Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFβ pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility of this system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CP specification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.

The neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

Disorders of gut-brain interaction (DGBIs), formerly known as functional gastrointestinal disorders, are extremely common and historically difficult to manage. This is largely because their cellular and molecular mechanisms have remained poorly understood and understudied. One approach to unravel the molecular underpinnings of complex disorders such as DGBIs is performing genome wide association studies (GWASs). However, due to the heterogenous and non-specific nature of GI symptoms, it has been difficult to accurately classify cases and controls. Thus, to perform reliable studies, we need to access large patient populations which has been difficult to date. Here, we leveraged the UK Biobank (UKBB) database, containing genetic and medical record data of over half a million individuals, to perform GWAS for five DGBI categories: functional chest pain, functional diarrhea, functional dyspepsia, functional dysphagia, and functional fecal incontinence. By applying strict inclusion and exclusion criteria, we resolved patient populations and identified genes significantly associated with each condition. Leveraging multiple human single-cell RNA-sequencing datasets, we found that the disease associated genes were highly expressed in enteric neurons, which innervate and control GI functions. Further expression and association testing-based analyses revealed specific enteric neuron subtypes consistently linked with each DGBI. Furthermore, protein-protein interaction analysis of each of the disease associated genes revealed protein networks specific to each DGBI, including hedgehog signaling for functional chest pain and neuronal function and neurotransmission for functional diarrhea and functional dyspepsia. Finally, through retrospective medical record analysis we found that drugs that inhibit these networks are associated with an increased disease risk, including serine/threonine kinase 32B drugs for functional chest pain, solute carrier organic anion transporter family member 4C1, mitogen-activated protein kinase 6, and dual serine/threonine and tyrosine protein kinase drugs for functional dyspepsia, and serotonin transporter drugs for functional diarrhea. This study presents a robust strategy for uncovering the tissues, cell types, and genes involved in DGBIs, presenting novel predictions of the mechanisms underlying these historically intractable and poorly understood diseases.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a global health crisis, and yet our understanding of the disease pathophysiology and potential treatment options remains limited. SARS-CoV-2 infection occurs through binding and internalization of the viral spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane. Lethal complications are caused by damage and failure of vital organs that express high levels of ACE2, including the lungs, the heart and the kidneys. Here, we established a high-throughput drug screening strategy to identify therapeutic candidates that reduce ACE2 levels in human embryonic stem cell (hESC) derived cardiac cells. Drug target analysis of validated hit compounds, including 5 alpha reductase inhibitors, revealed androgen signaling as a key modulator of ACE2 levels. Treatment with the 5 alpha reductase inhibitor dutasteride reduced ACE2 levels and internalization of recombinant spike receptor binding domain (Spike-RBD) in hESC-derived cardiac cells and human alveolar epithelial cells. Finally, clinical data on coronavirus disease 2019 (COVID-19) patients demonstrated that abnormal androgen states are significantly associated with severe disease complications and cardiac injury as measured by blood troponin T levels. These findings provide important insights on the mechanism of increased disease susceptibility in male COVID-19 patients and identify androgen receptor inhibition as a potential therapeutic strategy.

The enteric nervous system (ENS) is a tantalizing frontier in neuroscience. With the recent emergence of single cell transcriptomic technologies, this rare and poorly understood tissue has begun to be better characterized in recent years. A precise functional mapping of enteric neuron diversity is critical for understanding ENS biology and enteric neuropathies. Nonetheless, this pursuit has faced considerable technical challenges. By leveraging different methods to compare available primary mouse and human ENS datasets, we underscore the urgent need for careful identity annotation, achieved through the harmonization and advancements of wet lab and computational techniques. We took different approaches including differential gene expression, module scoring, co-expression and correlation analysis, unbiased biological function hierarchical clustering, data integration and label transfer to compare and contrast functional annotations of several independently reported ENS datasets. These analyses highlight substantial discrepancies stemming from an overreliance on transcriptomics data without adequate validation in tissues. To achieve a comprehensive understanding of enteric neuron identity and their functional context, it is imperative to expand tissue sources and incorporate innovative technologies such as multiplexed imaging, electrophysiology, spatial transcriptomics, as well as comprehensive profiling of epigenome, proteome, and metabolome. Harnessing human pluripotent stem cell (hPSC) models provides unique opportunities for delineating lineage trees of the human ENS, and offers unparalleled advantages, including their scalability and compatibility with genetic manipulation and unbiased screens. We encourage a paradigm shift in our comprehension of cellular complexity and function in the ENS by calling for large-scale collaborative efforts and research investments.

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.

Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation.

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.

Schwann cells (SCs) are the primary glia of the peripheral nervous system. SCs are involved in many debilitating disorders, including diabetic peripheral neuropathy (DPN). Here, we present a strategy for deriving SCs from human pluripotent stem cells (hPSCs) that enables comprehensive studies of SC development, physiology, and disease. hPSC-derived SCs recapitulate the molecular features of primary SCs and are capable of in vitro and in vivo myelination. We established a model of DPN that revealed the selective vulnerability of SCs to high glucose. We performed a high-throughput screen and found that an antidepressant drug, bupropion, counteracts glucotoxicity in SCs. Treatment of hyperglycemic mice with bupropion prevents their sensory dysfunction, SC death, and myelin damage. Further, our retrospective analysis of health records revealed that bupropion treatment is associated with a lower incidence of neuropathy among diabetic patients. These results highlight the power of this approach for identifying therapeutic candidates for DPN.

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19.

Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.

Regenerative therapies based on resident human cardiac progenitor cells (hCPCs) are a promising alternative to medical treatments for patients with myocardial infarction. However, hCPCs are rare in human heart and finding efficient source and proper surface marker for isolation of these cells would make them a good candidate for therapy.

Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown.

The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and limitations in robust in vitro derivation methods. We developed a dual SHOX2:GFP; MYH6:mCherry knockin human embryonic stem cell (hESC) reporter line, which allows the identification and purification of SAN-like cells. Using this line, we performed several rounds of chemical screens and developed an efficient strategy to generate and purify hESC-derived SAN-like cells (hESC-SAN). The derived hESC-SAN cells display molecular and electrophysiological characteristics of bona fide nodal cells, which allowed exploration of their transcriptional profile at single-cell level. In sum, our dual reporter system facilitated an effective strategy for deriving human SAN-like cells, which can potentially be used for future disease modeling and drug discovery.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse event associated with treatment with paclitaxel and other chemotherapeutic agents. The prevention and treatment of CIPN are limited by a lack of understanding of the molecular mechanisms underlying this toxicity. In the current study, a human induced pluripotent stem cell-derived sensory neuron (iPSC-SN) model was developed for the study of chemotherapy-induced neurotoxicity. The iPSC-SNs express proteins characteristic of nociceptor, mechanoreceptor, and proprioceptor sensory neurons and show Ca2+ influx in response to capsaicin, α,β-meATP, and glutamate. The iPSC-SNs are relatively resistant to the cytotoxic effects of paclitaxel, with half-maximal inhibitory concentration (IC50 ) values of 38.1 µM (95% confidence interval (CI) 22.9-70.9 µM) for 48-hour exposure and 9.3 µM (95% CI 5.7-16.5 µM) for 72-hour treatment. Paclitaxel causes dose-dependent and time-dependent changes in neurite network complexity detected by βIII-tubulin staining and high content imaging. The IC50 for paclitaxel reduction of neurite area was 1.4 µM (95% CI 0.3-16.9 µM) for 48-hour exposure and 0.6 µM (95% CI 0.09-9.9 µM) for 72-hour exposure. Decreased mitochondrial membrane potential, slower movement of mitochondria down the neurites, and changes in glutamate-induced neuronal excitability were also observed with paclitaxel exposure. The iPSC-SNs were also sensitive to docetaxel, vincristine, and bortezomib. Collectively, these data support the use of iPSC-SNs for detailed mechanistic investigations of genes and pathways implicated in chemotherapy-induced neurotoxicity and the identification of novel therapeutic approaches for its prevention and treatment.