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We present a TALEN-based workflow to generate and maintain dual-edited (IL-15+/+/TGFβR2-/-) iPSCs that produce enhanced iPSC-derived natural killer (iNK) cells for cancer immunotherapy. It involves using a cell lineage promoter for knocking in (KI) gene(s) to minimize the potential effects of expression of any exogenous genes on iPSCs. As a proof-of-principle, we KI IL-15 under the endogenous B2M promoter and show that it results in high expression of the sIL-15 in iNK cells but minimal expression in iPSCs. Furthermore, given that it is known that knockout (KO) of TGFβR2 in immune cells can enhance resistance to the suppressive TGF-β signaling in the tumor microenvironment, we develop a customized medium containing Nodal that can maintain the pluripotency of iPSCs with TGFβR2 KO, enabling banking of these iPSC clones. Ultimately, we show that the dual-edited IL-15+/+/TGFβR2-/- iPSCs can be efficiently differentiated into NK cells that show enhanced autonomous growth and are resistant to the suppressive TGF-β signaling.

Several lines of evidence indicate the involvement of neuroinflammatory processes in the pathophysiology of schizophrenia (SCZ). Microglia are brain resident immune cells responding toward invading pathogens and injury-related products, and additionally, have a critical role in improving neurogenesis and synaptic functions. Aberrant activation of microglia in SCZ is one of the leading hypotheses for disease pathogenesis, but due to the lack of proper human cell models, the role of microglia in SCZ is not well studied. We used monozygotic twins discordant for SCZ and healthy individuals to generate human induced pluripotent stem cell-derived microglia to assess the transcriptional and functional differences in microglia between healthy controls, affected twins and unaffected twins. The microglia from affected twins had increased expression of several common inflammation-related genes compared to healthy individuals. Microglia from affected twins had also reduced response to interleukin 1 beta (IL1β) treatment, but no significant differences in migration or phagocytotic activity. Ingenuity Pathway Analysis (IPA) showed abnormalities related to extracellular matrix signaling. RNA sequencing predicted downregulation of extracellular matrix structure constituent Gene Ontology (GO) terms and hepatic fibrosis pathway activation that were shared by microglia of both affected and unaffected twins, but the upregulation of major histocompatibility complex (MHC) class II receptors was observed only in affected twin microglia. Also, the microglia of affected twins had heterogeneous response to clozapine, minocycline, and sulforaphane treatments. Overall, despite the increased expression of inflammatory genes, we observed no clear functional signs of hyperactivation in microglia from patients with SCZ. We conclude that microglia of the patients with SCZ have gene expression aberrations related to inflammation response and extracellular matrix without contributing to increased microglial activation.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Standard single-cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to single-cell physiology. Here, a scPatch-Clamp/Proteomics platform is developed on single neurons generated from hiPSCs bearing an Alzheimer's disease (AD) genetic mutation and compares them to isogenic wild-type controls. This approach provides both current and voltage electrophysiological data plus detailed proteomics information on single-cells. With this new method, the authors are able to observe hyperelectrical activity in the AD hiPSC-neurons, similar to that observed in the human AD brain, and correlate it to ≈1400 proteins detected at the single neuron level. Using linear regression and mediation analyses to explore the relationship between the abundance of individual proteins and the neuron's mutational and electrophysiological status, this approach yields new information on therapeutic targets in excitatory neurons not attainable by traditional methods. This combined patch-proteomics technique creates a new proteogenetic-therapeutic strategy to correlate genotypic alterations to physiology with protein expression in single-cells.

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

Statin drugs lower blood cholesterol levels for cardiovascular disease prevention. Women are more likely than men to experience adverse statin effects, particularly new-onset diabetes (NOD) and muscle weakness. Here we find that impaired glucose homeostasis and muscle weakness in statin-treated female mice are associated with reduced levels of the omega-3 fatty acid, docosahexaenoic acid (DHA), impaired redox tone, and reduced mitochondrial respiration. Statin adverse effects are prevented in females by administering fish oil as a source of DHA, by reducing dosage of the X chromosome or the Kdm5c gene, which escapes X chromosome inactivation and is normally expressed at higher levels in females than males. As seen in female mice, we find that women experience more severe reductions than men in DHA levels after statin administration, and that DHA levels are inversely correlated with glucose levels. Furthermore, induced pluripotent stem cells from women who developed NOD exhibit impaired mitochondrial function when treated with statin, whereas cells from men do not. These studies identify X chromosome dosage as a genetic risk factor for statin adverse effects and suggest DHA supplementation as a preventive co-therapy.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 gene in induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations on plasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Amyotrophic Lateral Sclerosis (ALS), like many other neurodegenerative diseases, is highly heritable, but with only a small fraction of cases explained by monogenic disease alleles. To better understand sporadic ALS, we report epigenomic profiles, as measured by ATAC-seq, of motor neuron cultures derived from a diverse group of 380 ALS patients and 80 healthy controls. We find that chromatin accessibility is heavily influenced by sex, the iPSC cell type of origin, ancestry, and the inherent variance arising from sequencing. Once these covariates are corrected for, we are able to identify ALS-specific signals in the data. Additionally, we find that the ATAC-seq data is able to predict ALS disease progression rates with similar accuracy to methods based on biomarkers and clinical status. These results suggest that iPSC-derived motor neurons recapitulate important disease-relevant epigenomic changes.

Several genetic risk factors for Alzheimers disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimers disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimers disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimers disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aβ induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimers disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimers disease.

Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.

Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo.

In Alzheimer's disease (AD), dysfunctional mitochondrial metabolism is associated with synaptic loss, the major pathological correlate of cognitive decline. Mechanistic insight for this relationship, however, is still lacking. Here, comparing isogenic wild-type and AD mutant human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons (hiN), evidence is found for compromised mitochondrial energy in AD using the Seahorse platform to analyze glycolysis and oxidative phosphorylation (OXPHOS). Isotope-labeled metabolic flux experiments revealed a major block in activity in the tricarboxylic acid (TCA) cycle at the α-ketoglutarate dehydrogenase (αKGDH)/succinyl coenzyme-A synthetase step, metabolizing α-ketoglutarate to succinate. Associated with this block, aberrant protein S-nitrosylation of αKGDH subunits inhibited their enzyme function. This aberrant S-nitrosylation is documented not only in AD-hiN but also in postmortem human AD brains versus controls, as assessed by two separate unbiased mass spectrometry platforms using both SNOTRAP identification of S-nitrosothiols and chemoselective-enrichment of S-nitrosoproteins. Treatment with dimethyl succinate, a cell-permeable derivative of a TCA substrate downstream to the block, resulted in partial rescue of mitochondrial bioenergetic function as well as reversal of synapse loss in AD-hiN. These findings have therapeutic implications that rescue of mitochondrial energy metabolism can ameliorate synaptic loss in hiPSC-based models of AD.

Understanding metabolic heterogeneity is the key to uncovering the underlying mechanisms of metabolic-related diseases. Current metabolic imaging studies suffer from limitations including low resolution and specificity, and the model systems utilized often lack human relevance. Here, we present a single-cell metabolic imaging platform to enable direct imaging of lipid metabolism with high specificity in various human-derived 2D and 3D culture systems. Through the incorporation of an azide-tagged infrared probe, selective detection of newly synthesized lipids in cells and tissue became possible, while simultaneous fluorescence imaging enabled cell-type identification in complex tissues. In proof-of-concept experiments, newly synthesized lipids were directly visualized in human-relevant model systems among different cell types, mutation status, differentiation stages, and over time. We identified upregulated lipid metabolism in progranulin-knockdown human induced pluripotent stem cells and in their differentiated microglia cells. Furthermore, we observed that neurons in brain organoids exhibited a significantly lower lipid metabolism compared to astrocytes.

LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

Interactions between adipose tissue, liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model, we establish an interconnected microphysiological system (MPS) containing white adipocytes, hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS, we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function, whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance, corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases.

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.

Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreichs ataxia.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

Lithium (Li) is one of the most effective drugs for treating bipolar disorder (BD), however, there is presently no way to predict response to guide treatment. The aim of this study is to identify functional genes and pathways that distinguish BD Li responders (LR) from BD Li non-responders (NR). An initial Pharmacogenomics of Bipolar Disorder study (PGBD) GWAS of lithium response did not provide any significant results. As a result, we then employed network-based integrative analysis of transcriptomic and genomic data. In transcriptomic study of iPSC-derived neurons, 41 significantly differentially expressed (DE) genes were identified in LR vs NR regardless of lithium exposure. In the PGBD, post-GWAS gene prioritization using the GWA-boosting (GWAB) approach identified 1119 candidate genes. Following DE-derived network propagation, there was a highly significant overlap of genes between the top 500- and top 2000-proximal gene networks and the GWAB gene list (Phypergeometric = 1.28E-09 and 4.10E-18, respectively). Functional enrichment analyses of the top 500 proximal network genes identified focal adhesion and the extracellular matrix (ECM) as the most significant functions. Our findings suggest that the difference between LR and NR was a much greater effect than that of lithium. The direct impact of dysregulation of focal adhesion on axon guidance and neuronal circuits could underpin mechanisms of response to lithium, as well as underlying BD. It also highlights the power of integrative multi-omics analysis of transcriptomic and genomic profiling to gain molecular insights into lithium response in BD.

Background: Type 1 diabetes, an autoimmune disorder leading to the destruction of pancreatic β-cells, requires lifelong insulin therapy. Islet transplantation offers a promising solution but faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and have the capability for large-scale production. However, current differentiation protocols, predominantly conducted in hybrid or 2D settings, lack scalability and optimal conditions for suspension culture. Methods: We examined a range of bioreactor scaleup process parameters and quality target product profiles that might affect the differentiation process. This investigation was conducted using an optimized High Dimensional Design of Experiments (HD-DoE) protocol designed for scalability and implemented in 0.5L (PBS-0.5 Mini) vertical wheel bioreactors. Results: A three stage suspension manufacturing process is developed, transitioning from adherent to suspension culture, with TB2 media supporting iPSC growth during scaling. Stage-wise optimization approaches and extended differentiation times are used to enhance marker expression and maturation of iPSC-derived islet-like clusters. Continuous bioreactor runs were used to study nutrient and growth limitations and impact on differentiation. The continuous bioreactors were compared to a Control media change bioreactor showing metabolic shifts and a more β-cell-like differentiation profile. Cryopreserved aggregates harvested from the runs were recovered and showed maintenance of viability and insulin secretion capacity post-recovery, indicating their potential for storage and future transplantation therapies. Conclusion: This study demonstrated that stage time increase and limited media replenishing with lactate accumulation can increase the differentiation capacity of insulin producing cells cultured in a large-scale suspension environment. [ABSTRACT FROM AUTHOR]

Our study develops a generative adversarial network (GAN)-based method that generates faithful synthetic image data of human cardiomyocytes at varying stages in their maturation process, as a tool to significantly enhance the classification accuracy of cells and ultimately assist the throughput of computational analysis of cellular structure and functions. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were cultured on micropatterned collagen coated hydrogels of physiological stiffnesses to facilitate maturation and optical measurements were performed for their structural and functional analyses. Control groups were cultured on collagen coated glass well plates. These image recordings were used as the real data to train the GAN model. The results show the GAN approach is able to replicate true features from the real data, and inclusion of such synthetic data significantly improves the classification accuracy compared to usage of only real experimental data that is often limited in scale and diversity. The proposed model outperformed four conventional machine learning algorithms with respect to improved data generalization ability and data classification by incorporating synthetic data. This work demonstrates the importance of integrating synthetic data in situations where there are limited sample sizes and thus, effectively addresses the challenges imposed by data availability. [ABSTRACT FROM AUTHOR]

Direct cell-type conversion of somatic cells into cell types of interest has garnered great attention because it circumvents rejuvenation and preserves the hallmarks of cellular aging (unlike induced pluripotent stem cells [iPSCs]) and is more suitable for modeling diseases with strong age-related and epigenetic contributions. Fibroblasts are commonly used for direct conversion; however, obtaining these cells requires highly invasive skin biopsies. Urine-derived cells (UDCs) are an alternative cell source and can be obtained via noninvasive procedures. Herein, induced motor neuron-like cells (iMNs) were generated from UDCs by transducing transcription factors involved in motor neuron (MN) differentiation. iMNs exhibited neuronal morphology, upregulation of pan-neuron and MN markers, and MN functionality, including spontaneous calcium oscillation and bungarotoxin-positive neuromuscular junction formation, when co-cultured with myotubes. Altogether, the findings of this study indicated that UDCs can be converted to functional MNs. This technology may allow us to understand disease pathogenesis and progression and discover biomarkers and drugs for MN-related diseases at the population level. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that causes muscle atrophy and primarily targets motor neurons (MNs). Approximately 20% of familial ALS cases are caused by gain-of-function mutations in superoxide dismutase 1 (SOD1), leading to MN degeneration and ion channel dysfunction. Previous studies have shown that n-Butylidenephthalide (BP) delays disease progression and prolongs survival in animal models of ALS. However, no studies have been conducted on models from human sources. Herein, we examined the protective efficacy of BP on MNs derived from induced pluripotent stem cells (iPSCs) of an ALS patient harboring the SOD1G85R mutation as well as on those derived from genetically corrected iPSCs (SOD1G85G). Our results demonstrated that the motor neurons differentiated from iPSC with SOD1G85R mutation exhibited characteristics of neuron degeneration (as indicated by the reduction of neurofilament expression) and ion channel dysfunction (in response to potassium chloride (KCl) and L-glutamate stimulation), in contrast to those derived from the gene corrected iPSC (SOD1G85G). Meanwhile, BP treatment effectively restored calcium ion channel function by reducing the expression of glutamate receptors including glutamate ionotropic receptor AMPA type subunit 3 (GluR3) and glutamate ionotropic receptor NMDA type subunit 1 (NMDAR1). Additionally, BP treatment activated autophagic pathway to attenuate neuron degeneration. Overall, this study supports the therapeutic effects of BP on ALS patient-derived neuron cells, and suggests that BP may be a promising candidate for future drug development. [ABSTRACT FROM AUTHOR]

Defects at the neuromuscular junction (NMJ) are among the earliest hallmarks of amyotrophic lateral sclerosis (ALS). According to the "dying-back" hypothesis, NMJ disruption not only precedes but also triggers the subsequent degeneration of motoneurons in both sporadic (sALS) and familial (fALS) ALS. Using human induced pluripotent stem cells (iPSCs), we show that the RNA-binding protein HuD (ELAVL4) contributes to NMJ defects and apoptosis in FUS-ALS. HuD overexpression mimics the severe FUSP525L mutation, while its knockdown rescues the FUSP525L phenotypes. In Drosophila, neuronal overexpression of the HuD ortholog, elav, induces motor dysfunction, and its knockdown improves motor function in a FUS-ALS model. Finally, we report increased HuD levels upon oxidative stress in human motoneurons and in sALS patients with an oxidative stress signature. Based on these findings, we propose that HuD plays a role downstream of FUS mutations in fALS and in sALS related to oxidative stress. Impairment at the neuromuscular junction (NMJ) is an early sign of ALS. Here, the authors show that the RNA-binding protein HuD causes NMJ and apoptosis phenotypes in human iPSC and Drosophila ALS models, highlighting a possible role in disease onset. [ABSTRACT FROM AUTHOR]

In Finland, the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We conducted a genome-wide association study in the Finnish population and identified rs570516915, a single nucleotide polymorphism highly enriched in Finns, as strongly associated with CP (P = 5.25 × 10−34, OR = 8.65, 95% CI 6.11–12.25), but not with CL/P (P = 7.2 × 10−5), with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in independent cohorts of CP cases from Finland (P = 8.82 × 10−28) and Estonia (P = 1.25 × 10−5). The risk allele of rs570516915 alters a conserved binding site for the transcription factor IRF6 within an enhancer (MCS-9.7) upstream of the IRF6 gene and diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6, suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP. Here, the authors perform a genome-wide study and identify a genetic variant enriched in the Finnish population that is strongly associated with isolated cleft palate. This finding suggests a genetic basis for the high prevalence of cleft palate in Finland. [ABSTRACT FROM AUTHOR]

Background: Periventricular nodular heterotopia (PVNH) is a neuronal migration disorder caused by the inability of neurons to move to the cortex. Patients with PVNH often experience epilepsy due to ectopic neuronal discharges. Most cases of PVNH are associated with variations in filamin A (FLNA), which encodes an actin-binding protein. However, the underlying pathological mechanisms remain unclear. Methods: Next-generation sequencing was performed to detect variants in the patient with PVNH, and the findings were confirmed using Sanger sequencing. Iterative threading assembly refinement was used to predict the structures of the variant proteins, and the search tool for the retrieval of interacting genes/proteins database was used to determine the interactions between FLNA and motility-related proteins. An induced pluripotent stem cell (iPSC) line was generated as a disease model by reprogramming human peripheral blood mononuclear cells. The FLNA expression in iPSCs was assessed using western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Immunofluorescence analysis was performed to determine the arrangement of F-actin. Results: A novel FLNA frameshift variant (NM_001456.3: c.1466delG, p. G489Afs*9) was identified in a patient with PVNH and epilepsy. Bioinformatic analysis indicated that this variation was likely to impair FLNA function. Western blot and qRT-PCR analysis of iPSCs derived from the patient's peripheral blood mononuclear cells revealed the absence of FLNA protein and mRNA. Immunofluorescence analysis suggested an irregular arrangement and disorganization of F-actin compared to that observed in healthy donors. Conclusion: Our findings indicate that the frameshift variant of FLNA (NM_001456.3: c.1466delG, p. G489Afs*9) impairs the arrangement and organization of F-actin, potentially influencing cell migration and causing PVNH. [ABSTRACT FROM AUTHOR]

The demand for skin models as alternatives to animal testing has grown due to ethical concerns and the need for accurate substance evaluation. These alternatives, known as New Approach Methodologies (NAMs), are increasingly used for regulatory decisions. Current skin models from primary human cells often rely on bovine collagen, raising ethical issues. This study explores self-assembled skin models (SASM) as a new method, utilizing hair follicle-derived keratinocytes reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into fibroblasts and keratinocytes. The model relies on the ability of fibroblasts to secrete collagen to produce a xeno-free dermal layer and on the differentiation of keratinocytes to create a functional epidermal layer. These layers exhibited confirmed metabolic activity and the capability to withstand test substances. The successful development of SASM underscores the significance of accurate alternatives in dermatological research, providing an ethical and reliable option for substance evaluation and regulatory testing. [ABSTRACT FROM AUTHOR]

Background Objective Methods Results Conclusion Parkinson's disease (PD) is the second most common, and the fastest‐growing neurodegenerative disorder with unclear etiology in most cases. Therefore, the identification of non‐genetic risk factors for PD pathology is crucial to develop effective preventative or therapeutic strategies. An increasing number of evidence suggests that central insulin resistance might have an essential role in PD pathology. Nevertheless, it is not clear whether insulin resistance arises from external factors/lifestyle, comorbidities such as type 2 diabetes or it can occur in a PD patient's brain independently from peripheral insulin resistance.We aimed to investigate insulin resistance and its role in GBA1 mutation‐associated PD pathogenesis and phenotype severity.Midbrain organoids, generated from induced pluripotent stem cells (iPSCs) of PD patients carrying the GBA1‐N370S heterozygous mutation (GBA‐PD) and healthy donors, were exposed to different insulin concentrations to modify insulin signaling function. Transcriptomics analysis was performed to explore insulin signaling gene expression patterns in GBA‐PD and to find a potential target for GBA‐PD‐associated phenotype rescue.The insulin signaling pathway genes show dysregulation in GBA‐PD. Particularly, we highlight that a knockdown of FOXO1 mitigates the loss of dopaminergic neurons and cellular death in GBA‐PD. Additionally, our findings suggest a promising therapeutic potential of the anti‐diabetic drug Pioglitazone in decreasing dopaminergic neuron loss associated with GBA‐PD.Local insulin signaling dysfunction plays a substantial role in GBA‐PD pathogenesis, exacerbating dopaminergic neuron death. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

The use of allogeneic induced pluripotent stem cell (iPSC)-derived cell therapies for regenerative medicine offers an affordable and realistic alternative to producing individual iPSC lines for each patient in need. Human Leukocyte Antigens (HLA)-homozygous iPSCs matched in hemi-similarity could provide cell therapies with reduced immune rejection covering a wide range of the population with a few iPSC lines. Several banks of HLA-homozygous iPSCs (haplobanks) have been established worldwide or are underway, to provide clinical grade starting material for cell therapies covering the most frequent HLA haplotypes for certain populations. Harmonizing quality standards among haplobanks and creating a global registry could minimize the collective effort and provide a much wider access to HLA-compatible cell therapies for patients with less frequent haplotypes. In this review we present all the current haplobank initiatives and their potential benefits for the global population. [ABSTRACT FROM AUTHOR]

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines, we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers, while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells play a vital role in innate immunity and show great promise in cancer immunotherapy. Traditional sources of NK cells, such as the peripheral blood, are limited by availability and donor variability. In addition, in vitro expansion can lead to functional exhaustion and gene editing challenges. This study aimed to harness induced pluripotent stem cell (iPSC) technology to provide a consistent and scalable source of NK cells, overcoming the limitations of traditional sources and enhancing the potential for cancer immunotherapy applications. We developed human placental–derived iPSC lines using reprogramming techniques. Subsequently, an optimized two‐step differentiation protocol was introduced to generate high‐purity NK cells. Initially, iPSCs were differentiated into hematopoietic‐like stem cells using spin‐free embryoid bodies (EBs). Subsequently, the EBs were transferred to ultra‐low attachment plates to induce NK cell differentiation. iPSC‐derived NK (iNK) cells expressed common NK cell markers (NKp46, NKp30, NKp44, CD16 and eomesodermin) at both RNA and protein levels. iNK cells demonstrated significant resilience to cryopreservation and exhibited enhanced cytotoxicity. The incorporation of a chimeric antigen receptor (CAR) construct further augmented their cytotoxic potential. This study exemplifies the feasibility of generating iNK cells with high purity and enhanced functional capabilities, their improved resilience to cryopreservation and the potential to have augmented cytotoxicity through CAR expression. Our findings offer a promising pathway for the development of potential cellular immunotherapies, highlighting the critical role of iPSC technology in overcoming challenges associated with traditional NK cell sources. [ABSTRACT FROM AUTHOR]

The document delves into multiple studies on Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration, focusing on topics such as disease diagnosis, patient stratification, and understanding disease mechanisms. It discusses the importance of biomarkers, autonomic nervous function, and astrocyte pathology in ALS research and clinical trials. Additionally, the research explores genetic risk factors, respiratory impairments, and the impact of genetic polymorphisms on survival in ALS and FTD patients, aiming to improve prognosis and management of these neurodegenerative diseases. [Extracted from the article]

Advances in the treatment of childhood cancer have significantly improved survival rates, with more than 80% of survivors reaching adulthood. However, gonadotoxic cancer treatments endanger future fertility, and prepubertal males have no option to preserve fertility by sperm cryopreservation. In addition, boys with cryptorchidism are at risk of compromised fertility in adulthood. To investigate current evidence for male fertility restoration strategies, explore barriers to clinical implementation, and outline potential steps to overcome these barriers, a scoping review was conducted. This knowledge synthesis is particularly relevant for prepubertal male cancer survivors and boys with cryptorchidism. The review was conducted after the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews criteria and previously published guidelines and examined studies using human testis tissue of prepubertal boys or healthy male adults. A literature search in PubMed was conducted, and 72 relevant studies were identified, including in vivo and in vitro approaches. In vivo strategies, such as testis tissue engraftment and spermatogonial stem cell transplantation, hold promise for promoting cell survival and differentiation. Yet, complete spermatogenesis has not been achieved. In vitro approaches focus on the generation of male germ cells from direct germ cell maturation in various culture systems, alongside human induced pluripotent stem cells and embryonic stem cells. These approaches mark significant advancements in understanding and promoting spermatogenesis, but achieving fully functional spermatozoa in vitro remains a challenge. Barriers to clinical implementation include the risk of reintroducing malignant cells and introduction of epigenetic changes. Male fertility restoration is an area in rapid development. On the basis of the reviewed studies, the most promising and advanced strategy for restoring male fertility using cryopreserved testis tissue is direct testis tissue transplantation. This review identifies persistent barriers to the clinical implementation of male fertility restoration. However, direct transplantation of frozen-thawed testis tissue remains a promising strategy that is on the verge of clinical application. [ABSTRACT FROM AUTHOR]

Transmembrane protein 182 (TMEM182) is notably abundant in muscle and adipose tissue, but its role in the heart remains unknown. This study examined the contribution of TMEM182 in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. For this, we generated hiPSCs overexpressing TMEM182 in a doxycycline‐inducible manner and induced their differentiation into cardiomyocytes. On Day 12 of differentiation, expression of the cardiomyocyte markers, TNNT2 and MYH6, was significantly decreased in TMEM182‐overexpressing cells. Additionally, we found that phosphorylation of GSK‐3β (Ser9) and β‐catenin (Ser552) was increased during TMEM182 overexpression, suggesting activation of Wnt/β‐catenin signaling. We further focused on integrin‐linked kinase (ILK) as the mechanism by which TMEM182 activates Wnt/β‐catenin signaling. Evaluation showed that ILK expression was increased in cells overexpressing TMEM182. These results suggest that TMEM182 maintains Wnt/β‐catenin signaling in an activated state after mesoderm formation by increasing ILK expression, thereby suppressing hiPSCs differentiation into cardiomyocytes. [ABSTRACT FROM AUTHOR]

A hexanucleotide GGGGCC repeat expansion in the non-coding region of the C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR/Cas13d, a powerful RNA-targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR/Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR/Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR/Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD. [ABSTRACT FROM AUTHOR]

Angelman syndrome (AS) and Prader-Willi syndrome (PWS), two distinct neurodevelopmental disorders, result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele, respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless, gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences, reprogramming methods, and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here, we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally, these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. [ABSTRACT FROM AUTHOR]

To investigate intestinal health and its potential disruptors in vitro, representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However, the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently, the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge, IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model, while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately, comparison to in vivo transcriptomics data showed limited similarities, which could be explained by essential differences in the study setups. Altogether, hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro. [ABSTRACT FROM AUTHOR]

Stem cell therapies are progressively redefining the treatment landscape for a spectrum of neurological and age-related disorders. This review discusses the molecular and functional attributes of stem cells, emphasizing the roles of neural stem cells and mesenchymal stem cells in the context of neurological diseases such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, Parkinson's disease, and Alzheimer's disease. The review also explores the potential of stem cells in addressing the aging process. The paper analyzes stem cells' intrinsic properties of self-renewal, differentiation, and paracrine effects, alongside the importance of laboratory-modified stem cells like induced pluripotent stem cells and transgenic stem cells. Insights into disease-specific stem cell treatments are offered, reviewing both successes and challenges in the field. This includes the translational difficulties from rodent studies to human trials. The review concludes by acknowledging the uncharted territories that warrant further investigation, emphasizing the potential roles of stem cell-derived exosomes and indole-related molecules, and aiming at providing a basic understanding of stem cell therapies. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) results in permanent neurological dysfunction and neuropathic pain. To address this pathology, we recently conducted a clinical study in which we transplanted neural precursor cells (NPCs) derived from human induced pluripotent stem cells into patients during the subacute phase of SCI. One of the therapeutic mechanisms of cell transplantation is the formation of synaptic connections with the host’s neural tissues, which we demonstrated using a chemogenetic tool. In addition, we have developed innovative strategies to enhance the effectiveness of cell transplantation through gene therapy. Moreover, our current study is focused on developing cell therapy for chronic SCI, a more challenging pathology characterized by the formation of cavities and scar tissue. In such situations, transplanting NPCs with neurogenic properties could effectively penetrate scar tissue and form functional synapses with the host neurons. To improve the outcomes of cell transplantation alone, we have found that incorporating rehabilitation is beneficial. In animal models of SCI, we have established an effective rehabilitative training program in which NPCs were transplanted during the chronic phase. Robotic rehabilitation has demonstrated improvements in gait ability and trunk function in clinical situations. Therefore, regenerative medicine shows promise for chronic SCI, particularly when rehabilitation strategies are incorporated. [ABSTRACT FROM AUTHOR]

Humans can be exposed to per- and polyfluoroalkyl substances (PFASs) via many exposure routes, including diet, which may lead to several adverse health effects. So far, little is known about PFAS transport across the human intestinal barrier. In the current study, we aimed to assess the transport of 5 PFASs (PFOS, PFOA, PFNA, PFHxS and HFPO-DA) in a human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cell (IEC) model. This model was extensively characterized and compared with the widely applied human colonic adenocarcinoma cell line Caco-2 and a human primary IEC-based model, described to most closely resemble in vivo tissue. The hiPSC-derived IEC layers demonstrated polarized monolayers with tight junctions and a mucus layer. The monolayers consisted of enterocytes, stem cells, goblet cells, enteroendocrine cells, and Paneth cells that are also present in native tissue. Transcriptomics analysis revealed distinct differences in gene expression profiles, where the hiPSC-derived IECs showed the highest expression of intestinal tissue-specific genes relative to the primary IEC-based model and the Caco-2 cells clustered closer to the primary IEC-based model than the hiPSC-derived IECs. The order of PFAS transport was largely similar between the models and the apparent permeability (Papp) values of PFAS in apical to basolateral direction in the hiPSC-derived IEC model were in the following order: PFHxS > PFOA > HFPO-DA > PFNA > PFOS. In conclusion, the hiPSC-derived IEC model highly resembles human intestinal physiology and is therefore a promising novel in vitro model to study transport of chemicals across the intestinal barrier for risk assessment of chemicals. [ABSTRACT FROM AUTHOR]

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–associated genome editing (GE) components (e.g., nucleases, guide RNAs (gRNAs), and plasmids) are used to genetically modify cells during development of ex vivo genome-edited cell therapies. Prolonged presence of GE components may increase the risk of unintended genome modifications (e.g., off-target editing and chromosomal rearrangements). This risk is a function of the stability of the GE components, culture conditions (i.e., culture length, media changes, etc.), and the nature of the GE component (i.e., only plasmids can be integrated into a cell's genome). Testing for residual GE components on ex vivo genetically edited drug products is generally recommended in current regulatory guidance (CBER 2024). For allogenic cell therapies derived from induced pluripotent stem cells (iPSC), cells typically undergo clonal selection and extensive culturing following completion of genome editing. This post-engineering clonal selection substantially reduces the amount of residual GE components while the long-duration cell culture significantly reduces the presence of active residual GE components. Here we present a case in which the need for testing of the drug product for residual GE components has been eliminated. In silico modeling was used to estimate clearance mechanisms across a variety of relevant assumptions, including disposition of extracellular GE components via media changes and dilution of intracellular GE components via cell expansion. Determining the ability of each GE component—alone or in complex with other GE components—to modify genomic material was assessed by a series of both in vitro and ex vivo (i.e., engineering cells) studies. For the in vitro studies, a DNA cutting assay was developed to assess the ability of the component to cut a representative DNA strand. For the ex vivo modification of cells, an assessment of the knock-out of the relevant gene was completed by flow cytometry specifically assessing the presence or absence of protein expression on the modified cells. The persistence and stability of GE components were examined under cell-mimicking conditions and in ex vivo modified cells. The components were stressed under multiple conditions mimicking a range of culture conditions and tested in the aforementioned DNA cutting assay. The presence of residual gRNA was directly assessed in the ex vivo modified cells via a gRNA-specific digital droplet polymerase chain reaction (ddPCR) assay. Simulations estimating genome editing residual clearance via dilution for extracellular residuals (via media changes) or intracellular residuals (via cell doubling) demonstrate clearance of measurable residuals within 28 days of cell culture. Studies simulating the stability of genome editing residuals estimate less than 7 days for the nuclease, gRNA and ribonucleoprotein (RNP) complex. gRNA was undetectable by 8 days post-engineering under actual engineering conditions. Additionally, without gRNA present, CRISPR Cas12a nucleases did not demonstrate evidence of cutting. In contrast, plasmid DNA can be randomly integrated into the genome and free plasmid is highly stable under cell culture-like conditions (50+ days). Additionally, plasmid DNA integrated in cells will propagate during mitosis, leading to the additional risk of expansion of an unintentional integration event. Both the gRNA and nuclease in the RNP complex are required for DNA cutting. Neither individual component nor the complex are stable beyond 7 days in culture-mimicking conditions. These findings suggest that the risk of unplanned genomic modification resulting from residual gRNA or nuclease is minimal for processes in which extensive culture is performed after the completion of genome editing and clonal selection. However, the risk of residual plasmid DNA integration is significantly higher regardless of the manufacturing process. The residual plasmid itself is quite stable (at least 50 days) and the risk of random, off-target integration is present. By establishing the stability of these components, we have demonstrated that testing for residual gRNA or nuclease is not warranted for clonally derived allogeneic cell therapies. [Display omitted] • Ribonucleoprotein (RNP) complex is required for CRISPR-mediated gene modification. • RNP complex and individual components are inactivated quickly in cell culture • Single-cell cloning can act as a primary elimination route of gene modification agents. • Long culture times further reduce the risk of unintended modification. • Unintended gene modification risk is cell therapy manufacturing process dependent. [ABSTRACT FROM AUTHOR]

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes. [ABSTRACT FROM AUTHOR]

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α−SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis. [ABSTRACT FROM AUTHOR]