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Human blastocyst-derived,pluripotent cell lines are described that have normal karyotypes,express high levels of telomerase activity,and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months,these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers,including gut epithelium (endoderm); cartilage,bone,smooth muscle,and striated muscle (mesoderm); and neural epithelium,embryonic ganglia,and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology,drug discovery,and transplantation medicine. View Publication

INTRODUCTION Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors--OCT4,SOX2,KLF4,and cMYC--via integrating vectors. Here,we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). METHODS We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit. RESULTS After verification of OCT4 expression,we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-,KLF4-,and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. Reverse transcription-polymerase chain reaction and immunostaining confirmed induction of endogenous pluripotency-associated marker genes,such as SSEA-4,TRA-1-60,and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers,teratoma formation,and guided differentiation into beating cardiomyocytes. CONCLUSIONS MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials,our MV vector system provides an RNA-based,non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation. View Publication

Alzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening,we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons,seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks,without affecting general cell health. To validate our model,activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model,highly suitable to screen for compounds that modulate TAU aggregation. View Publication

Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors,histone-modification enzymes,and mRNA processing proteins. Recent evidence suggests that nucleoporins,well known components that control nucleo-cytoplasmic trafficking,have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation,which initially has been described in fungi and flies,also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition,we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively,genes that are highly induced can interact with NUP98 in the nuclear interior,away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation,revealing a role of a nuclear pore protein in regulating developmental gene expression programs. View Publication

Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1),a GPI-anchored cell surface molecule,specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a,a bona-fide mesDA lineage marker,during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development,as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3,whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. View Publication

When transmitted through the oral route,Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication,as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However,due to a lack of tractable infection models,we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However,the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here,we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular,we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization,and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with,and support replication of,T. gondii. Using quantitative label-free mass spectrometry,we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism,extracellular exosomes,intermicrovillar adhesion,and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses,indicating that de novo synthesis may support,but is not required for,parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection. View Publication

Idorn et al. characterized a mouse strain harboring a mutation identified in an HSE patient. Defective IFN-driven antiviral responses led to hyperactivation of inflammatory responses,which contributed to disease development. The study identifies immunopathology as an important contributor to HSE pathogenesis. View Publication

The COVID-19 pandemic has created a global health crisis,with challenges arising from the ongoing evolution of the SARS-CoV-2 virus,the emergence of new strains,and the long-term effects of COVID-19. Aiming to overcome the development of viral resistance,our study here focused on developing broad-spectrum pan-coronavirus antiviral therapies by targeting host protein quality control mechanisms essential for viral replication. Screening an in-house compound library led to the discovery of three candidate compounds targeting cellular proteostasis. The three compounds are (1) the nucleotide analog cordycepin,(2) a benzothiozole analog,and (3) an acyldepsipeptide analog initially developed as part of a campaign to target the mitochondrial ClpP protease. These compounds demonstrated dose-dependent efficacy against multiple coronaviruses,including SARS-CoV-2,effectively inhibiting viral replication in vitro as well as in lung organoids. Notably,the compounds also showed efficacy against SARS-CoV-2 delta and omicron strains. As part of this work,we developed a BSL2-level cell-integrated SARS-CoV-2 replicon,which could serve as a valuable tool for high-throughput screening and studying intracellular viral replication. Our study should aid in the advancement of antiviral drug development efforts. Subject terms: High-throughput screening,Small molecules View Publication

Introduction: Pathogenic variants in canonical transforming growth factor β (TGFβ) signaling genes predispose patients to thoracic aortic aneurysm and dissection (TAAD),predominantly in aortic root. Although TAAD pathogenesis associated with TGFβ receptor defects is well characterized,distinct and redundant mechanisms of TGFβ isoforms in TAAD incidence and severity remain elusive. Objective: Here we examined the biological role of TGFB2 in smooth muscle cell (SMC) differentiation and investigated how TGFB2 defects can lead to regional TAAD manifestations. Methods: To characterize the role of TGFB2 in SMC differentiation and function,we employed human-induced pluripotent stem cell (hiPSC)-derived SMC differentiation,CRISPR/Cas9 gene editing,three-dimensional SMC constructs,and human aortic tissue samples. Results: Despite the similar effects of different TGFβ isoforms on hiPSC-derived SMC differentiation,siRNA experiments revealed that TGFB2 distinctively displays TGFBR3 dependence for signal transduction,an understudied TGFβ receptor in TAAD. Molecular evaluation of different thoracic aorta regions suggested TGFB2 and TGFBR3 enrichment in the aortic root tunica media. TGFB2 haploinsufficiency (TGFB2KO/+) and TGFB2 neutralization impaired the differentiation of second heart field-derived SMCs. TGFBR3KO/KO prevented the molecular rescue of TGFB2KO/+ by TGFB2 supplementation indicating the involvement of TGFBR3 in TGFB2-mediated SMC differentiation. Lastly,a missense TGFB2 variant (TGFB2G276R/+) caused mechanical defects in SMC tissue ring constructs that were rescued by TGFB2 supplementation or genetic correction. Conclusion: Our data suggests the distinct regulation and action of TGFB2 in SMCs populating the aortic root,while redundant activities of TGFβ isoforms provide implications about the milder TAAD aggressiveness of pathogenic TGFB2 variants. View Publication

We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ,MMEJ),and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1),affecting several key DNA repair proteins,such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling,gene therapy,chimeric antigen receptor immunotherapy,and cancer treatment. DNA repair pathways shape CRISPR editing outcomes. Here,authors identified FDA approved drugs that can be repurposed as repair modulators or to induce synthetic lethality,and uncovered new roles for ESR2 and AOX1 in DNA repair,enhancing editing and offering potential therapeutic applications. View Publication

Chronic neuroinflammation plays a key role in the progression of Alzheimer’s disease (AD),and the cytosolic calcium-dependent phospholipase A2 (cPLA2) enzyme is a critical mediator of inflammatory lipid signaling pathways. Here we investigate the therapeutic potential of novel cPLA2 inhibitors in modulating neuroinflammation in AD. By leveraging the giga-scale V-SYNTHES2 virtual screening in on-demand chemical space and conducting two rounds of optimization for potency and selectivity,we have identified BRI-50460,achieving an IC50 of 0.88 nM in cellular assays that measure cPLA2-mediated arachidonic acid release. In vivo studies revealed favorable brain-to-plasma ratios,highlighting the ability of BRI-50460 to penetrate the central nervous system,modulating neuroinflammatory pathways,and restoring lipid homeostasis. In astrocytes and neurons derived from human induced pluripotent stem cells,BRI-50460 mitigates the effects of amyloid beta 42 oligomers on cPLA2 activation,tau hyperphosphorylation,and synaptic loss. Our results support that small molecule inhibitors of cPLA2 can modulate the downstream inflammatory signaling,offering a promising therapeutic strategy for neurodegenerative diseases. View Publication