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Results for “Human induced pluripotent stem cells”

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At least 19 recordsLinked to original sources
PubMed2027

Generation of Transgene-Free Naive Human Induced Pluripotent Stem Cells from Somatic Cells Using a Modified Temperature-Sensitive Sendai Virus System.

The Sendai virus (SeV) vector system offers an efficient, nonintegrating approach to reprogram somatic cells into either naive or primed human induced pluripotent stem cells (iPSCs). Here, we describe a protocol to generate transgene-free naive iPSCs from human dermal fibroblasts (HDFs) and peripheral blood mononuclear cells (PBMCs) using a modified, temperature-sensitive SeV system. The method leverages LMYC in place of cMYC and an optional H1FOO-DD factor to enhance efficiency and uniformity, and employs a controlled temperature shift to facilitate vector clearance.

Humans↗
PubMed2026

CRISPR/Cpf1-mediated knockout of FLG in human induced pluripotent stem cells generates a model for studying epidermal barrier dysfunction.

Loss of filaggrin (FLG) function impairs skin barrier formation and contributes to common inflammatory skin diseases. In this study, we established a FLG knockout human induced pluripotent stem cell (iPSC) line based on KOLF2.1 J using CRISPR/Cas12a (Cpf1)-mediated genome editing. A guide RNA targeting exon 2 introduced a homozygous mutation, which was confirmed by sequencing. The edited cells maintained typical pluripotent stem cell morphology, expressed key undifferentiated markers, and retained the ability to differentiate into all three germ layers. Karyotype and copy number variation (CNV) analyses confirmed genomic stability and parental origin; the cells were free of mycoplasma. This cell line enables studies of FLG-associated skin biology and pathology.

Humans↗
PubMed2026

Generation of Aneuploid Human Induced Pluripotent Stem Cells from Primary Amniotic Fluid Cells via Episomal Plasmid Electroporation.

The generation of patient-specific induced pluripotent stem cells (iPSCs) from amniotic fluid cells (AFCs) carrying defined chromosomal aneuploidies provides a powerful platform for modeling genetic disorders. However, establishing a reliable and reproducible reprogramming pipeline for aneuploid AFCs remains technically challenging due to the intrinsic genomic instability and variable proliferative capacity of these cells. Here, we present a comprehensive, non-integrating method for generating aneuploid human iPSCs from primary AFCs using episomal plasmid electroporation. This protocol details the complete workflow, encompassing cell thawing and expansion with a gradual media adaptation strategy, optimized plasmid delivery via electroporation system, sequential post-electroporation culture with mesenchymal-to-epithelial transition (MET)-directed media changes, and mechanical colony picking based on defined morphological criteria. We further describe validation procedures, including immunofluorescence staining for core pluripotency markers, G-banding karyotype analysis to confirm aneuploid karyotype maintenance, and PCR-based episomal vector clearance verification. This feeder-free, integration-free protocol yields aneuploid iPSC lines suitable for disease modeling, drug screening, and studies of chromosome biology.

Humans↗
PubMed2026

Establishment of human induced pluripotent stem cell lines and isogenic gene-corrected controls from three patients with prolidase deficiency.

Prolidase deficiency is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the PEPD gene. To date, close to 200 patients have been reported worldwide with a poorly understood pathomechanism. The PEPD gene encodes an enzyme that is involved in the final steps of collagen degradation. Urine amino acid analysis or specific dipeptide analysis can establish the biochemical diagnosis. In this study, we reprogrammed peripheral blood mononuclear cells (PBMCs) from three prolidase deficient patients into induced pluripotent stem cell (iPSC) lines and additionally generated isogenic controls using CRISPR-Cas9 genome editing. The pathogenic PEPD variants identified in our patients were NP_000276.2:p.? (NIHTVBi032-A), NP_000276.2:p.(Ile415Asn)/NP_000276.2:p.(Trp326Ter) (NIHTVBi033-A), and NP_000276.2:p.(Arg265Ter) (NIHTVBi034-A). These iPSC lines are valuable models to help investigate the pathomechanism of prolidase deficiency.

Humans↗
PubMed2026

Establishment of a human induced pluripotent stem cell line, KMUGMCi011-A, from a patient bearing a frameshift mutation in the KMT2D gene leading Kabuki syndrome 1.

Kabuki syndrome 1 is a rare genetic disorder typically characterized by facial abnormalities, cognitive impairment, developmental delay and organ dysfunction. It is caused by a loss-of-function mutation in the KMT2D gene. The peripheral blood mononuclear cells from a patient carrying frameshift mutation in the KMT2D gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. This frameshift mutation results in a truncated protein. This established human induced pluripotent cell line will allow proper in vitro disease modelling of Kabuki syndrome 1.

Journal Article↗
PubMed2025

Pre-clinical safety and efficacy of human induced pluripotent stem cell-derived products for autologous cell therapy in Parkinson's disease.

Human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic cells (mDACs) represent a promising source for autologous cell therapy in Parkinson's disease (PD), but standardized regulatory criteria are essential for clinical translation. In this pre-clinical study, we generated multiple clinical-grade hiPSC lines from freshly biopsied fibroblasts of four sporadic PD patients using episomal reprogramming and differentiated them into mDACs using a refined 21-day protocol. Rigorous evaluations included whole-genome/exome sequencing, RNA sequencing, and in vivo studies, including a 39-week Good Laboratory Practice-compliant mouse safety study. While mDACs from all lines met safety criteria, mDACs from one patient failed to improve rodent behavioral outcomes, underscoring inter-individual variability. Importantly, in vitro assessments did not reliably predict in vivo efficacy, identifying dopaminergic fiber density as a key efficacy criterion. These findings support comprehensive quality control guidelines for autologous cell therapy and pave the way for a clinical trial with eight sporadic PD patients, scheduled to commence in 2025.

Humans↗
PubMed2025

High-Sensitivity Top-Down Proteomics Reveals Enhanced Maturation of Micropatterned Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for disease modeling, drug discovery, and precision medicine, yet their utility is often limited by their immature phenotype. One promising maturation strategy involves using micropatterned substrates that mimic native cardiomyocytes' organizational growth and stiffness. However, the maturity of this model has not fully been assessed, and there is currently no method to extract proteins from micropatterned hiPSC-CMs for top-down proteomic analysis. Herein, we present a high-sensitivity protein extraction protocol for top-down proteomic analysis of hiPSC-CMs. Through this method, we assessed the maturation of micropatterned hiPSC-CMs compared to traditional monoculture and coculture monolayers at the proteoform level. We found that micropatterned hiPSC-CMs display molecular signatures of cardiomyocyte maturation including increased expression of ventricular myosin light chain isoforms, reduced expression of the fetal troponin T isoform, and decreased phosphorylation of alpha-tropomyosin. This high-sensitivity approach enables robust top-down proteomics from limited, heterogeneous cell populations and identifies the micropattern hiPSC-CM as a more adult-like CM model, broadening the utility of structured culture systems for cardiac disease modeling and translational research. Source data for this manuscript are available via MassIVE repository at massive.ucsd.edu with identifier: MSV000097864.

Myocytes, Cardiac↗
PubMed2026

Ligand-based directed differentiation to produce granulosa-like cells expressing steroidogenic enzyme genes.

The ovarian granulosa cells are responsible for producing hormones and supporting oocytes through maturation and meiotic resumption. There is a need to generate granulosa-like cells (GLCs) from human induced pluripotent stem cells (hiPSCs) to better model human gonadal development and to test the effects of exogenous or pharmaceutical compounds on the ovary. Here we report a rapid ligand-based protocol for differentiating hiPSCs into cells that express markers of the transient developmental lineages and steroidogenic pathway genes. Single-cell RNA-sequencing (scRNA-seq) analysis identified canonical granulosa cell genes were expressed in a subset of cells and identified new genes of interest that were significantly associated with computationally modeled pseudotime. HSD17B1 was expressed in resulting GLCs but at low levels, suggesting an immature granulosa cell phenotype. The GLCs were produced using a simple culture method that could be augmented for granulosa cell functions such as sustaining oocyte growth. Producing GLCs through protocols such as this one is a first step toward designing large-scale ovarian endocrinology assays and developing personalized cell-based fertility and hormone restoration technologies in the future. This rapid protocol produced cells that express steroidogenic enzyme genes etoc blurb. Kubo and colleagues present a 5-day rapid protocol to generate immature granulosa-like cells from hiPSCs. Cells differentiated with inhibition of DKK1, a WNT signaling target gene, expressed gonadal ridge markers and FOXL2 transcripts and protein. Additionally, steroidogenic enzyme genes were expressed. A small population of differentiated cells were identified as expressing early-stage granulosa cell genes by single-cell RNA-seq.

Female↗
PubMed2026

Generation of an induced pluripotent stem cell line, LGMi002-A, from a Bardet-Biedl Syndrome patient with a BBS5 homozygous pathogenic variant.

The human induced pluripotent stem cell (iPSC) line, iPSC-BBS5stbg1, derived from a patient with a Bardet-Biedl Syndrome (BBS) phenotype and carrying a BBS5 homozygous pathogenic variant: c.123delA, p.Gly42Glufs*11 is described. The reprogramming of the patient's dermal fibroblasts was achieved using the non-integrative Sendai virus system delivering the OCT4, SOX2, KLF4 and c-MYC (OSKM) transcription factors. The established iPSC line iPSC-BBS5stbg1 displays typical iPSC morphology, maintains genomic stability, and demonstrates the ability to differentiate into cell types representative of the three embryonic germ layers. This iPSC line constitutes robust and relevant cellular model for studying BBS-associated disease mechanisms and ciliary dysfunction.

Humans↗
PubMed2027

Systematic Dissection of Key Driver Perturbation Signatures in Single Cells via ECCITE-seq.

CRISPR screens, such as expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq), enable the simultaneous measurement of transcriptomes, gRNA identity, and cell-surface protein expression at single-cell resolution to systematically interrogate gene function. This platform provides a powerful and scalable experimental approach for validating disease-associated regulators identified by large-scale association studies and other computational methods, including network-based analyses of multi-omics data. Here, as an example application, we describe an ECCITE-seq framework to characterize the transcriptomic consequences of perturbing multiple neuronal key driver genes associated with Alzheimer's disease (AD) in human-induced pluripotent stem cell (hiPSC)-derived neurons. More broadly, by integrating customized pooled gRNA libraries with different CRISPR effectors across multiple cell types, this approach allows for the assessment of the regulatory impact of candidate genes implicated in development and disease processes.

Humans↗
PubMed2026

Generation of induced pluripotent stem cell line NTUHi003-A from a patient with premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is characterized by impaired ovarian function before 40 years of age and is associated with heterogeneous etiologies. Herein, we established a human induced pluripotent stem cell (hiPSC) line, NTUHi003-A, from the peripheral blood mononuclear cells (PBMCs) of a patient with POI. The generated hiPSC line exhibited a normal 46, XX karyotype and demonstrated confirmed pluripotency. This cell line provides a valuable cellular platform for disease modeling and mechanistic studies of POI.

Humans↗
PubMed2027

A Standardized Protocol for Generating iPSC-Derived Human Microglia for Functional Genomic Assays.

Human induced pluripotent stem cell (iPSC)-derived microglia (iMG) provide an in vitro experimental system for studying human microglial biology, neuroinflammation, and genetic risk mechanisms associated with neurological disease. This chapter describes a standardized, scalable, and reproducible protocol for the differentiation of human iPSCs into functional microglia-like cells, with particular emphasis on applications in transcriptional and epigenomic network analysis. The protocol supports high-viability floating iMG production, compatibility with pooled CRISPR perturbation approaches, and downstream multiomic profiling, including single-cell RNA sequencing, chromatin accessibility assays, and proteomics. Detailed procedures are provided for iPSC maintenance, hematopoietic progenitor cell generation, microglial maturation, functional genomics integration, and quality control.

Humans↗
PubMed2026

Establishment and characterization of two human pluripotent stem cell lines from patients with ATX-FGF14/spinocerebellar ataxia 27A (SCA27A).

Spinocerebellar ataxia 27A (SCA27A) is a rare inherited ataxia arising from heterozygous pathogenic loss-of-function variants in FGF14. Autosomal recessive FGF14-related cerebellar ataxia has also been reported in a single individual to date. Here, we describe the generation and characterization of human induced pluripotent stem cell (iPSC) lines derived from two individuals with FGF14-related ataxia (ATX-FGF14): one with SCA27A and one with autosomal recessive disease. Given the predominantly neuronal expression of FGF14, these iPSC lines represent a valuable resource for investigating the cellular and molecular consequences of FGF14 deficiency in disease-relevant neuronal populations following directed differentiation.

Humans↗
PubMed2026

Establishment of induced pluripotent stem cell line TRNDi045-A-38 carrying homozygous DOK7-related Congenital Myasthenia patient-mutation knock-in variant from parental KOLF2.1J.

DOK7-related Congenital Myasthenic Syndrome (CMS) is a rare genetic neuromuscular junction disorder. This is one of the most common of the recessive forms of CMS, often presenting with more static proximal weakness (hence also referred to as limb girdle CMS). Whole-genome sequencing of affected patients implicates frameshift duplication mutations in DOK7 as drivers of impaired neuromuscular-junction signaling. In this study, we generated a human induced pluripotent stem cell (hiPSC) line TRNDi045-A-38 from the KOLF2.1J reference line, engineered to carry homozygous DOK7 c.1124_1127dupTGCC mutation knock-in using CRISPR/Cas9. This iPSC line could be used for in vitro disease modeling to study disease pathophysiology and for therapeutic development.

Humans↗
PubMed2026

CRISPR/Cpf1-mediated editing of DNM1L in induced pluripotent stem cells.

The dynamin-1-like protein (DNM1L), also termed DRP1, is essential for mitochondrial fission. Mutations in DNM1L are associated with neurological disorders and cardiac dysfunction. To decipher the role of DNM1L in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with a mutation by targeting exon 18 of the DNM1L gene. The generated compound heterozygous (biallelic) DNM1L mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers meso-, endo-, and ectoderm.

Journal Article↗
PubMed2026

CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage.

Intervertebral disc (IVD) degeneration, a leading cause of chronic lower back pain, is associated with loss of vacuolated notochordal cells (NCs) and fibrotic remodeling of the nucleus pulposus. Emerging therapies increasingly focus on NCs, which are rare but therapeutically relevant cells for regenerating degenerated IVDs. In this study, we used CRISPR-based transactivation (CRISPRa) to direct the differentiation of human induced pluripotent stem cells (iPSCs) into the NC lineage. We tested CRISPRa-mediated activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9, coupled with single-cell sequencing of Aggrecan-2A-mScarlet reporter iPSCs. This approach identified the SOX5/6/9 combination (SOX-trio) as critical for promoting NC lineage commitment. The SOX-trio yielded the largest cell population expressing a range of genes previously associated with NC identity, including SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, and KRT18. Our study demonstrates the integration of CRISPRa with single-cell technologies as a powerful platform for investigating and enriching iPSC-derived NCs, supporting future regenerative strategies across various fields.

Humans↗
PubMed2025

The role of iPSC research for insight into inherited arrhythmia conditions.

Human induced pluripotent stem cells (iPSCs) have emerged as a transformative platform for modeling inherited cardiac arrhythmia syndromes and uncovering human-specific disease mechanisms. However, the promise of iPSC-derived cardiomyocytes lies beyond the recapitulation of arrhythmogenic phenotypes and channelopathies. In this review, we explore recent works which have enabled mechanistic interrogation and therapeutic insight for inherited arrhythmia syndromes, beyond the capabilities of traditional animal models. Such studies have leveraged iPSCs to elucidate the role of splice variants, transcriptional regulation, and mitochondrial stress in arrhythmogenesis. Further, iPSC systems have proven important for reclassifying variants of uncertain significance and in modeling idiopathic arrhythmias where genotype-phenotype links are elusive. Advances in directed differentiation now permit chamber-specific cardiac cell generation, allowing for atrial and ventricular disease modeling and revealing critical cell-cell interactions. iPSCs also serve as high-fidelity precursor platforms for drug testing, offering predictive insight into mutation-specific responses to pharmacologic and genetic therapies. Though limitations in maturation and scalability persist, ongoing efforts for integration with tissue engineering, multi-cellular models, and computational frameworks are evolving to improve model reliability. iPSC-based systems now occupy a critical role in arrhythmia research, bridging basic discovery with translational applications, thereby contributing to personalizing care and advancing therapeutics in inherited and idiopathic arrhythmic syndromes.

Humans↗
PubMed2026

Generation of an NKX2-1-EGFP reporter iPSC line with inducible Cas9 for lung progenitor cell tracing.

NK2 homeobox 1 (NKX2-1), a master regulator robustly expressed in lung, thyroid, and forebrain, is indispensable for specifying lung epithelial fate and serves as a definitive marker of lung progenitors. Here, we generated a human induced Pluripotent Stem Cell (iPSC) line harboring a doxycycline (dox)-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair. This dual-function line combines inducible genome editing with real-time tracing of early lung progenitors, enabling their prospective isolation and screening for stage-specific maturation regulators. Therefore, this engineered iCas9-NKX2-1 EGFP line is a key resource for dissecting human lung development, modeling pulmonary disease, and advancing regenerative therapies.

Humans↗