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Background: Alzheimer's disease (AD) is characterized by progressive amyloid beta (Aβ) deposition in the brain,with eventual widespread neurodegeneration. While the cell-specific molecular signature of end-stage AD is reasonably well characterized through autopsy material,less is known about the molecular pathways in the human brain involved in the earliest exposure to Aβ. Human model systems that not only replicate the pathological features of AD but also the transcriptional landscape in neurons,astrocytes and microglia are crucial for understanding disease mechanisms and for identifying novel therapeutic targets. Methods: In this study,we used a human 3D iPSC-derived neurosphere model to explore how resident neurons,microglia and astrocytes and their interplay are modified by chronic amyloidosis induced over 3-5 weeks by supplementing media with synthetic Aβ1 - 42 oligomers. Neurospheres under chronic Aβ exposure were grown with or without microglia to investigate the functional roles of microglia. Neuronal activity and oxidative stress were monitored using genetically encoded indicators,including GCaMP6f and roGFP1,respectively. Single nuclei RNA sequencing (snRNA-seq) was performed to profile Aβ and microglia driven transcriptional changes in neurons and astrocytes,providing a comprehensive analysis of cellular responses. Results: Microglia efficiently phagocytosed Aβ inside neurospheres and significantly reduced neurotoxicity,mitigating amyloidosis-induced oxidative stress and neurodegeneration following different exposure times to Aβ. The neuroprotective effects conferred by the presence of microglia was associated with unique gene expression profiles in astrocytes and neurons,including several known AD-associated genes such as APOE. These findings reveal how microglia can directly alter the molecular landscape of AD. Conclusions: Our human 3D neurosphere culture system with chronic Aβ exposure reveals how microglia may be essential for the cellular and transcriptional responses in AD pathogenesis. Microglia are not only neuroprotective in neurospheres but also act as key drivers of Aβ-dependent APOE expression suggesting critical roles for microglia in regulating APOE in the AD brain. This novel,well characterized,functional in vitro platform offers unique opportunities to study the roles and responses of microglia to Aβ modelling key aspects of human AD. This tool will help identify new therapeutic targets,accelerating the transition from discovery to clinical applications. View Publication

Backgroundc-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers,c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development,its impact on nervous system development in higher mammals,especially for some deep structures,for example,thalamus in diencephalon,remains unclear.MethodsTo investigate the influence of c-JUN on early nervous system development,c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs),cerebral organoids (COs),and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining,omics analysis,and loss/gain of function analysis.ResultsAt embryonic day 14.5,c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6+/NESTIN+ exterior layer but lacked a fibers-connected core. Moreover,the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably,in guided ThOs,c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2,WNT8B,MXRA5,HSPG2 and PLAGL1 while 24 genes including MSX1,CYP1B1,LMX1B,NQO1 and COL2A1 were significantly up-regulated.ConclusionOur findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01303-8. View Publication

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors,enabling the translation of this approach to clinical trials,are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM,MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect,replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells,but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly,ZIKV rapidly replicated in GBM,MED,and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover,ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether,our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However,such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects,suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV. View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study,we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs,this method prioritizes functional noncoding variants using deep learning,links cis-regulatory elements (CREs) to target genes using epigenomics data,and integrates these data through gene-level burden tests to identify survival-modifying variants,CREs,and genes. We apply this approach to analyze 6,715 ALS genomes,and pinpoint four novel rare noncoding variants associated with survival,including chr7:76,009,472:C>T linked to CCDC146. CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes,including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO),showing no toxicity,completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72. ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases. View Publication

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research,with great potential for the use of hPSC-derived RGCs for studies of human retinal development,in vitro disease modeling,drug discovery,as well as their potential use for cell replacement therapeutics. Of all these possibilities,the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention,due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies,many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus,to further advance this field of research,in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein,which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK,along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs,and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs,while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together,these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1,contributing to the degeneration of RGCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01872-2. View Publication

ObjectiveThyroid hormone (TH) action is mediated by thyroid hormone receptor (THR) isoforms. While THR?1 is likely the main isoform expressed in liver,its role in human hepatocytes is not fully understood.MethodsTo elucidate the role of THR?1 action in human hepatocytes we used CRISPR/Cas9 editing to knock out THR?1 in induced pluripotent stem cells (iPSC). Following directed differentiation to the hepatic lineage,iPSC-derived hepatocytes were then interrogated to determine the role of THR?1 in ligand-independent and -dependent functions.ResultsWe found that the loss of THR?1 promoted alterations in proliferation rate and metabolic pathways regulated by T3,including gluconeogenesis,lipid oxidation,fatty acid synthesis,and fatty acid uptake. We observed that key genes involved in liver metabolism are regulated through both T3 ligand-dependent and -independent THR?1 signaling mechanisms. Finally,we demonstrate that following THR?1 knockout,several key metabolic genes remain T3 responsive suggesting they are THR? targets.ConclusionsThese results highlight that iPSC-derived hepatocytes are an effective platform to study mechanisms regulating TH signaling in human hepatocytes. Graphical abstractImage 1 Highlights•THR?1 is essential for T3 effects in human iPSC-derived hepatocytes (iHEPs).•THR?1 knockout reduces iPSC and progenitor cell proliferative capacity.•T3 regulates key genes involved in lipid and carbohydrate metabolism through THR?1.•THR?1 plays a strong ligand-independent role. View Publication

Diabetic retinopathy (DR) is the leading cause of visual impairment,particularly in the proliferative form (proliferative DR [PDR]). The impact of the PDR microenvironment on microglia,which are the resident immune cells in the central nervous system,and the specific pathological changes it may induce remain unclear. This study aimed to investigate the role of microglia in the progression of PDR under hypoxic and inflammatory conditions. We performed a comprehensive gene expression analysis using human-induced pluripotent stem cell-derived microglia under different stimuli (dimethyloxalylglycine (DMOG),lipopolysaccharide (LPS),and DMOG + LPS) to mimic the hypoxic inflammatory environment characteristic of PDR. Principal component analysis revealed distinct gene expression profiles,with 76 genes synergistically upregulated under combined stimulation. Notably,prostaglandin-endoperoxide synthase 2 (encoding cyclooxygenase (COX)-2) exhibited the most pronounced increase,leading to elevated prostaglandin E2 (PGE2) levels and driving pathological angiogenesis and inflammation via the COX-2/PGE2/PGE receptor 2 signaling axis. Additionally,the upregulation of the fibrogenic genes snail family transcriptional repressor 1 and collagen type I alpha 1 chain suggested a role for microglia in fibrosis. These findings underscore the critical involvement of microglia in PDR and suggest that targeting both the angiogenic and fibrotic pathways may present new therapeutic strategies for managing this condition. View Publication

Genetic and experimental evidence suggests that Alzheimer’s disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages,including microglia,to damage of lipid-rich tissues like the brain. Recently,sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues,we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors,BHLHE40/41,in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia,resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing,increased cholesterol efflux and storage,and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues. Factors regulating lipid and lysosomal clearance in microglia and peripheral macrophage are not known. Here,authors nominate and validate transcription factors BHLHE40 and BHLHE41 as regulators of these processes in health and disease. View Publication

IntroductionEmerging biotechnologies are increasingly being explored for food production,including the development of cell-cultivated meat. Conventional approaches typically rely on satellite cell (SC) biopsies,which present challenges in scalability. Bovine induced pluripotent stem cells (biPSCs) represent a promising alternative due to their capacity for self-renewal and developmental plasticity.MethodsThis study utilized both lentiviral (integrating) and episomal (non-integrating) reprogramming strategies to generate biPSCs suitable for myogenic differentiation. Bovine fetal fibroblasts (bFFs) were reprogrammed using episomal vectors pMaster K and pCXB-EBNA1,leading to the emergence of putative iPSC colonies 13 days post-nucleofection. A clonal line,bFF-iPSCs pMK,was selected for further analysis.ResultsThe bFF-iPSCs pMK line expressed key pluripotency markers including alkaline phosphatase (AP),OCT4,SOX2,and NANOG,and was stably maintained for over 33 passages,although episomal plasmids remained detectable. in vitro myogenic differentiation was assessed by comparing this line to a previously established lentiviral reprogrammed line (bFF-iPSCs mOSKM). Both lines exhibited downregulation of pluripotency markers and upregulation of the early myogenic marker PAX3. By day 30,the bFF-iPSCs pMK line formed elongated,multinucleated cells characteristic of myotubes and displayed a corresponding gene expression profile.DiscussionThese results provide new insights into bovine in vitro myogenesis and its application in cultured meat production. While promising,the study also highlights the difficulty in achieving complete myogenic differentiation,indicating a need for further optimization of differentiation protocols. Graphical abstract View Publication

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm,leading to death by respiratory failure. Human experimental models to study phMNs are lacking,hindering the understanding of the mechanisms of phMN degeneration in ALS. Here,we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development,phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels,under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine,followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines,offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research,it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest. View Publication

Mutations in GBA1 encoding the lysosomal enzyme ?-glucocerebrosidase (GCase) are among the most prevalent genetic susceptibility factors for Parkinson’s disease (PD),with 10–30% of carriers developing the disease. To identify genetic modifiers contributing to the incomplete penetrance,we examined the effect of 1634 human transcription factors (TFs) on GCase activity in lysates of an engineered human glioblastoma line homozygous for the pathogenic GBA1 L444P variant. Using an arrayed CRISPR activation library,we uncovered 11 TFs as regulators of GCase activity. Among these,activation of MITF and TFEC increased lysosomal GCase activity in live cells,while activation of ONECUT2 and USF2 decreased it. While MITF,TFEC,and USF2 affected GBA1 transcription,ONECUT2 might control GCase trafficking. The effects of MITF,TFEC,and USF2 on lysosomal GCase activity were reproducible in iPSC-derived neurons from PD patients. Our study provides a systematic approach to identifying modulators of GCase activity and deepens our understanding of the mechanisms regulating GCase. View Publication

CRISPR-Cas9-mediated gene editing has vast applications in basic and clinical research and is a promising tool for several disorders. Our lab previously developed a non-integrating RNA virus,measles virus (MeV),as a single-cycle reprogramming vector by replacing the viral attachment protein with the reprogramming factors for induced pluripotent stem cell generation. Encouraged by the MeV reprogramming vector efficiency,in this study,we develop a single-cycle MeV vector to deliver the gRNA(s) and Cas9 nuclease to human cells for efficient gene editing. We show that the MeV vector achieved on-target gene editing of the reporter (mCherry) and endogenous genes (HBB and FANCD1) in human cells. Additionally,the MeV vector achieved precise knock-in via homology-directed repair using a single-stranded oligonucleotide donor. The MeV vector is a new and flexible platform for gene knock-out and knock-in modifications in human cells,capable of incorporating new technologies as they are developed. Graphical abstract Devaux and colleagues developed a novel single-cycle measles vector allowing gene editing of human cells. They show that Measles can express the CRISPR-Cas9 and gRNA from one genome. Finally,they demonstrate that these vectors can efficiently perform KO and knock-in in human cells without excessive off-target effects. View Publication