Recently, we showed that ketoconazole, a known anti-fungal inhibitor of CYP51, stabilized TAR DNA-binding protein 43 (TDP-43) native self-interactions, reduced TDP-43 pathology and rescued TDP-43-induced SREBP2 downregulation. Despite its promising effects, ketoconazole is not viable for repurposing for ALS due to liver toxicity side effects that occur when orally delivered. To address this, we tested the activities of seven additional known azole-based CYP51 inhibitors in order identify a viable alternative to ketoconazole. Using our established TDP-43 mislocalization and aggregation assay in HEK293T cells, we identified posaconazole, an FDA-approved, CNS-penetrant and orally delivered anti-fungal, as the strongest inhibitor of TDP-43 pathology. Posaconazole was able to reduce insoluble TDP-43 and restore SREBP2 levels, outperforming ketoconazole. Mechanism of action (MOA) experiments suggest posaconazole is able to outperform ketoconazole by inducing a significantly stronger activation of autophagy and upregulation of heat shock proteins known to clear TDP-43. Further MOA experiments show that the effects of posaconazole on TDP-43 are dependent on its known ability to lower cellular cholesterol levels. By correlating our experimental results on the eight CYP51 inhibitors tested, we show that predicted affinity towards human CYP51 strongly correlates with the inhibitors' ability to lower TDP-43 aggregation and mislocalization. Finally, we tested posaconazole in a low dose sodium arsenite ALS model in iPSC-derived motor neurons, showing that it is efficacious at inhibiting TDP-43 pathology in the nanomolar range. Altogether, these results support the repurposing of posaconazole for ALS/FTD as a means to prevent TDP-43 pathology.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is associated with multiple risk factors and involves death of the retinal pigment epithelium (RPE). We investigated how the Y402H polymorphism of Complement Factor H (CFH) and cigarette smoke extract (CSE), major AMD genetic and environmental risks, affect lipid metabolism in RPE differentiated from induced pluripotent stem cells (iPSC-RPE) that were derived from human donors genotyped for low-risk (LR) or high-risk (HR) CFH. Results from discovery-based (lipidomics, proteomics) and targeted (mitochondrial fatty acid oxidation (FAO)) assays found significant genotype-dependent differences under basal conditions include higher free fatty acids and cholesterol esters in HR cells. CSE induced differences in proteins regulating lipid handling, lipolysis, and inflammation. Lower FAO in HR cells was observed in multiple donors and pairs of parent/isogenic edited lines compared with LR lines. CSE induced lipid accumulation, lipid composition remodeling, and upregulation of proteins involved in lipid synthesis/hydrolysis, production of bioactive lipid mediators, and metabolism of ceramide and cholesterol. These results elucidate putative mechanisms driving pathology in RPE harboring CFH Y402H.

Objective: There is currently no long-term treatment for the repair of damaged cartilage and osteoarthritis (OA). Induced pluripotent stem cells (iPSCs) are an ideal cell source for screening platforms due to their ability to self-renew and differentiate to cell types that would otherwise require invasive surgeries to obtain, such as chondrocytes and mesenchymal stromal cells (MSCs). Here, we developed an iPSC-based screening platform and tested previously described pro-chondrogenic small molecule compounds, to determine their potential to identify hits. Design: iPSC derived chondroprogenitors (iCPs) and neural crest cell (NCC) derived MSCs (iNCC-MSCs) were generated, and their chondrogenic potential was confirmed. The iPSC derived cells and a primary bone marrow derived MSC (BM-MSC) line were cultured as pellets and treated with different concentrations of small molecule compounds, in the presence of chondrogenic inducing growth factors, over 14 days at 2 ​% O2. Glycosaminoglycan (GAG) synthesis was quantified by a 1,9- dimethylmethylene blue (DMMB) assay. Results: After 14 days of chondrogenesis, forskolin, baicalin and sesamin enhanced GAG synthesis in the iCPs, and forskolin enhanced GAG synthesis in the iNCC-MSCs, while no small molecule compounds enhanced GAG synthesis in the BM-MSCs. Conclusion: Our findings further demonstrate how the small molecules pro-chondrogenic effects are dependent on the screening platform conditions, including the cell type, molecule concentration, 3D culture, hypoxia, and the inclusion of additional growth factors. The iPSC-based screening platform developed has the potential to identify disease modifying OA drugs (DMOADs) in novel compound screening libraries.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable cell type for studying human cardiac health and disease in vitro. However, it is not known whether hiPSC-CMs display sex dimorphism and therefore whether sex should be incorporated as a biological variable in in vitro studies that include this cell type. To date, the vast majority of studies that utilize hiPSC-CMs do not include both male and female sex nor stratify results based on sex because it is challenging to amass such a cohort of cells. Here, we generated 3 female and 3 male hiPSC lines from adult left ventricular cardiac fibroblasts as a resource for studying sex differences in in vitro cardiac models. We used this resource to generate hiPSC-CMs and maintained them in basal media without exogenous hormones. Functional assessment of CMs showed enhanced calcium handling in female-derived hiPSC-CMs relative to male. Bulk RNA sequencing revealed over 300 differentially expressed genes (DEGs) between male and female hiPSC-CMs. Gene ontology analysis of DEGs showed distinct differences in pathways related to cardiac pathology including cell-cell adhesion, metabolic processes, and response to ischemic stress. Differential expression of the sodium channel auxiliary unit SCN3B was found and validated through patch-clamp measurements of sodium currents, showing increased peak amplitude and window current in female hiPSC-CMs. These findings highlight the importance of considering sex as a variable when conducting studies to evaluate aspects of human cardiac health and disease related to CM function.

Autophagy is one of the main intracellular recycling systems and its impairment is considered a primary hallmark of the aging process. Defective macroautophagy in the retinal pigment epithelium (RPE) has been described in age-related macular degeneration (AMD), a blindness-causing disease that affects roughly 200 million patients worldwide. The relevance of chaperone-mediated autophagy (CMA), a selective type of autophagy for proteins containing a KFERQ-like motif, in RPE cell biology and homeostasis remains to be elucidated. Here we describe decreased CMA activity in the RPE of AMD patients compared to healthy age-matched controls, along with accumulation of substrate proteins, and in donor-derived iPSC-RPE cells, which we used to further characterize AMD-associated alterations of cellular homeostasis derived from proteotoxicity. Treatment with CA77.1 (CMA activator) restores proteostasis and remodels specific subsets of the proteome in cells from healthy and AMD donors. CA77.1-treated AMD iPSC-RPE display reduced oxidative stress and improved mitochondrial function. These findings may explain the specific vulnerability of the RPE during AMD and shed light on CMA as a new druggable target for this as-of-now incurable disease.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored. hiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium l-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function. hiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90

A key goal of evolutionary genomics is to harness molecular data to draw inferences about selective forces that have acted on genomes. The field progresses in large part through the development of advanced molecular-evolution analysis methods. Here we explored the intersection between classical sequence-based tests for selection and an empirical expression-based approach, using stem cells from Mus musculus subspecies as a model. Using a test of directional, cis -regulatory evolution across genes in pathways, we discovered a unique program of induction of translation genes in stem cells of the Southeast Asian mouse M. m. castaneus relative to its sister taxa. We then mined population-genomic sequences to pursue underlying regulatory mechanisms for this expression divergence, finding robust evidence for alleles unique to M. m. castaneus at the upstream regions of the translation genes. We interpret our data under a model of changes in lineage-specific pressures across Mus musculus in stem cells with high translational capacity. Our findings underscore the rigor of integrating expression and sequence-based methods to generate hypotheses about evolutionary events from long ago.

Age-related macular degeneration (AMD), the leading cause of central vision loss in the elderly, involves death of the retinal pigment epithelium (RPE) and light-sensing photoreceptors. This multifactorial disease includes contributions from both genetic and environmental risk factors. The current study examined the effect of the Y402H polymorphism of Complement Factor H (CFH, rs1061170) and cigarette smoke, predominant genetic and environmental risk factors associated with AMD. We used targeted and discovery-based approaches to identify genotype-dependent responses to chronic oxidative stress induced by cigarette smoke extract (CSE) in RPE differentiated from induced pluripotent stem cells (iPSC) derived from human donors harboring either the low risk (LR) or high risk (HR) CFH genotype. Chronic CSE altered the metabolic profile in both LR and HR iPSC-RPE and caused a dose-dependent reduction in mitochondrial function despite an increase in mitochondrial content. Notably, cells with the HR CFH SNP showed a greater reduction in maximal respiration and ATP production. Significant genotype-dependent changes in the proteome were observed for HR RPE at baseline (cytoskeleton, MAPK signaling) and after CSE exposure, where a less robust upregulation of the antioxidants and significant downregulation in proteins involved in nucleic acid metabolism and membrane trafficking were noted compared to LR cells. In LR cells, uniquely upregulated proteins were involved in lipid metabolism and chemical detoxification. These genotype-dependent differences at baseline and in response to chronic CSE exposure suggest a broader role for CFH in modulating the response to oxidative stress in RPE and provides insight into the interaction between environmental and genetic factors in AMD pathogenesis.

Numerous interventions have been explored in animal models using cells differentiated from human induced pluripotent stem cells (iPSCs) in the context of neural injury with some success. Our work seeks to transplant cells that are generated from hiPSCs into regionally specific spinal neural progenitor cells (sNPCs) utilizing a novel accelerated differentiation protocol designed for clinical translation. We chose a xenotransplantation model because our laboratory is focused on the behaviour of human cells in order to bring this potential therapy to translation. Cells were transplanted into adult immunodeficient rats after moderate contusion spinal cord injury (SCI). Twelve weeks later, cells derived from the transplanted sNPCs survived and differentiated into neurons and glia that filled the lesion cavity and produced a thoracic spinal cord transcriptional program in vivo. Furthermore, neurogenesis and ionic channel expression were promoted within the adjacent host spinal cord tissue. Transplanted cells displayed robust integration properties including synapse formation and myelination by host oligodendrocytes. Axons from transplanted hiPSC sNPC-derived cells extended both rostrally and caudally from the SCI transplant site, rostrally approximately 6 cm into supraspinal structures. Thus, iPSC-derived sNPCs may provide a patient-specific cell source for patients with SCI that could provide a relay system across the site of injury.

Primary cultures of retinal pigment epithelium (RPE) from human adult donors (haRPE) and induced pluripotent stem cell derived-RPE (iPSC-RPE) are valuable model systems for gaining mechanistic insight and for testing potential therapies for age-related macular degeneration (AMD). This study evaluated the treatment response of haRPE and iPSC-RPE to oxidative stress and potential therapeutics addressing mitochondrial defects. haRPE and iSPC-RPE were derived from donors with or without AMD. Mitochondrial function was measured after treatment with menadione, AICAR, or trehalose and the response to treatment was compared between cell models and by disease status. In a subset of samples, haRPE and iPSC-RPE were generated from the same human donor to make a side-by-side comparison of the two cell models' response to treatment. Disease-specific responses to all three treatments was observed in the haRPE. In contrast, iPSC-RPE had a similar response to all treatments irrespective of disease status. Analysis of haRPE and iPSC-RPE generated from the same human donor showed a similar response for donors without AMD, but there were significant differences in treatment response between cell models generated from AMD donors. These results support the use of iPSC-RPE and haRPE when investigating AMD mechanisms and new therapeutics but indicates that attention to experimental conditions is required.

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. No universally effective treatments exist for atrophic or “dry” AMD, which results from loss of the retinal pigment epithelium (RPE) and photoreceptors and accounts for ≈80% of all AMD patients. Prior studies provide evidence for the involvement of mitochondrial dysfunction in AMD pathology. This study used induced pluripotent stem cell (iPSC) RPE derived from five AMD patients to test the efficacy of three drugs (AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), Metformin, trehalose) that target key processes in maintaining optimal mitochondrial function. The patient iPSC-RPE lines were used in a proof-of-concept drug screen, utilizing an analysis of RPE mitochondrial function following acute and extended drug exposure. Results show considerable variability in drug response across patient cell lines, supporting the need for a personalized medicine approach for treating AMD. Furthermore, our results demonstrate the feasibility of using iPSC-RPE from AMD patients to develop a personalized drug treatment regime and provide a roadmap for the future clinical management of AMD.

Differentiation of human induced pluripotent stem cells (hiPSCs) generates cell phenotypes valuable for cell therapy and personalized medicine. Successful translation of these hiPSC-derived therapeutic products will rely upon effective cryopreservation at multiple stages of the manufacturing cycle. From the perspective of cryobiology, we attempted to understand how the challenge of cryopreservation evolves between cell phenotypes along an hiPSC-to-sensory neuron differentiation trajectory. Cells were cultivated at three different stages to represent intermediate, differentiated, and matured cell products. All cell stages remained ≥90% viable in a dimethyl sulfoxide (DMSO)-free formulation but suffered ≥50% loss in DMSO before freezing. Raman spectroscopy revealed higher sensitivity to undercooling in hiPSC-derived neuronal cells with lower membrane fluidity and higher sensitivity to suboptimal cooling rates in stem cell developmental stages with larger cell bodies. Highly viable and functional sensory neurons were obtained following DMSO-free cryopreservation. Our study also demonstrated that dissociating adherent cultures plays an important role in the ability of cells to survive and function after cryopreservation.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is characterized by loss of the retinal pigment epithelium (RPE). While the disease mechanism remains unclear, prior studies have linked AMD with RPE mitochondrial defects and genetic polymorphisms in the complement pathway. This study used RPE generated from induced pluripotent stem cells (iPSC-RPE), which were derived from human donors with or without AMD and genotyped for the complement factor H (CFH) AMD high-risk allele (rs1061170, Y402H) to investigate whether donor disease state or genotype had a detrimental effect on mitochondrial function and inflammation. Results show that cells derived from donors with AMD display decreased mitochondrial function under conditions of stress and elevated expression of inflammatory markers compared to iPSC-RPE from individuals without AMD. A more pronounced reduction in mitochondrial function and increased inflammatory markers was observed in CFH high-risk cells, irrespective of disease state. These results provide evidence for a previously unrecognized link between CFH and mitochondrial function that could contribute to RPE loss in AMD patients harboring the CFH high-risk genotype.

Background There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. Methods The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. Results Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. Conclusions This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

This research is the first to produce induced pluripotent stem cell-derived inner ear sensory neurons in the Neurog1 +/- heterozygote mouse using blastocyst complementation. Additionally, this approach corrected non-sensory deficits associated with Neurog1 heterozygosity, indicating that complementation is specific to endogenous Neurog1 function. This work validates the use of blastocyst complementation as a tool to create novel insight into the function of developmental genes and highlights blastocyst complementation as a potential platform for generating chimeric inner ear cell types that can be transplanted into damaged inner ears to improve hearing.

Despite concerted efforts to identify CNS regeneration strategies, an incomplete understanding of how the needed molecular machinery is regulated limits progress. Here we use models of lateral compression and FEJOTA clip contusion-compression spinal cord injury (SCI) to identify the thrombin receptor (Protease Activated Receptor 1 (PAR1)) as an integral facet of this machine with roles in regulating neurite growth through a growth factor- and cholesterol-dependent mechanism. Functional recovery and signs of neural repair, including expression of cholesterol biosynthesis machinery and markers of axonal and synaptic integrity, were all increased after SCI in PAR1 knockout female mice, while PTEN was decreased. Notably, PAR1 differentially regulated HMGCS1, a gene encoding a rate-limiting enzyme in cholesterol production, across the neuronal and astroglial compartments of the intact versus injured spinal cord. Pharmacologic inhibition of cortical neuron PAR1 using vorapaxar in vitro also decreased PTEN and promoted neurite outgrowth in a cholesterol dependent manner, including that driven by suboptimal brain derived neurotrophic factor (BDNF). Pharmacologic inhibition of PAR1 also augmented BDNF-driven HMGCS1 and cholesterol production by murine cortical neurons and by human SH-SY5Y and iPSC-derived neurons. The link between PAR1, cholesterol and BDNF was further highlighted by demonstrating that the deleterious effects of PAR1 over-activation are overcome by supplementing cultures with BDNF, cholesterol or by blocking an inhibitor of adenylate cyclase, Gαi. These findings document PAR1-linked neurotrophic coupling mechanisms that regulate neuronal cholesterol metabolism as an important component of the machinery regulating CNS repair and point to new strategies to enhance neural resiliency after injury.