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Human pluripotent stem cell (hPSC)-based therapies offer promise but pose potential risks due to culture-acquired genetic variants, some of which have been linked with cancer. An international workshop addressed these concerns, highlighting the need for improved strategies to stratify variants and chart a path toward definitive guidelines in hPSC-based therapy.
Mammalian embryonic development is a highly orchestrated process initiated by the fusion of the oocyte with sperm to generate the zygote. In humans, the zygote remains transcriptionally quiescent until the major wave of zygotic genome activation (ZGA) occurs around the eight-cell (8C) stage (day 3 after fertilization). These cells and the derived morula cells are totipotent: they have the capacity to form a whole individual. Our understanding of human totipotency is very limited because of ethical concerns using embryos and the scarcity of material available for research. Recently, we established a controllable transgene-free methodology to generate totipotent eight-cell embryo-like cells (8CLCs) from human pluripotent stem cells (PSCs) in vitro. These 8CLCs are produced using a novel medium, formulated by us, containing specific chemical compounds and cytokines. Here, we provide a detailed protocol for inducing, isolating and characterizing 8CLCs generated with this medium. The induction process can be done either in a stepwise manner (primed-naive-8CLC) that requires only 5 d starting from naive PSCs or directly from primed PSCs, which takes ~7 d. The resulting 8CLCs exhibit transcriptional and epigenetic features resembling those of human 8C embryo cells. On the basis of our experience, we expect that an individual with ~1 year of experience working with human PSC culture would be able to carry out this protocol. Our approach provides a valuable model for studying human early embryogenesis, particularly the molecular events surrounding ZGA.
BACKGROUND: Cost-effective, practical, and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However, the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover, we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly, the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. METHODS: We formulated B8 with commercially available, growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry, and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability, genome editing efficiency, single-cell cloning, and differentiation in both monolayer and organoids. Finally, we validated key findings using male (H1) and female (H9) human embryonic stem cells. RESULTS: hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast, cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time, B8+ led to gene expression changes indicative of marked lineage priming, reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state, consistent with intermittent media starvation. CONCLUSIONS: Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8, integrating some features of B8 while resembling cE8, emerges as a robust and practical option with limited compromises. B8+, though advantageous in some contexts, warrants caution due to lineage priming effects that may impact differentiation outcomes.
Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant cerebellar ataxia caused by an intronic GAA•TTC repeat expansion in the FGF14 gene. Here, we report the generation and validation of three human induced pluripotent stem cell (iPSC) lines derived from unrelated individuals with SCA27B, including two lines carrying a monoallelic pathogenic GAA•TTC repeat expansion in FGF14 and one line with biallelic expansions. These iPSC lines constitute a valuable resource, particularly given the predominantly neuronal expression of FGF14, and enable the investigation of disease mechanisms in relevant cell types following neuronal differentiation.
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.
Human pluripotent stem cells offer a scalable platform to study genetic and signalling mechanisms governing cell lineage decisions during differentiation. Genome-wide and single-cell transcriptomics technologies likewise offer high-throughput analysis of heterogeneous cell differentiation states. While in vivo development has been extensively characterised using these technologies, there remains a need for comprehensive single-cell transcriptomic profiling of stem cell differentiation from pluripotency. Understanding gene expression changes governing differentiation in vitro is key to developing high fidelity differentiation protocols and understanding fundamental mechanisms of development. We generated a single-cell RNA sequencing time course to study the role of developmental signalling pathways on multilineage diversification from pluripotency in vitro. The combined dataset of over 60,000 cells spans cell types from a time course of differentiation across all germ layers, ranging from gastrulation cell states to progenitor and committed cell types. These data provide a diverse benchmarking reference point to compare against in vivo development and advance understanding of signalling regulation of differentiation, providing insights into protocol development, drug screening, and regenerative medicine applications.
The induction of tissue-specific vessels in in vitro living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b+ putative liver sinusoidal progenitors by dictating developmental pathways. By devising an inverted multilayered air-liquid interface culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organize to generate and sustain hepatocyte-like cells neighboured by divergent endothelial subsets composed of CD32blowCD31high, LYVE1+STAB1+CD32bhighCD31lowTHBD-vWF- and LYVE1-THBD+vWF+ cells. WNT2 mediates sinusoidal-to-hepatic intercellular crosstalk potentiating hepatocyte differentiation and branched endothelial network formation. Intravital imaging reveals the iPS-cell-derived putative liver sinusoidal endothelial progenitor develops fully perfused human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enable correction of in vitro clotting time with Factor V-, VIII-, IX- and XI-deficient plasma, and rescues the severe bleeding phenotype in haemophilia A mice on transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.
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.
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.
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.
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.
Human gastruloids are a powerful class of stem cell-derived models that recapitulate key features of early embryonic development, including symmetry breaking and the emergence of three germ layers1-3. However, they lack anterior embryonic structures and coordinated axial organization4-6. To address this limitation, we pre-patterned human pluripotent stem cells (hPSCs) by exposing them to either anterior (FGF2) or posterior (CHIR99021 [CHIR] & retinoic acid [RA]) cues. Upon mixing, these dual-patterned hPSCs interacted and self-organized into elongated structures with both anterior and posterior features-which we term anterior-posterior (AP) human gastruloids. Anteriorly pre-treated cells robustly intercalated into posteriorly pre-treated cells, collectively giving rise to a continuum of neural tissues-including a brain-like domain, a neural tube-like structure, and neuro-mesodermal progenitors (NMPs)-with segmented somites arrayed bilaterally. Single cell RNA sequencing (scRNA-seq) revealed that human AP gastruloids contain cell types resembling the midbrain-hindbrain boundary (MHB), regionalized hindbrain structures (i .e. rhombomeres 1-8), regionalized neural crest (i.e. cranial, vagal, trunk)7,8 and head mesoderm. Transcriptomic comparisons to primate embryos revealed that human AP gastruloids most closely resemble Carnegie stage 11 (CS11) embryos. While they lack a notochord and full dorsal-ventral polarity, human AP gastruloids recapitulate key spatial and temporal features of early neurulation and somitogenesis. Perturbation of folic acid metabolism or rho-associated kinase (ROCK) signaling induced spinal cord defects, phenocopying aspects of spina bifida and other neural tube defects, highlighting this model's potential for studying congenital disorders9. AP gastruloids may serve as a simple, robust, scalable platform for modeling coordinated human AP body axis development. More broadly, our results suggest that controlled interactions between differentially prepatterned progenitors can initiate self-organization of complex body axis features. The "pattern-and-mix" strategy may serve as a generalizable framework for assembling spatially organized stem cell models of mammalian development.
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.
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.
Organoids are considered a novel modeling platform for studying human biology and advancing health research. With the ability to demonstrate complex 3D structure and multicellular interactions, organoids have advanced studies in all major organs as a reliable model. In this study, we generated an advanced cardiovascular organoid by using a genome-edited human pluripotent stem cell line with inducible SOX17 expression, enabling controlled endothelial specification, adjustable cell-type composition, and human heart-like morphology. Our organoids recapitulated the cardiotoxic phenotypes of FDA-approved chemotherapeutic doxorubicin, manifesting as decreased cell viability and diminished contractile activity. Cryoinjury-induced myocardial infarction in our organoids led to reduced beating, viability, and α-actinin expression, along with increased fibroblast formation, which were mitigated by Captopril. Lastly, isoproterenol treatment increased peak Ca2+ transient amplitude and shortened APD50 in our organoids, consistent with previously reported β-adrenergic responses. In summary, we established a protocol for generating in vitro 3D cardiovascular organoids with controllable cellular composition and heart-like structures, providing a robust and easy-to-produce platform for future studies of human heart disease.
Blastoids are a promising model for studying early human embryogenesis, but current models have limitations in post-implantation development and lack comprehensive epigenetic assessments, especially regarding genomic imprinting. These issues can lead to failures in accurately modeling early embryonic development. In this study, we developed a high-fidelity blastoid model using 4 chemicals + leukemia inhibitory factor (LIF) (4CL) naive human pluripotent stem cells (hPSCs) (4CL blastoids). 4CL blastoids closely resemble human blastocysts in morphology and transcriptional profiles, exhibiting similar DNA methylation and gene imprinting patterns. By extending the 3D culture to 14 days, these blastoids mimic early gastrulation, demonstrating the specification and migration of cells. They also show the transcriptional signature of hemogenic angioblast (HAB) cells at Carnegie stage 6 (CS6). This model bridges pre- and post-implantation stages, offering valuable insights into early tissue formation and human development.
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.
Over the past three decades, advances in human pluripotent stem cell (hPSC) technologies, including induced pluripotent stem cells, gene editing, and 2D/3D models, have transformed biomedical research. These powerful tools have revolutionized disease modeling, drug discovery, and the development of advanced therapy medicinal products (ATMPs), while driving the establishment of stem cell core facilities. By providing specialized expertise, standardized workflows, and access to advanced technologies, these facilities support both fundamental and translational research, promote rigor and reproducibility, and foster collaboration. This manuscript highlights their role as hubs of excellence and discusses current challenges and future opportunities for the global stem cell community.