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Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle,two orthogonal strategies for reducing off-target cleavage,truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs),could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally,we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing. View Publication

Conventional generation of stem cells from human blastocysts produces a developmentally advanced,or primed,stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However,whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here,we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration,global gene expression,and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. View Publication

Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic,stiffer matrices that mimic muscle are myogenic,and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture,reprogramming of these lineages is possible with addition of soluble induction factors,but after several weeks in culture,the cells commit to the lineage specified by matrix elasticity,consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells. View Publication

Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However,existing approaches to cardiomyocyte production from human iPS cells are inefficient,limiting the application of iPS cells in basic and translational cardiac research. Furthermore,strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging,we showed that these induced cardiomyocytes expressed typical sarcomeric markers,exhibited normal rhythmic Ca(2+) transients,and responded to both β-adrenergic and electric stimulation. Furthermore,human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus,modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct,rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface,and their removal from CM impairs proliferation. Finally,we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography,immunoblotting,and mass spectrometry-based proteomic analysis,we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture. View Publication

Although a number of growth factors have been shown to be involved in neurogenesis,the role of inflammatory cytokines remains relatively unexplored in the normal brain. Here we investigated the effect of interferon gamma (IFNgamma) in the regulation of neural precursor (NP) activity in both the developing and the adult mouse brain. Exogenous IFNgamma inhibited neurosphere formation from the wild-type neonatal and adult subventricular zone (SVZ). More importantly,however,these effects were mirrored in vivo,with mutant mice lacking endogenous IFNgamma displaying enhanced neurogenesis,as demonstrated by an increase in proliferative bromodeoxyuridine-labeled cells in the SVZ and an increased percentage of newborn neurons in the olfactory bulb. Furthermore,NPs isolated from IFNgamma null mice exhibited an increase in self-renewal ability and in the capacity to produce differentiated neurons and oligodendrocytes. These effects resulted from the direct action of IFNgamma on the NPs,as determined by single-cell assays and the fact that nearly all the neurospheres were derived from cells positive for major histocompatibility complex class I antigen,a downstream marker of IFNgamma-mediated activation. Moreover,the inhibitory effect was ameliorated in the presence of SVZ-derived microglia,with their removal resulting in almost complete inhibition of NP proliferation. Interestingly,in contrast to the results obtained in the adult,exogenous IFNgamma treatment stimulated neurosphere formation from the embryonic brain,an effect that was mediated by sonic hedgehog. Together these findings provide the first direct evidence that IFNgamma acts as a regulator of the active NP pool in the non-inflammatory brain. View Publication

Hantaviruses are zoonotically transmitted from rodents to humans through the respiratory route,with no currently approved antivirals or widely available vaccines. The recent discovery of interhuman-transmitted Andes virus (ANDV) necessitates the systematic identification of cell tropism,infective potential,and potent therapeutic agents. We utilized human primary lung endothelial cells,various pluripotent stem cell-derived heart and brain cell types,and established human lung organoid models to evaluate the tropisms of Old World Hantaan (HTNV) and New World ANDV and Sin Nombre (SNV) viruses. ANDV exhibited broad tropism for all cell types assessed. SNV readily infected pulmonary endothelial cells,while HTNV robustly amplified in endothelial cells,cardiomyocytes,and astrocytes. We also provide the first evidence of hantaviral infection in human 3D distal lung organoids,which effectively modeled these differential tropisms. ANDV infection transcriptionally promoted cell injury and inflammatory responses,and downregulated lipid metabolic pathways in lung epithelial cells. Evaluation of selected drug candidates and pharmacotranscriptomics revealed that the host-directed small molecule compound urolithin B inhibited ANDV infection and restored cellular metabolism with minimal changes in host transcription. Given the scarcity of academic BSL-4 facilities that enable in vivo hantaviral studies,this investigation presents advanced human cell-based model systems that closely recapitulate host cell tropism and responses to infection,thereby providing critical platforms to evaluate potential antiviral drug candidates. Author summaryHantaviruses are fatal human pathogens that cause hemorrhagic fevers and are classified into either Old World or New World groups. Though most hantaviruses utilize zoonotic transmission,the New World Andes virus (ANDV) is unique in its ability to spread between humans. This distinct transmission mode underscores the need to investigate its cell tropism,pathogenicity,and therapeutic targets. Thus,we performed a systems-level comparison of the Old World Hantaan virus (HTNV) and New World hantaviruses,ANDV and Sin Nombre virus (SNV),using human lung,heart,and brain cell models,alongside lipidomic and transcriptomic profiling. We observed that ANDV exhibits broad tropism,infecting all tested cell types,including lung epithelial cells. HTNV replicated in lung endothelial,heart,and brain cells,whereas SNV replication was largely confined to lung endothelial cells. Notably,ANDV infection induced stronger host transcriptional changes,promoted cell injury and inflammatory responses,and suppressed lipid metabolic pathways in lung epithelial cells. Further drug testing and pharmacotranscriptomic analysis identified effective inhibitors of ANDV infection,including urolithin B,that restored cellular metabolism with minimal transcriptional disruption. This study provides a comparative framework for understanding hantavirus cell tropism and host responses and highlights potential antiviral candidates for treating these severe viral infections. View Publication

IntroductionAmid the persistent threat of future pandemics,the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions,highlighting the need for rapid and adaptable approaches to respond to immune escape variants.MethodsHere,we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.ResultsImmortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors,yielding transduction efficiencies of 67.5% for PBMCs and 50.2% for tonsil-derived cells. Analysis revealed that immortalized B cell libraries produced with this method retain diverse immunoglobulin isotype representations. Through high-throughput functional screening of approximately 40,000 B cells per library,we identified 12 unique clones with neutralization activity for SARS-CoV-2,leading to selection of monoclonal antibodies with robust neutralization activity against Delta and BA.5 variants. We applied our directed evolution approach to libraries generated by ex vivo AID-induced somatic hypermutation (SHM) of immortalized B cell clones to enhance the affinity and cross-reactivity,resulting in improved binding and neutralization potency to escape variants such as EG.5.1 and JN.1. Furthermore,we engineered a bi-paratopic antibody combining KBA2401,a broadly neutralizing antibody binding to highly conserved epitope on Spike-RBD,and KBA2402,a broadly binding non-neutralizing antibody,resulting in enhanced potency against SARS-CoV-2 variant JN.1 and KP.3.DiscussionOur findings illustrate the use of immortalized B cell libraries for development of therapeutics that adapt to viral evolution and highlight the application of ex vivo directed evolution in refining antibody responses against emerging immune escape SARS-CoV-2 variants. The approach here described offers a promising pathway for rapid therapeutic development in the face of evolving viral threats. View Publication

The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here,we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo,thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies,we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes,resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin,we show resistance of engrafted,multiplex edited human cells in vivo,and a 2-fold enrichment for edited cells in vitro. Together,our results highlight the potential of adenine base editors for improved immune and gene therapies. Subject terms: Haematopoietic stem cells,Bone marrow transplantation,Cell biology View Publication

Reduced serum testosterone (T),or hypogonadism,affects millions of men and is associated with many pathologies,including infertility,cardiovascular diseases,metabolic syndrome,and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However,TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus,there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs),proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs,the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively,human induced pluripotent stem cells (hiPSCs),which are expandable in culture and have the potential to differentiate into all somatic cell types,have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis,synthesized T rather than cortisol,secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol,and displayed ultrastructural features resembling LCs. By contrast,hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration. View Publication

Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy,and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro,in vivo (in female mice),and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors,with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore,STINGa ADCs induce type III interferons,which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs. Activation of the STING pathway can promote anti-tumor immunity. Here the authors generate tumor cell-directed STING agonist antibody-drug conjugates that activate STING in tumor and myeloid cells,promoting anti-tumor innate immune responses in preclinical cancer models. View Publication