Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

Inflammation-associated suppression of metabolic gene networks in acute and chronic liver disease.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.

APEX1 Nuclease and Redox Functions are Both Essential for Adult Mouse Hematopoietic Stem and Progenitor Cells.

Self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) are carefully controlled by extrinsic and intrinsic factors, to ensure the lifelong process of hematopoiesis. Apurinic/apyrimidinic endonuclease 1 (APEX1) is a multifunctional protein implicated in DNA repair and transcriptional regulation. Although previous studies have emphasized the necessity of studying APEX1 in a lineage-specific context and its role in progenitor differentiation, no studies have assessed the role of APEX1, nor its two enzymatic domains, in supporting adult HSPC function. In this study, we demonstrated that complete loss of APEX1 from murine bone marrow HSPCs (induced by CRISPR/Cas9) caused severe hematopoietic failure following transplantation, as well as a HSPC expansion defect in culture conditions maintaining in vivo HSC functionality. Using specific inhibitors against either the nuclease or redox domains of APEX1 in combination with single cell transcriptomics (CITE-seq), we found that both APEX1 nuclease and redox domains are regulating mouse HSPCs, but through distinct underlying transcriptional changes. Inhibition of the APEX1 nuclease function resulted in loss of HSPCs accompanied by early activation of differentiation programs and enhanced lineage commitment. By contrast, inhibition of the APEX1 redox function significantly downregulated interferon-stimulated genes and regulons in expanding HSPCs and their progeny, resulting in dysfunctional megakaryocyte-biased HSPCs, as well as loss of monocytes and lymphoid progenitor cells. In conclusion, we demonstrate that APEX1 is a key regulator for adult regenerative hematopoiesis, and that the APEX1 nuclease and redox domains differently impact proliferating HSPCs.

T cells, more than antibodies, may prevent symptoms developing from respiratory syncytial virus infections in older adults.

Introduction: The immune mechanisms supporting partial protection from reinfection and disease by the respiratory syncytial virus (RSV) have not been fully characterized. In older adults, symptoms are typically mild but can be serious in patients with comorbidities when the infection extends to the lower respiratory tract. Methods: This study formed part of the RESCEU older-adults prospective-cohort study in Northern Europe (2017-2019; NCT03621930) in which a thousand participants were followed over an RSV season. Peripheral-blood samples (taken pre-season, post-season, during illness and convalescence) were analyzed from participants who (i) had a symptomatic acute respiratory tract infection by RSV (RSV-ARTI; N=35) or (ii) asymptomatic RSV infection (RSV-Asymptomatic; N=16). These analyses included evaluations of antibody (Fc-mediated-) functional features and cell-mediated immunity, in which univariate and machine-learning (ML) models were used to explore differences between groups. Results: Pre-RSV-season peripheral-blood biomarkers were predictive of symptomatic RSV infection. T-cell data were more predictive than functional antibody data (area under receiver operating characteristic curve [AUROC] for the models were 99% and 76%, respectively). The pre-RSV season T-cell phenotypes which were selected by the ML modelling and which were more frequent in RSV-Asymptomatic group than in the RSV-ARTI group, coincided with prominent phenotypes identified during convalescence from RSV-ARTI (e.g., IFN-γ+, TNF-α+ and CD40L+ for CD4+, and IFN-γ+ and 4-1BB+ for CD8+). Conclusion: The evaluation and statistical modelling of numerous immunological parameters over the RSV season suggests a primary role of cellular immunity in preventing symptomatic RSV infections in older adults.

The RSVPreF3-AS01 vaccine elicits broad neutralization of contemporary and antigenically distant respiratory syncytial virus strains.

The RSVPreF3-AS01 vaccine, containing the respiratory syncytial virus (RSV) prefusion F protein and the AS01 adjuvant, was previously shown to boost neutralization responses against historical RSV strains and to be efficacious in preventing RSV-associated lower respiratory tract diseases in older adults. Although RSV F is highly conserved, variation does exist between strains. Here, we characterized variations in the major viral antigenic sites among contemporary RSV sequences when compared with RSVPreF3 and showed that, in older adults, RSVPreF3-AS01 broadly boosts neutralization responses against currently dominant and antigenically distant RSV strains. RSV-neutralizing responses are thought to play a central role in preventing RSV infection. Therefore, the breadth of RSVPreF3-AS01-elicited neutralization responses may contribute to vaccine efficacy against contemporary RSV strains and those that may emerge in the future.

HiPSC-Derived Hepatocyte-like Cells Can Be Used as a Model for Transcriptomics-Based Study of Chemical Toxicity.

Traditional toxicity risk assessment approaches have until recently focussed mainly on histochemical readouts for cell death. Modern toxicology methods attempt to deduce a mechanistic understanding of pathways involved in the development of toxicity, by using transcriptomics and other big data-driven methods such as high-content screening. Here, we used a recently described optimised method to differentiate human induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs), to assess their potential to classify hepatotoxic and non-hepatotoxic chemicals and their use in mechanistic toxicity studies. The iPSC-HLCs could accurately classify chemicals causing acute hepatocellular injury, and the transcriptomics data on treated HLCs obtained by TempO-Seq technology linked the cytotoxicity to cellular stress pathways, including oxidative stress and unfolded protein response (UPR). Induction of these stress pathways in response to amiodarone, diclofenac, and ibuprofen, was demonstrated to be concentration and time dependent. The transcriptomics data on diclofenac-treated HLCs were found to be more sensitive in detecting differentially expressed genes in response to treatment, as compared to existing datasets of other diclofenac-treated in vitro hepatocyte models. Hence iPSC-HLCs generated by transcription factor overexpression and in metabolically optimised medium appear suitable for chemical toxicity detection as well as mechanistic toxicity studies.

PU.1 drives specification of pluripotent stem cell-derived endothelial cells to LSEC-like cells.

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFß-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System.

Astrocytes, the main supportive cell type of the brain, show functional impairments upon ageing and in a broad spectrum of neurological disorders. Limited access to human astroglia for pre-clinical studies has been a major bottleneck delaying our understanding of their role in brain health and disease. We demonstrate here that functionally mature human astrocytes can be generated by SOX9 overexpression for 6 days in pluripotent stem cell (PSC)-derived neural progenitor cells. Inducible (i)SOX9-astrocytes display functional properties comparable to primary human astrocytes comprising glutamate uptake, induced calcium responses and cytokine/growth factor secretion. Importantly, electrophysiological properties of iNGN2-neurons co-cultured with iSOX9-astrocytes are indistinguishable from gold-standard murine primary cultures. The high yield, fast timing and the possibility to cryopreserve iSOX9-astrocytes without losing functional properties makes them suitable for scaled-up production for high-throughput analyses. Our findings represent a step forward to an all-human iPSC-derived neural model for drug development in neuroscience and towards the reduction of animal use in biomedical research.

Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.

Dystrophin deficiency leads to dysfunctional glutamate clearance in iPSC derived astrocytes.

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.