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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

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

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

The differentiation of pluripotent stem cells into neurons is an essential area of biomedical research,with significant implications for understanding neural development and treating neurological diseases. This study compares neural cultures derived from a common induced pluripotent stem cell line (KYOU-DXR0109B) generated by two widely adopted methods: DUAL SMAD inhibition and exogenous NGN2 overexpression. The DUAL SMAD inhibition method,which differentiates through the neural stem cell stage,produces heterogeneous cultures containing a mix of neurons,neural precursors,and glial cells. Conversely,NGN2 overexpression generates more homogeneous cultures composed predominantly of mature neurons. Transcriptomic analysis revealed significant differences in neural gene markers expression profiles,with cultures from the DUAL SMAD inhibition method enriched in neural stem cell and glial markers,while NGN2 overexpression cultures showed elevated markers for cholinergic and peripheral sensory neurons. This study underscores the importance of choosing appropriate differentiation protocols based on the desired cell types,as each method yields neural cultures with distinct cellular compositions. Understanding these differences can help optimize protocols for specific research and therapeutic applications. 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

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

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases,including those with genetic origins. Currently,primary ECs are the main source for disease modelling in this field. However,they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level,we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor,known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level,which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly,the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability,while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary,we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09925-9. View Publication

A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle,midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved,post-mitotic iPSC-mDA neurons retained high viability with gene,protein,and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy,cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth,supporting the translational development of pluripotent cell-based therapies in PD. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines,we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene,and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines,even in the absence of patient tissue. 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

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) have greater potential for generating chondrocytes without hypertrophic and fibrotic phenotypes compared to bone marrow-derived mesenchymal stem/stromal cells (BMSCs). However,there is a lack of research demonstrating the use of autologous iMSCs for repairing articular chondral lesions in large animal models. In this study,we aimed to evaluate the effectiveness of autologous miniature pig (minipig) iMSC-chondrocyte (iMSC-Ch)-laden implants in comparison to autologous BMSC-chondrocyte (BMSC-Ch)-laden implants for cartilage repair in porcine femoral condyles. iMSCs and BMSCs were seeded into fibrin glue/nanofiber constructs and cultured with chondrogenic induction media for 7 days before implantation. To assess the regenerative capacity of the cells,19 skeletally mature Yucatan minipigs were randomly divided into microfracture control,acellular scaffold,iMSC,and BMSC subgroups. A cylindrical defect measuring 7 mm in diameter and 0.6 mm in depth was created on the articular cartilage surface without violating the subchondral bone. The defects were then left untreated or treated with acellular or cellular implants. Both cellular implant-treated groups exhibited enhanced joint repair compared to the microfracture and acellular control groups. Immunofluorescence analysis yielded significant findings,showing that cartilage treated with iMSC-Ch implants exhibited higher expression of COL2A1 and minimal to no expression of COL1A1 and COL10A1,in contrast to the BMSC-Ch-treated group. This indicates that the iMSC-Ch implants generated more hyaline cartilage-like tissue compared to the BMSC-Ch implants. Our findings contribute to filling the knowledge gap regarding the use of autologous iPSC derivatives for cartilage repair in a translational animal model. Moreover,these results highlight their potential as a safe and effective therapeutic strategy. The online version contains supplementary material available at 10.1186/s13287-025-04215-7. View Publication