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UNLABELLED Among the known genetic risk factors for Parkinson disease,mutations in GBA1,the gene responsible for the lysosomal disorder Gaucher disease,are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics,we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease,two with and two without parkinsonism,and one patient with Type 2 (acute neuronopathic) Gaucher disease,and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine,demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein,a protein present as aggregates in Parkinson disease and related synucleinopathies,were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607,a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme,restored glucocerebrosidase activity and protein levels,and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons,indicating its potential for treating neuronopathic Gaucher disease. In addition,NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism,suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease,dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity,reduced lysosomal glucocerebrosidase levels,and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype,the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone,which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition,the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons,indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease. View Publication

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However,methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately,these methods entail numerous challenges,including poor efficiency,variable cell type heterogeneity,and lengthy,expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol,these lines can be inducibly differentiated into either cortical (i3 Neurons) or lower motor neurons (i3 LMN) in a rapid,efficient,and scalable manner (Wang et al.,2017). In this manuscript,we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3 Neurons or i3 LMNs,and we present neuronal culture conditions for various experimental applications. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy,but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with {\textgreater}85{\%} purity in 10 days,using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes,we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies,including in vivo tissue regeneration models and drug screening. View Publication

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had textgreater1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization,two iPSC lines from each subject were selected,differentiated into postmitotic neurons,and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs,iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover,repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism. View Publication

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM),an embryonic germ layer that gives rise to kidneys. In order to achieve this goal,establishing an efficient,stable and low-cost method for differentiating IM cells using small molecules is required. In this study,we identified two retinoids,AM580 and TTNPB,as potent IM inducers by high-throughput chemical screening,and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator,CHIR99021,combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys,and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step,directly inducing IM cells by activating Wnt,retinoic acid (RA),and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy. 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

Mohr-Tranebjaerg syndrome (MTS) is a rare X-linked recessive neurodegenerative disorder caused by mutations in the Translocase of Inner Mitochondrial Membrane 8A (TIMM8A) gene,which encodes TIMM8a,a protein localized to the mitochondrial intermembrane space (IMS). The pathophysiology of MTS remains poorly understood. To investigate the molecular mechanisms underlying MTS,we established induced pluripotent stem cells (iPSCs) from a male MTS patient carrying a novel TIMM8A mutation (c.225-229del,p.Q75fs95*),referred to as MTS-iPSCs. To generate an isogenic control,we introduced the same mutation into healthy control iPSCs (CTRL-iPSCs) using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9),resulting in mutant iPSCs (MUT-iPSCs). We differentiated the three iPSC lines into neurons and evaluated their mitochondrial function and neuronal development. Both MTS- and MUT-iPSCs exhibited impaired neuronal differentiation,characterized by smaller somata,fewer branches,and shorter neurites in iPSC-derived neurons. Additionally,these neurons showed increased susceptibility to apoptosis under stress conditions,as indicated by elevated levels of cytochrome c and cleaved caspase-3. Mitochondrial function analysis revealed reduced protein levels and activity of complex IV,diminished ATP synthesis,and increased reactive oxygen species (ROS) generation in MTS- and MUT-neurons. Furthermore,transmission electron microscopy revealed mitochondrial fragmentation in MTS-neurons. RNA sequencing identified differentially expressed genes (DEGs) involved in axonogenesis,synaptic activity,and apoptosis-related pathways. Among these DEGs,coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2),which encodes a mitochondrial IMS protein essential for mitochondrial homeostasis,was significantly downregulated in MTS-neurons. Western blot analysis confirmed decreased CHCHD2 protein levels in both MTS- and MUT-neurons. Overexpression of CHCHD2 rescued mitochondrial dysfunction and promoted neurite elongation in MTS-neurons,suggesting that CHCHD2 acts as a downstream effector of TIMM8a in the pathogenesis of MTS. In summary,loss-of-function of TIMM8a leads to a downstream reduction in CHCHD2 levels,collectively impairing neurogenesis by disrupting mitochondrial homeostasis. TIMM8a mutation (p.Q75fs95*) leads to mitochondrial dysfunction and neuronal defects in iPSC-derived neurons from patient with Mohr-Tranebjaerg syndrome,which are rescued by overexpression of CHCHD2. TIMM8a translocase of inner mitochondrial membrane 8a,CHCHD2 coiled-coil-helix-coiled-coil-helix domain-containing protein 2,MTS Mohr-Tranebjaerg syndrome,I mitochondrial complex I,II mitochondrial complex II,III mitochondrial complex III,IV mitochondrial complex IV,Q coenzyme Q10,Cyt c cytochrome c. 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

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however,their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study,we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles,which,with a capacity of up to 36 kb,can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version,both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency,with sustained transgene expression observed for up to 110 days post-transduction. Moreover,we found efficient transduction of photoreceptors and Müller glial cells,without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However,an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction,suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery,further investigations are required to assess their long-term safety and efficacy. View Publication

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types,such as cardiac fibroblasts,into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore,the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs,incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis. View Publication

BackgroundSchizophrenia (SCZ) is a severe psychiatric disorder associated with alterations in early brain development. Details of underlying pathomechanisms remain unclear,despite genome and transcriptome studies providing evidence for aberrant cellular phenotypes and pathway deregulation in developing neuronal cells. However,mechanistic insight at the protein level is limited.MethodsHere,we investigate SCZ-specific protein expression signatures of neuronal progenitor cells (NPC) derived from patient iPSC in comparison to healthy controls using high-throughput Western Blotting (DigiWest) in a targeted proteomics approach.ResultsSCZ neural progenitors displayed altered expression and phosphorylation patterns related to Wnt and MAPK signaling,protein synthesis,cell cycle regulation and DNA damage response. Consistent with impaired cell cycle control,SCZ NPCs also showed accumulation in the G2/M cell phase and reduced differentiation capacity. Furthermore,we correlated these findings with elevated p53 expression and phosphorylation levels in SCZ patient-derived cells,indicating a potential implication of p53 in hampering cell cycle progression and efficient neurodevelopment in SCZ.ConclusionsThrough targeted proteomics we demonstrate that SCZ NPC display coherent mechanistic alterations in regulation of DNA damage response,cell cycle control and p53 expression. These findings highlight the suitability of iPSC-based approaches for modeling psychiatric disorders and contribute to a better understanding of the disease mechanisms underlying SCZ,particularly during early development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12888-024-06127-x. View Publication