Differentiation of human embryonic stem cells (hESCs) to specific functional cell types can be achieved using methods that mimic in vivo embryonic developmental programs. Current protocols for generating hepatocytes from hESCs are hampered by inefficient differentiation procedures that lead to low yields and large cellular heterogeneity. We report here a robust and highly efficient process for the generation of high-purity (70%) hepatocyte cultures from hESCs that parallels sequential hepatic development in vivo. Highly enriched populations of definitive endoderm were generated from hESCs and then induced to differentiate along the hepatic lineage by the sequential addition of inducing factors implicated in physiological hepatogenesis.The differentiation process was largely uniform, with cell cultures progressively expressing increasing numbers of hepatic lineage markers, including GATA4, HNF4␣, ␣-fetoprotein, CD26, albumin, ␣-1-antitrypsin, Cyp7A1, and Cyp3A4. The hepatocytes exhibited functional hepatic characteristics, such as glycogen storage, indocyanine green uptake and release, and albumin secretion. In a mouse model of acute liver injury, the hESC-derived definitive endoderm differentiated into hepatocytes and repopulated the damaged liver. The methodology described here represents a significant step toward the efficient generation of hepatocytes for use in regenerative medicine and drug discovery.

These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.

Metastatic melanoma is a deadly skin cancer with limited therapeutic options. Although targeted-and immuno-therapeutics led to a paradigm shift in melanoma management, resistance to these drugs necessitates identifying new targets. We demonstrated that constitutive activation of NLRP inflammasome is responsible for autoinflammation in human melanoma. Autoinflammation has been linked to tumorigenesis and drug resistance in cancers. Among NLRP proteins, NLRP1 is unique because it contains the caspase recruitment domain (CARD), a homotypic protein interaction motif. Knocking down NLRP1 revealed a tumor promoting property of NLRP1 in metastatic melanoma in vitro and in vivo. While NLRP1 did not affect cell cycle, it inhibited caspase-2,-9, and-3/7 activities and apoptosis in melanoma cells. NLRP1, caspase-2 and-9 all contain a CARD which facilitates protein binding through CARD homodimerization. Co-IP confirmed the interaction of NLRP1 with caspase-2 and-9 in melanoma. We activated NLRP1 inflammasome because its activation has been reported to induce apoptosis and pyroptosis in macrophages. Whereas NLRP1 inflammasome activation increased caspase-2,-9 and-3/7 activities and induced pyroptosis and apoptosis in monocytic THP-1 cells, it reduced apoptotic caspase activities and provided protection against cell death in melanoma cells. To understand the differential effects of active NLRP1 inflammasome on apoptosis, we evaluated cellular localization of NLRP1. Unlike THP-1 with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm where NLRP1 inflammasome is assembled with inflammatory caspase-1, and NLPR1 interacts with apoptotic caspase-9. Thus, we demonstrated that melanoma NLRP1 has an anti-apoptotic activity, leading to tumor promotion and cell survival in metastatic melanoma.

An alternative for voriconazole-induced photocarcinogenesis for at-risk patients has not been well studied. Here we present a patient who has been successfully treated with posaconazole after developing severe voriconazole-induced photocarcinogenesis. An elderly light-skinned female had been receiving voriconazole and low-dose corticosteroids for suppression of chronic Exophiala dermatitidis meningitis for the last 5 years. She had no history of hematologic malignancy, solid organ transplantation or other immunosuppressed state. Several months after starting voriconazole, the patient developed a severe photosensitivity reaction, which progressed to extensive actinic keratosis (AK) development. The patient subsequently required 3 Mohs surgeries for large, aggressive squamous cell carcinomas (SCC) on her extremities, had 348 AKs treated, and 32 of 41 biopsies positive for SCC over 21 months. We compared the number of AKs treated during six months preceding the switch with a six-month period after, allowing for a "transition" time in between. AKs trended down, decreasing from 22.2 (SD¼13.0) to 6.6 (SD¼8.2) and no subsequent Mohs surgery was required. Treatment options for Exophiala dermatitidis, a dematiaceous fungi with melaninlike pigment in the cell wall, are not well documented, but voriconazole is widely used for treatment and prophylaxis of fungal infections. However, approximately 8-10% of patients experience dermatologic side effects including phototoxicity, new-onset squamous cell carcinoma, pseudoporphyria, discoid lupus erythematosus, and accelerated photoaging. These adverse events have not been reported with posaconazole and a recent meta-analysis ranked posaconazole superior to voriconazole in preventing invasive fungal infections (Zhao et al. 2015). Considering that posaconazole has a favorable side effect profile and has shown good efficacy in invasive fungal infection prophylaxis and treatment, it should be considered more often as a first line drug in at-risk patients.