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Introduction: Africa,home to 1.4 billion people and the highest genetic diversity globally,harbors unique genetic variants crucial for understanding complex diseases like neurodegenerative disorders. However,African populations remain underrepresented in induced pluripotent stem cell (iPSC) collections,limiting the exploration of population-specific disease mechanisms and therapeutic discoveries. Methods: To address this gap,we established an open-access African Somatic and Stem Cell Bank. Results: In this initial phase,we generated 10 rigorously characterized iPSC lines from fibroblasts representing five Nigerian ethnic groups and both sexes. These lines underwent extensive profiling for pluripotency,genetic stability,differentiation potential,and Alzheimer's disease and Parkinson's disease risk variants. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology was used to introduce frontotemporal dementia-associated MAPT mutations (P301L and R406W). Discussion: This collection offers a renewable,genetically diverse resource to investigate disease pathogenicity in African populations,facilitating breakthroughs in neurodegenerative research,drug discovery,and regenerative medicine. Highlights: We established an open-access African Somatic and Stem Cell Bank. 10 induced pluripotent stem cell lines from five Nigerian ethnic groups were rigorously characterized. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology was used to introduce frontotemporal dementia-causing MAPT mutations. The African Somatic and Stem Cell Bank is a renewable,genetically diverse resource for neurodegenerative research. View Publication

BackgroundSpecific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases,including Parkinson’s disease (PD). However,the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here,according to the multiple-hit hypothesis,which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors,we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency,a genetic cause of PD,during lipopolysaccharide (LPS)-induced inflammation.MethodsUsing a combination of single-cell RNA-sequencing,bulk RNA-sequencing,multicolor flow cytometry and immunofluorescence analyses,we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives,we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs).ResultsBy excluding the contribution of other immune brain resident and peripheral cells,we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype,specially related to type II interferon and DNA damage response signaling,when compared with wildtype microglia,in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level,with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice,both at 6 and 24 h after acute inflammation,as also observed in BMDMs.ConclusionsTaken together,our results show that,under inflammatory conditions,PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses,which may play a causative role in PD onset and progression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03164-x. View Publication

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. Scalable CRISPRa screening of cis-regulatory elements in non-cancer cell lines has proved challenging. Here,the authors describe a scalable,CRISPR activation screening framework to identify regulatory element-gene pairs in diverse cell types including cancer cells and neurons. View Publication

Neutrophils are critical for host defense against fungi. However,the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here,we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation,antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal ?-glucan,including an upregulation of the pentose phosphate pathway (PPP),similar to primary human neutrophils in vitro. Furthermore,we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively,these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections,and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation,limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus,demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively,these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity. View Publication

Evolutionary innovations can be driven by changes in the rates of RNA translation and the emergence of new genes and small open reading frames (sORFs). In this study,we characterized the transcriptional and translational landscape of the hearts of four primate and two rodent species through integrative ribosome and transcriptomic profiling,including adult left ventricle tissues and induced pluripotent stem cell-derived cardiomyocyte cell cultures. We show here that the translational efficiencies of subunits of the mitochondrial oxidative phosphorylation chain complexes IV and V evolved rapidly across mammalian evolution. Moreover,we discovered hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution in the heart,including 551 genes,504 sORFs and 76 evolutionarily conserved genes displaying human-specific cardiac-enriched expression. Overall,our work describes the evolutionary processes and mechanisms that have shaped cardiac transcription and translation in recent primate evolution and sheds light on how these can contribute to cardiac development and disease. Ruiz-Orera et al. used comparative transcriptomics and translatomics to analyze the cardiac evolution in primates and discovered species-specific and lineage-specific genomic innovations that might contribute to cardiac development and disease. View Publication

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 Friedreich’s ataxia. View Publication

Diabetes mellitus,a chronic and non-transmissible disease,triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ?-like cells (P?LCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways,directing the differentiation of iPSCs into pancreatic ? cells. Despite significant progress and improved protocols,the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here,we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into P?LCs. Our protocol has shown to be effective,with the resulting cells exhibiting key functional properties of pancreatic ? cells,including the expression of crucial molecular markers (pdx1,nkx6.1,ngn3) and the capability to secrete insulin in response to glucose. Furthermore,the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ? cell genes. View Publication

SummaryCell size is a crucial physical property that significantly impacts cellular physiology and function. However,the influence of cell size on stem cell specification remains largely unknown. Here,we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly,cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore,the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements,we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically,the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation,which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together,our study has established a novel connection between cell size diminution and DE differentiation,which is mediated by AMOT nuclear translocation. Additionally,our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation. Graphical abstract Highlights•Cell size decreases during the differentiation of human pluripotent stem cells into endoderm•Hypertonic pressure is conducive to the differentiation of human definitive endoderm•Actomyosin contributes to both size diminution and endoderm promotion under hypertonic pressure•Cell size diminution represses YAP activity via promoting AMOT nuclear translocation Jiang and colleagues show that cell size exhibits a gradual decrease during human endoderm differentiation. The application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced endoderm differentiation. This enhancement is reliant on actomyosin activity and achieved by promoting the nuclear translocation of AMOT,thereby repressing YAP activity. View Publication

Disease modeling with isogenic Induced Pluripotent Stem Cell (iPSC)-differentiated organoids serves as a powerful technique for studying disease mechanisms. Multiplexed coculture is crucial to mitigate batch effects when studying the genetic effects of disease-causing variants in differentiated iPSCs or organoids,and demultiplexing at the single-cell level can be conveniently achieved by assessing natural genetic barcodes. Here,to enable cost-efficient time-series experimental designs via multiplexed bulk and single-cell RNA-seq of hybrids,we introduce a computational method in our Vireo Suite,Vireo-bulk,to effectively deconvolve pooled bulk RNA-seq data by genotype reference,and thereby quantify donor abundance over the course of differentiation and identify differentially expressed genes among donors. Furthermore,with multiplexed scRNA-seq and bulk RNA-seq,we demonstrate the usefulness and necessity of a pooled design to reveal donor iPSC line heterogeneity during macrophage cell differentiation and to model rare WT1 mutation-driven kidney disease with chimeric organoids. Our work provides an experimental and analytic pipeline for dissecting disease mechanisms with chimeric organoids. IPSC-derived organoids model diseases. Multiplexed coculture and demultiplexing natural genetic barcodes aid in studying genetic effects. Here,authors introduce Vireo-bulk to deconvolve bulk RNA-seq data,quantify donor abundance and identify differentially expressed genes. View Publication

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors,CoREST1,CoREST2,or CoREST3 (encoded by RCOR1,RCOR2,and RCOR3,respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST,in various neural and glial cell types during different developmental stages. However,there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap,we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons,as well as mature functional NGN2-induced neurons. Additionally,a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary,our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically,RCOR3 expression increases in neurons,while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans. Graphical abstractImage 1 Highlights•REST and RCOR genes display cell-type specific expression patterns in neural cells.•RCOR3 (encodes CoREST3) is upregulated during neuronal and astrocyte differentiation.•RCOR2 (encodes CoREST2) is downregulated during differentiation of astrocytes.•Evidence of potential cell-type specific functions of the CoREST family. View Publication

High-throughput massively parallel reporter assays (MPRAs) and phenotype-rich in vivo transgenic mouse assays are two potentially complementary ways to study the impact of noncoding variants associated with psychiatric diseases. Here,we investigate the utility of combining these assays. Specifically,we carry out an MPRA in induced human neurons on over 50,000 sequences derived from fetal neuronal ATAC-seq datasets and enhancers validated in mouse assays. We also test the impact of over 20,000 variants,including synthetic mutations and 167 common variants associated with psychiatric disorders. We find a strong and specific correlation between MPRA and mouse neuronal enhancer activity. Four out of five tested variants with significant MPRA effects affected neuronal enhancer activity in mouse embryos. Mouse assays also reveal pleiotropic variant effects that could not be observed in MPRA. Our work provides a catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays. MPRAs and in vivo transgenic mouse assays are two potentially complementary ways to assay the impact of noncoding variants. Here,authors find a strong and specific correlation between the assays in neural cells. Mouse assays also reveal pleiotropic effects not observed in MPRA. View Publication

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants,including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons,which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS,and TDP-43 modulates several mitochondrial transcripts. In this study,we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons,linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected,which correlated with downregulation of the motor protein complex,DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport,which can be partially rescued by DCTN1 overexpression. Dafinca et al. show that mutations in TDP-43 lead to decreased mitochondrial oxidative phosphorylation,partially due to interactions with the ATP production machinery and COX5A. These have direct effects on axonal transport,which is reduced in amyotrophic lateral sclerosis motor neurons,and overexpression of dynactin-1 significantly increases retrograde mitochondrial dynamics. View Publication