搜索结果: 'ipsc'

The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this,we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that,although iHTNs maintain a fetal identity,they respond appropriately to metabolic hormones ghrelin and leptin. Notably,OBS iHTNs retained disease signatures and phenotypes of high BMI,exhibiting dysregulated respiratory function,ghrelin-leptin signaling,axonal guidance,glutamate receptors,and endoplasmic reticulum (ER) stress pathways. Thus,human iHTNs provide a powerful platform to study obesity and gene-environment interactions. View Publication

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However,marker-free genome editing using standard protocols remains inefficient,yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing,so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA),donor DNA and piggyBac transposase resulted in efficient,targeted genome editing and concurrent scarless transgene excision. Using this approach,in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%. View Publication

Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons,cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids,T lymphocytes,and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly,nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac,neurological,or other disease associations. Overall,PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling,and variant-preferred healthy control lines were identified for specific disease settings. View Publication

The molecular basis of the earliest neuronal changes that lead to Alzheimer's disease (AD) is unclear. Here,we analyze neural cells derived from sporadic AD (SAD),APOE4 gene-edited and control induced pluripotent stem cells (iPSCs). We observe major differences in iPSC-derived neural progenitor (NP) cells and neurons in gene networks related to neuronal differentiation,neurogenesis,and synaptic transmission. The iPSC-derived neural cells from SAD patients exhibit accelerated neural differentiation and reduced progenitor cell renewal. Moreover,a similar phenotype appears in NP cells and cerebral organoids derived from APOE4 iPSCs. Impaired function of the transcriptional repressor REST is strongly implicated in the altered transcriptome and differentiation state. SAD and APOE4 expression result in reduced REST nuclear translocation and chromatin binding,and disruption of the nuclear lamina. Thus,dysregulation of neural gene networks may set in motion the pathologic cascade that leads to AD. View Publication

Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However,to date,most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently,both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably,these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements,suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient,cost effective and scalable way. In summary,our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy. View Publication

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE),a cell monolayer essential for photoreceptor survival,and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD,which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD,including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins,and those from the AMD donors show significantly increased complement and inflammatory factors,which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening,combined with transcriptome analysis,we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome,ribosome,and chromatin-modifying genes. Thus,targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD. View Publication

This study describes the characterization of one induced pluripotent stem cell line (iPSC) from a healthy female. It is crucial to use iPSCs derived from healthy individuals as controls in genetic disease studies. Thus,we established a human iPSC cell line derived from healthy people. The iPSC cell line was generated in our lab from the peripheral blood mononuclear cells (PBMCs) of a 28-year-old girl. The generated hiPSC line is free of episomal vectors,has a normal karyotype,expresses pluripotency markers and can differentiate into three germ layers in vivo. View Publication

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN),the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4,a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein,thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells,the developmental ontology of the limb bud,or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs,lenalidomide,and pomalidomide,dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide,lenalidomide,and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen,all-trans retinoic acid (atRA). Finally,disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide,lenalidomide,and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents. View Publication

Becker muscular dystrophy (BMD) is caused by in-frame mutations in dystrophin gene,leading to progressive muscle weakness,and cardiac and respiratory complications. Currently,there is no cure. We have recently identified the importance of poly-ubiquitination in regulating dystrophin stability through the binding of lncRNA H19 to the dystrophin C-terminal zinc-finger domain (ZNF),inhibiting TRIM63-mediated poly-ubiquitination. We also demonstrated that BMD mutations lead to conformational changes in ZNF domain,reduced lncRNA H19 binding and increased dystrophin ubiquitination. Here we used BMD iPSCs to investigate the in vitro myogenic potential of BMD myogenic cells,as well as in vitro and in vivo studies to evaluate the therapeutic efficacy of three candidate molecules targeting dystrophin ubiquitination pathway. In vitro assays indicated significant deficiencies in myogenic cell differentiation of BMD iPSCs,including reduced proliferation,cell-cycle arrest,increased apoptosis,senescence,and membrane damage,and impaired myotube formation. In vivo engraftment demonstrated significant improvement in BMD iPSC myogenic cell survival and dystrophin expression in the animals treated with two molecules: a TRIM63 inhibitor and an ?-synuclein aggregation inhibitor. These findings provide promising evidence for the potential therapeutic efficacy of these ubiquitination pathway inhibitors to improve muscle progenitor cell survival and dystrophin expression in BMD patients. Graphical abstract Regulation of dystrophin stability via poly-ubiquitination is crucial in Becker muscular dystrophy (BMD). BMD mutations impair lncRNA H19 binding,increasing dystrophin ubiquitination. Darabi and colleagues’ studies,using BMD iPSCs and in vivo models,demonstrate that inhibiting TRIM63 or ?-synuclein aggregation improves myogenic cell survival and dystrophin expression,suggesting promising therapeutic avenues for BMD. View Publication

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor screens have enabled efficient production of a few cell types; however,engineering cell types that require complex transcription factor combinations remains challenging. Here,we report an iterative,high-throughput single-cell transcription factor screening method that enables the identification of transcription factor combinations for specialized cell differentiation,which we validated by differentiating human microglia-like cells. We found that the expression of six transcription factors,SPI1,CEBPA,FLI1,MEF2C,CEBPB,and IRF8,is sufficient to differentiate human iPSC into cells with transcriptional and functional similarity to primary human microglia within 4 days. Through this screening method,we also describe a novel computational method allowing the exploration of single-cell RNA sequencing data derived from transcription factor perturbation assays to construct causal gene regulatory networks for future cell fate engineering. Liu et al. developed a platform to identify transcription factors (TFs) that turn stem cells into desired cell types. They discovered six key TFs that produce microglia efficiently,enhancing cell differentiation methods. View Publication

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson’s disease,however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5’-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson’s patient midbrain cultures accumulate glucose and uridine-5’-triphosphate,while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme,GFPT2,through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate,while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological ?-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity,and its failure in Parkinson’s disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement. Reduced glucose flux via the hexosamine pathway contributes to lysosomal dysfunction and protein accumulation in Parkinson patient iPSC-neurons. Enhancing the hexosamine pathway rescues lysosome activity and restores proteostasis. View Publication

Microglia are the immune cells in the central nervous system (CNS) and become pro-inflammatory/activated in Alzheimer’s disease (AD). Cell surface glycosylation plays an important role in immune cells; however,the N-glycosylation and glycosphingolipid (GSL) signatures of activated microglia are poorly understood. Here,we study comprehensively combined transcriptomic and glycomic profiles using human induced pluripotent stem cells-derived microglia (hiMG). Distinct changes in N-glycosylation patterns in amyloid-? oligomer (A?O) and LPS-treated hiMG were observed. In A?O-treated cells,the relative abundance of bisecting N-acetylglucosamine (GlcNAc) N-glycans decreased,corresponding with a downregulation of MGAT3. The sialylation of N-glycans increased in response to A?O,accompanied by an upregulation of genes involved in N-glycan sialylation (ST3GAL4 and 6). Unlike A?O-induced hiMG,LPS-induced hiMG exhibited a decreased abundance of complex-type N-glycans,aligned with downregulation of mannosidase genes (MAN1A1,MAN2A2,and MAN1C1) and upregulation of ER degradation related-mannosidases (EDEM1-3). Fucosylation increased in LPS-induced hiMG,aligned with upregulated fucosyltransferase 4 (FUT4) and downregulated alpha-L-fucosidase 1 (FUCA1) gene expression,while sialofucosylation decreased,aligned with upregulated neuraminidase 4 (NEU4). Inhibition of sialylation and fucosylation in A?O- and LPS-induced hiMG alleviated pro-inflammatory responses. However,the GSL profile did not exhibit significant changes in response to A?O or LPS activation,at least in the 24-hour stimulation timeframe. A?O- and LPS- specific glycosylation changes could contribute to impaired microglia function,highlighting glycosylation pathways as potential therapeutic targets for AD.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-96596-1. View Publication