Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of α-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

Solvent-based and mechanical recycling technology approaches were compared with respect to each process's decontamination efficiency. Herein, post-consumer polystyrene (PS) feedstock was recycled by both technologies, yielding recycled PS resins (rPS). The process feedstock was subjected to four recycling cycles in succession to assess the technology perennity. The physico-chemical and mechanical properties of the rPS were then evaluated to discern the advantages and drawbacks of each recycling approach. The molecular weight of the mechanically recycled resin was found to decrease by 30% over the reprocessing cycles. In contrast, the solvent-base recycling technology yielded a similar molecular weight regarding the feedstock. This consistency in the rPS product is critical for consumer applications. Further qualitative and quantitative analyses on residual organic compounds and inorganic and particulate contaminants were investigated. It was found that the solvent-based technology is very efficient for purifying deeply contaminated feedstock in comparison to mechanical recycling, which is limited to well-cleaned and niche feedstocks.

We examined the effects of spinal cord injury (SCI) on alterations in gene expression and respective protein products in human skeletal muscle 2 days and 5 days post-SCI. Biopsies were taken from skeletal muscle of 9 men and 1 woman (n = 10) (43.9 +/- 6.7 years) 2 days and 5 days post-SCI and from 5 healthy young men who served as controls (20.4 +/- 0.5 years). Global changes in gene expression were analysed using Affymetrix GeneChips on a subsample of subjects (n = 3). Candidate genes were then pursued via qRT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes. Immunohistochemistry (IHC) was used to localize proteins. Groups of transcripts showing the greatest percentage of altered expression, the most robust fold-changes, and indicative of involvement of an entire pathway using the GeneChip included genes involved in the ubiquitin proteasome pathway (UPP), metallothionein function, and protease inhibition. qRT-PCR analysis confirmed increases in gene expression for UPP components (UBE3C, Atrogin-1, MURF1, and PSMD11), the metallothioneins (MT1A, MT1F, MT1H), and the protease inhibitor, SLPI (P < 0.05) at 2 days and 5 days post-SCI. Protein levels of the proteasome subunit (PSMD11) and the metallothioneins were increased 5 days post-SCI. Protein levels of UBE3C, Atrogin-1, MURF1 and SLPI were unchanged (P > 0.05). IHC showed increased staining for PSMD11 and the metallothioneins 5 days post-SCI, along the peripheral region of the cells. IHC also showed altered staining for Atrogin-1 at 5 days post-SCI along the membrane region. Thus, there was a profound increase in gene expression of UPP components, the metallothioneins, and the protease inhibitor, SLPI, within 5 days of SCI. Increased protein levels for PSMD11 and the metallothioneins 5 days post-SCI, specifically along the cell periphery, indicate that proteins in this region may be early targets for degradation post-SCI.

The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.