Induced pluripotent stem cells (iPSCs) are increasingly used as disease models to accelerate drug repurposing, especially for rare diseases. While ethical and regulatory issues in iPSC research have been widely discussed, little is known about how these challenges are addressed in practice. To provide insights into the current practice, challenges, and opportunities in the governance of research on iPSC-based drug repurposing for rare neurological disorders, semi-structured interviews were conducted with various experts from eight countries in the context of the SIMPATHIC project. The results indicate that this research context requires improved information provision for minors and individuals with cognitive impairment and more clarity regarding commercial use and reporting of findings. Uncertainty in governance procedures hinders collaboration, validation, and clinical translation in iPSC-based drug repurposing. Responsible (re)use of iPSCs requires dynamic, transparent, and participatory governance structures based on shared decision-making with all stakeholders involved.
Japan’s Act on the Safety of Regenerative Medicine entered into force in 2014 and was revised in 2025 to ensure the appropriate provision of unproven cell and gene therapies. However, two fatal cases occurred after the revision. This article examines practical challenges and proposes measures to better protect patient safety.
SetDB1 is best known for catalyzing H3K9me3, but it also influences H3K27me3 deposition, CTCF-binding, and DNA methylation (DNAme). Given the interplay between DNAme and the other epigenetic features, we profiled DNAme following Setdb1 knockout (KO) in ground-state and serum-grown mouse embryonic stem cells (ESCs) to illuminate DNAme-dependent and -independent functions of SetDB1. Time-course whole-genome bisulfite sequencing of serum-grown ESCs shows that nearly half of SetDB1 binding sites are enriched with DNAme and H3K9me3, primarily at retrotransposons. Upon Setdb1 KO, both H3K9me3 and DNAme are reduced, with DNAme rapidly removed at many sites by TET enzymes. Some retrotransposons, primarily IAPs, are TET-resistant and lose DNAme slowly via passive dilution. Notably, SetDB1-mediated regulation of H3K27me3, CTCF-binding, and SMAD3 are uncoupled from the DNAme-H3K9me3 axis, and from each other. AlphaFold modeling and co-immunoprecipitation mass spectrometry suggest this uncoupling involves competitive binding to distinct SetDB1 protein domains, highlighting the complex coordination underlying SetDB1 functions.
The placental labyrinth is the primary site of maternal-fetal exchange, and its disruption contributes to placental insufficiency and fetal growth restriction (FGR). Here, we identify insulin receptor substrate 2 (IRS2) as a key regulator of mouse placental labyrinth development. Irs2 is expressed in the syncytiotrophoblast (SynT) layers of the labyrinth, where it integrates growth factor signaling during placental morphogenesis. Loss of IRS2 disrupts labyrinth organization, causing defective SynT-II differentiation, impaired trophoblast fusion, and reduced fetal vascularization. Using CRISPR-Cas9-engineered Irs2-/- trophoblast stem cells, we show that IRS2 deficiency impairs upregulation of the SynT-II regulators Pparγ and Gcm1, disrupting SynT-II differentiation and sinusoidal trophoblast giant cell specification. Mechanistically, IRS2 relays insulin-like growth factor (IGF) signals through phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways to coordinate trophoblast proliferation, differentiation, and vascular morphogenesis. Human trophoblast stem cell differentiation and organoid analyses reveal IRS2 enrichment in the human syncytiotrophoblast (STB), indicating conserved expression at the human maternal-fetal interface.
Understanding age-driven bone weakening mechanisms is essential for developing precision therapeutic strategies. This study systematically investigated skeletal aging, focusing on the critical decline of SIRT1 and its impact on type H vessels and osteogenic cells. We found that SIRT1 markedly enhanced both endothelial tube formation and osteogenic differentiation in senescent bone marrow-derived mesenchymal stem cell (BMSC)/human umbilical vein endothelial cell (HUVEC) co-cultures. In vivo validation studies revealed that pharmacological activation of SIRT1 significantly improved bone regeneration in age-related osteoporotic defects in both femoral and mandibular sites. Mechanistically, β-catenin served as a downstream mediator of these effects in our experimental systems, with SIRT1 promoting β-catenin deacetylation and nuclear translocation to activate Wnt signaling. By demonstrating that SIRT1 enhances both endothelial and osteogenic functions in the aged bone microenvironment, this work provides a rationale for targeted bone rejuvenation strategies and mechanistically informed therapeutic innovation against geriatric osteoporosis.
Human pluripotent stem cells (hPSCs) are a promising cell source for producing T cells for regenerative medicine and immunotherapy. However, it is challenging to develop a scalable suspension culture system for generating functional T lymphocytes from hPSCs. Here, we developed a Matrigel-based artificial thymic organoid (Gel-ATO) system by encapsulating hPSC-derived induced hematopoietic progenitor cells (iHPCs) with MS5-hDLL4 stromal cells. The resulting Gel-ATOs produced functional iT cells capable of antigen-specific cytotoxicity in vitro and tumor suppression in vivo. Building on this system, we further established a rotating suspension culture to robustly generate iT lymphocytes with nearly 20-fold enhancement of iT cells production compared to stationary suspension culture. Importantly, these rotating suspension culture-derived iT cells were functional, exhibiting cytokine secretion, proliferation, and cytotoxicity. By addressing the dual requirements of scalability and function, our work paves the way for future clinical production of T cells for T cell-based cell therapy.
Natural Killer (NK) cells generally exhibit dysfunction in tumor microenvironment (TME), significantly limiting their efficacy in antitumor therapy. Tumor cells display enhanced glycolysis, leading to lactic acid (LA) secretion and accumulation in the TME. Using human-induced pluripotent stem cell (hiPSC)-derived NK cells (iNKs) as a model, we demonstrate that LA induces substantial iNK dysfunction, including reduced survival, impaired IFN-γ secretion, and diminished tumor-killing capacity due to severely compromised mitochondrial function. To overcome LA-induced dysfunction, we knocked in an expression cassette for lactate dehydrogenase B (LDHB) into hiPSCs (LDHB-hiPSCs), enabling conversion of cellular lactate to pyruvate. iNKs derived from LDHB-hiPSCs (LDHB-iNKs) largely resisted LA-induced dysfunction, exhibiting enhanced cytotoxicity and improved survival in high-LA tumor tissues. Importantly, LDHB-iNKs show superior suppression of solid tumor formation in vivo. Our study provides a novel strategy to enhance NK cell therapy against solid tumors by reshaping the metabolic pathways.
Mesenchymal stromal cell (MSC) injection has afforded heterogeneous outcomes in knee osteoarthritis (KOA). Herein, a framework that dually correlates KOA patient responsiveness with baseline autologous bone marrow-derived MSC(M) donor batch attributes and baseline clinical and biomarker features is provided. Using clinical trial data (NCT02351011), we demonstrated that MSC(M) with increased immunomodulatory potency are more efficacious. Multivariable MSC(M) genes correlated strongly with responder status and 12- and 24-month improvements in Knee Injury and Osteoarthritis Outcome Scores. Responder MSC(M) donor batches had unique microRNA expression and ability to polarize CD14+ monocytes in vitro. KOA responders had lower baseline physical activity and trended toward more severe baseline KOA. Baseline local but not systemic biomarkers showed trending correlations with patient responsiveness. 42% of KOA patients were responders at 24 months, emphasizing durability of single MSC(M) injections. Together, our analytical methodology defines critical quality attributes of potent MSC(M) donor batches and identifies putative KOA patient theratypes to MSC treatments.
Immune-epithelial interactions are essential for intestinal homeostasis, yet organoid monocultures lack immune components and fail to model how immune cells shape epithelial identity. Here, we establish a macrophage-organoid co-culture integrating bone marrow-derived macrophages (BMDMs) into the intestinal niche to create a controlled, immune-competent system. Under homeostatic conditions, the optimized 5k configuration of BMDMs preserves crypt-like architecture and maintains stem cell localization and secretory lineage organization. TNFα stimulation induces coordinated epithelial remodeling characterized by reduced canonical stem cell marker expression and density-dependent shifts in macrophage phenotype. Transcriptomic profiling confirms enrichment of immune-associated and epithelial stress pathways. Comparative analysis with a DSS-induced murine inflammation model reveals overlapping inflammatory and epithelial remodeling features, supporting physiological relevance. This platform provides a reproducible framework for studying epithelial-immune interactions and inflammatory remodeling under defined conditions, offering a controlled alternative to animal models.
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.
C9orf72 hexanucleotide repeat expansion (C9-HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). However, approximately half of the FTD patients are sporadic without a clear genetic background. To compare characteristics of microglia from different FTD subtypes, we generated induced pluripotent stem cell-derived microglia (iMG) from sporadic and C9-HRE-carrying behavioral variant FTD (bvFTD) patients and healthy controls. C9-HRE iMG displayed C9-HRE-associated RNA foci and dipeptide repeat proteins. All bvFTD iMG had fewer LAMP2-A-positive vesicles compared to control iMG. Additionally, C9-HRE iMG showed significantly increased LC3BII/I conversion after bafilomycin A1 treatment and altered phagocytic activity. The gene expression profile of C9-HRE iMG only modestly differed from the control iMG, but was greatly different from the sporadic bvFTD patient iMG. Our data show alterations in phagocytic and autophagosomal/lysosomal pathways and gene expression profiles between C9-HRE and sporadic bvFTD iMG for the first time.
Methyltransferase-like 5 (METTL5) catalyzes N6-methyladenosine (m6A) modification on 18S rRNA. In humans, loss-of-function mutations in METTL5 cause severe microcephaly and intellectual disability, whereas Mettl5 knockout (KO) animal models display inconsistent and milder phenotypes. To better model human disease, we generated METTL5-KO human-induced pluripotent stem cell (hiPSC)-derived cortical organoids, which exhibit impaired neural progenitor cell (NPC) proliferation and differentiation, leading to reduced ventricle-like structures and significant reductions in cortical organoid diameter. Mechanistically, Ribo-seq analysis revealed broad translational changes in METTL5-KO NPCs consistent with cellular stress responses rather than transcript-specific translational changes. Single-cell RNA-seq identified downregulation of coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a mitochondrial regulator of oxidative metabolism. Overexpression of CHCHD2 in METTL5-KO NPCs rescued proliferation and partially rescued oxidative metabolism in NPCs and ventricle formation in organoids. This highlights a previously uncharacterized connection between CHCHD2, oxidative metabolism, and METTL5-mediated regulation of human neurogenesis.
Stem-cell-based human embryo models offer an ethically tractable platform for studying early human development. This study employs somitoids, three-dimensional models of human somitogenesis, to investigate how transcriptional programs and culture conditions influence somite formation and segmentation. We show that pre-differentiation culture medium impacts the developmental potential of induced pluripotent stem cells (iPSCs), with StemFit medium and Matrigel embedding outperforming mTeSR Plus medium in generating robust somite-like structures. Strikingly, these differences arise despite only subtle changes in transcriptomic and time-resolved proteomic profiles. P300-based proximity labeling also reveals a largely overlapping set of chromatin-associated regulators across iPSC conditions. In somitoids, enhancer-associated profiling highlights factors linked to somitogenesis, including MESP2 and TBX6. Knockout of three identified regulators, BPTF, RBPJ, and CITED2, demonstrate their essential roles in somite formation. Together, these findings highlight how culture conditions and enhancer-associated networks influence early human development and demonstrate somitoids as a scalable system for functional genomics.
African swine fever virus (ASFV) causes a lethal hemorrhagic fever in pigs, and spread of this disease threatens many pig species (Suidae) globally. By contrast, ASFV infections in the natural evolved hosts, warthog and bushpig, are subclinical. The macrophage (Mϕ) is the primary target of ASFV, and species-dependent responses in Mϕs are presumed to influence disease susceptibility. In an attempt to model these differences in vitro, we generated transgene-regulated induced pluripotent stem cells (iPSCs) from domestic pig, wild boar, red river hog, and warthog and confirmed that their corresponding iPSC-derived Mϕs (iPSCdMs) supported infection and replication of ASFV. In contrast to the other species, however, warthog iPSCdMs did not induce interferon α upon infection by either virulent or attenuated ASFV. iPSCdMs may therefore represent an experimental system to understand how ASFV infection of Mϕs contributes to disease and aid development of strategies to combat this economically and environmentally devastating pathogen.
Paternally expressed gene 3 (Peg3) is an imprinted gene whose knockout (KO) causes placental and fetal growth deficits as well as maternal behavioral defects, but its molecular role remains poorly understood. Here, we report that Peg3-null trophoblast stem cells (TSCs) exhibit increased rates of mitotic errors and double-strand DNA breaks as a likely consequence of a global reduction in H3K9me3 and DNA methylation levels. Transcriptional profiling highlights a deficiency of KO cells to adequately silence genes, resulting in elevated expression levels of differentiation stage-inappropriate genes. Peg3 KO TSCs also exhibit a loss of DNA methylation and H3K9me3 at a subset of developmentally regulated CpG islands that likely rewire CTCF-regulated transcriptional networks. Collectively, our data identify PEG3 as an intrinsic component of the epigenetic repressive machinery, thus solidifying its putative role as a KRAB-ZFP protein that functions to regulate gene expression and genome integrity in the placental trophoblast lineage.
The immaturity of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and engineered cardiac tissues (ECTs) limits their use in regenerative therapies. Ventricular loading pressure plays a vital role in cardiac repair, prompting the hypothesis that hydrostatic pressure could enhance ECT maturation. ECTs were created by co-culturing hPSC-derived cardiovascular cells with extracellular matrix proteins and subjecting them to intermittent hydrostatic pressure (1 h/day at 50 kPa for 3 days after 2 weeks of pre-culture). This protocol improved cardiomyocyte alignment, mitochondrial content, and metabolic and functional maturation, evidenced by enhanced contractility, calcium flux, and single-cell RNA sequencing. Endothelial cells were critical for these effects, as their absence prevented maturation. Hydrostatic pressure combined with dynamic culture also promoted vascular network formation. Transplanted trained ECTs significantly improved ejection fraction in a rat myocardial infarction model. These findings demonstrate the potential of hydrostatic pressure training to advance ECT maturation and therapeutic application.
Various immunotherapies have been developed to treat malignant tumors, and autologous CAR-T cell therapy is clinically used for certain malignancies. However, their efficacy against solid tumors is limited. γδT cells are recognized for their potent tumor cytotoxicity and MHC-unrestricted activity. We successfully induced γδT cells from iPS cells and demonstrated their cytotoxicity against colorectal cancer cell lines in vitro. However, cancer cell lines often lack critical tumor characteristics such as heterogeneity and drug sensitivity. In contrast, patient-derived cancer organoids retain many features of the patient's tumor tissues. This study evaluated the cytotoxicity of allogeneic iγδT cells against cell lines and organoids, in vitro and in vivo. iγδT cells showed 70%-90% cytotoxicity against cell lines and organoids in vitro. In vivo, local and intravenous administration of iγδT cells suppressed tumor growth by 70%-100%, highlighting their potential as a novel immunotherapy for colorectal cancer.
While extensive research has examined the neuroinvasiveness of SARS-CoV-2, its relationship with brain maturation remains unclear. Using a multi-omics approach, we established cerebral organoid (CBO) at day 60 (EB60) and day 120 (EB120) to model immature and mature developmental stages. We found that enhanced corticogenesis and gliogenesis during maturation are associated with substantial alterations in lipid metabolism, functionally linking these processes to coordinated molecular and functional brain development. Leveraging this model, we provide the first quantitative insights into long-term viral propagation kinetics up to 20 days post-infection, revealing significantly higher infectivity in mature CBOs. Single-cell transcriptomics, RT-qPCR, and immunohistochemistry revealed the upregulation of lipid-associated genes in EB120, suggesting a key driver of increased susceptibility. Consistently, pharmacological lipid reduction attenuated SARS-CoV-2 infection. Our findings establish a mechanistic link between intrinsic brain maturation and viral susceptibility mediated by lipid remodeling, suggesting lipid-lowering strategies as potential candidates for therapeutic repurposing in COVID-19.
The marketing of unproven stem cell-based interventions raises public health, ethical, and regulatory challenges. This article examines online marketing in Panama, identifies new businesses, and argues that lack of enforcement has contributed to their expansion. Urgent actions are needed to mitigate this market, protect patients, and generate evidence-based treatments.
Glioblastoma multiforme (GBM) remains refractory to current treatment modalities. Differentiation-based approaches, which force cancer stem/progenitor cells to exit the cell cycle and adopt terminal fates, offer an alternative therapeutic strategy. Cell fate regulators operating during stem/progenitor cell divisions integrate proliferative and anti-proliferative cues and represent particularly attractive points of intervention. Here, we investigated the therapeutic potential of targeting mitotic kinesin KIF20A in GBM stem/progenitor cells. KIF20A is a crucial component of cytokinetic machinery and cooperates with a network of cell fate regulators to balance proliferative and differentiative divisions in neural stem/progenitor cells (NSPCs). Using complementary in vitro and in vivo models, including 2D cultures, 3D organoids, and intracranial xenografts, we show that inhibition of KIF20A drives cell cycle exit and induces a postmitotic/differentiated state in GBM stem/progenitor cells, resulting in a marked suppression of proliferation. Together, these findings establish KIF20A as a key vulnerability in GBM and a promising target for differentiation-based intervention.
