to FasL-induced apoptosis. Stat3+/- as well as c-Jun-/- cells exhibited increased Fas cell surface expression and higher sensitivity to FasL-mediated apoptosis. Suppression of Fas expression by Stat3 and c-Jun is uncoupled from Stat3-mediated transcriptional activation. Our findings indicate that Stat3 oncogenic activities could also be mediated through its cooperation with c-Jun, resulting in downregulation of Fas surface expression, which is implicated in the tumor's ability to resist therapy and metastasize.

The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%-80% of the cell population consisted of PDX1-positive (PDX1 + ) and NKX6.1 + PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%-98% endocrine cells and 1%-3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important b cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1214-1222 SIGNIFICANCEType 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin-producing cells in vitro and a new protocol for producing the cells, representing another potential cell source for a diabetes cell therapy. These cells can be loaded into a protective device that is implanted under the skin. The device is designed to protect the cells from immune rejection by the implant recipient. The implant can engraft and respond to glucose by secreting insulin, thus potentially replacing the b cells lost in patients with T1D.

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