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Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis,while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX,indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus,increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. View Publication

Electrospun scaffolds with varied stiffness promote distinct colony morphology of human induced pluripotent stem cells,which affects their subsequent differentiation. On soft scaffolds,induced pluripotent stem cells develop 3D colonies due to the pliability of the electrospun fibrous networks,leading to greater differentiation tendency to ectodermal lineage. View Publication

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and EGFR in determining cardiac differentiation in vitro as these receptor tyrosine kinases (RTKs) are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation,cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment towards neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (textgreater2-fold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/non-canonical Wnt signaling determines cardiogenic fate in hPSCs. This article is protected by copyright. All rights reserved. View Publication

Associations between chronic antigen stimulation,T cell dysfunction,and the expression of various inhibitory receptors are well characterized in several mouse and human systems. During chronic hepatitis B virus (HBV) infection (CHB),T cell responses are blunted with low frequencies of virus-specific T cells observed,making these parameters difficult to study. Here,using mass cytometry and a highly multiplexed combinatorial peptide-major histocompatibility complex (pMHC) tetramer strategy that allows for the detection of rare antigen-specific T cells,we simultaneously probed 484 unique HLA-A*1101-restricted epitopes spanning the entire HBV genome on T cells from patients at various stages of CHB. Numerous HBV-specific T cell populations were detected,validated,and profiled. T cells specific for two epitopes (HBVpol387 and HBVcore169) displayed differing and complex heterogeneities that were associated with the disease progression,and the expression of inhibitory receptors on these cells was not linearly related with their extent of T cell dysfunction. For HBVcore169-specific CD8+ T cells,we found cellular markers associated with long-term memory,polyfunctionality,and the presence of several previously unidentified public TCR clones that correlated with viral control. Using high-dimensional trajectory analysis of these cellular phenotypes,a pseudo-time metric was constructed that fit with the status of viral infection in corresponding patients. This was validated in a longitudinal cohort of patients undergoing antiviral therapy. Our study uncovers complex relationships of inhibitory receptors between the profiles of antigen-specific T cells and the status of CHB with implications for new strategies of therapeutic intervention. View Publication

Pluripotent stem cells possess a unique nuclear architecture characterized by a larger nucleus and more open chromatin,which underpins their ability to self-renew and differentiate. Here,we show that the nucleolus-specific RNA helicase DDX18 is essential for maintaining the pluripotency of human embryonic stem cells. Using techniques such as Hi-C,DNA/RNA-FISH,and biomolecular condensate analysis,we demonstrate that DDX18 regulates nucleolus phase separation and nuclear organization by interacting with NPM1 in the granular nucleolar component,driven by specific nucleolar RNAs. Loss of DDX18 disrupts nucleolar substructures,impairing centromere clustering and perinucleolar heterochromatin (PNH) formation. To probe this further,we develop NoCasDrop,a tool enabling precise nucleolar targeting and controlled liquid condensation,which restores centromere clustering and PNH integrity while modulating developmental gene expression. This study reveals how nucleolar phase separation dynamics govern chromatin organization and cell fate,offering fresh insights into the molecular regulation of stem cell pluripotency. Pluripotent stem cells depend on specialized nuclear organization for their function. Here,the authors show that DDX18 regulates nucleolar phase separation and chromatin architecture to preserve human embryonic stem cell pluripotency. View Publication

ABSTRACTHuman induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However,their differentiation into specific cell lineages,such as definitive endoderm (DE) and pancreatic progenitor (PP),often suffers from poor reproducibility,due partially to their pluripotency. In this work,we investigated the impact of iPSC confluency during cell self?renewal and seeding density on cell metabolic activity,glycolysis to oxidative phosphorylation shift,and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual?positive cells in PPs. Interestingly,we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. Graphical Abstract and Lay SummaryHuman induced pluripotent stem cell (iPSC) differentiation into specific cell lineages often shows poor reproducibility due to cell pluripotency. This study demonstrated impact of iPSC seeding strategy on metabolic activity and differentiation potential,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. View Publication

BackgroundMale factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However,the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here,we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of MFI.MethodsWe reprogrammed human dermal fibroblasts (hDFs) to hiPSCs,induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA,2-AG) by LC/MS,receptors (CB1R,CB2R,TRPV1,GPR55) by qPCR,flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA,CB65,CSP,ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to hSSCs.ResultshiPSCs from hDFs expressed the pluripotency markers OCT4,SOX2,NANOG,SSEA4 and TRA-1-60; and could be differentiated into ID4+,PLZF?+?hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10??10 ??10??9 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs,with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA,2-AG,ACPA,CSP and ML184. The EC50 of CB65 was determined to be 2.092?×?10??8 M for support of pluripotency and preservation of stemness on hiPSCs from 78 h. CB65 stimulation at EC50 also increased the yield of ID4?+?hSSCs,PLZF?+?SSPCs and SCP3?+?spermatocytes from day 10 to 12.ConclusionsWe demonstrated here for the first time that stimulation of CB2R results in an increased yield of hiPSCs and hiPSC-derived hSSCs. CB65 is a potent CB2R agonist that can be used to increase the yield of hiPSC-derived hSSCs offering an alternative source of autologous male germ cells for patients with MFI. Increasing the male germ/stem cell pool by CB65 supplementation could be part of the ART-associated protocols in MFI patients with complete germ cell aplasia.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40659-025-00596-4. View Publication

Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations,small insertions,and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA),which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs,especially when the genetic context precludes the screening of multiple pegRNAs,and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system,we generated other isogenic models of inherited retinal diseases (IRDs),including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure,such as plasmid concentrations,PE component ratios,and delivery method settings,showing that our improved workflow increased the hiPSC editing efficiency. View Publication

IntroductionHeme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation,and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors,and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation,that is,NOD-like receptor protein 3 (NLRP3),glutathione (GSH),GSH disulfide (GSSG),and interleukin 6 (IL-6).MethodsThe study population comprised three age groups: young adults (age range,19–23 years),older adults aged under 85 years (age range,73–84 years),and older adults aged over 85 years (age range,85–92 years). NLRP3,GSH,and GSSG concentrations were measured in serum,whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2,lipopolysaccharide (LPS),or phorbol 12-myristate 13-acetate (PMA) with ionomycin.ResultsThe analysis of serum NLRP3,GSH,and GSSG concentrations revealed no statistically significant differences among the studied age groups. However,some typical trends of aging were observed,such as a decrease in GSH concentration and an increase in both GSSG level,and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover,statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells.ConclusionsThese results showed that NK cells can express HO-1 at a basal level,which was significantly increased in activated cells,even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters. View Publication

Variants of phospholipase C gamma 2 (PLCG2),a key microglial immune signaling protein,are genetically linked to Alzheimer's disease (AD) risk. Understanding how PLCG2 variants alter microglial function is critical for identifying mechanisms that drive neurodegeneration or resiliency in AD. Induced pluripotent stem cell (iPSC) –derived microglia carrying the protective PLCG2 P522R or risk‐conferring PLCG2 M28L variants,or loss of PLCG2,were generated to ascertain the impact on microglial transcriptome and function. Protective PLCG2 P522R microglia showed significant transcriptomic similarity to isogenic controls. In contrast,risk‐conferring PLCG2 M28L microglia shared similarities with PLCG2 KO microglia,with functionally reduced TREM2 expression,blunted inflammatory responses,and increased proliferation and cell death. Uniquely,PLCG2 P522R microglia showed elevated cytokine secretion after lipopolysaccharide (LPS) stimulation and were protected from apoptosis. These findings demonstrate that PLCG2 variants drive distinct microglia transcriptomes that influence microglial functional responses that could contribute to AD risk and protection. Targeting PLCG2‐mediated signaling may represent a powerful therapeutic strategy to modulate neuroinflammation. The impact of Alzheimer's disease protective‐ and risk‐associated variants of phospholipase C gamma 2 (PLCG2) on the transcriptome and function of induced pluripotent stem cell (iPSC) –derived microglia was investigated. PLCG2 risk variant microglia exhibited a basal transcriptional profile similar to PLCG2‐deficient microglia but significantly different from isotype control and the transcriptionally similar PLCG2 protective variant microglia. PLCG2 risk variant and PLCG2‐deficient microglia show decreased levels of triggering receptor expressed on myeloid cells 2 (TREM2). The differential transcriptional pathways of protective and risk‐associated PLCG2 variant microglia functionally affect proliferation,apoptosis,and immune response. Protective PLCG2 microglia show resilience to apoptosis and increased cytokine/chemokine secretion upon exposure to lipopolysaccharide (LPS). View Publication

INTRODUCTION Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs),molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However,no currently accepted molecular target exists against TNBC and,moreover,TICs. Therefore,we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs. METHODS A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1),including its expression in breast cancer cell lines,effect on the CD44high/CD24-/low TIC subpopulation,growth inhibition,mammosphere formation,and apoptosis,as well as the activity of the PLK1 inhibitor,BI 2536. RESULTS Of the 85 kinases identified in the screen,28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC),BT474-M1 (ER+/HER2+,a metastatic variant),and HR5 (ER+/HER2+,a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly,12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines,representing all of the breast cancer subtypes,and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth,mammosphere formation,and apoptosis as did PLK1 siRNAs. Finally,whereas paclitaxel,doxorubicin,and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149,subsequent treatment with BI 2536 killed the emergent population,suggesting that it could potentially be used to prevent relapse. CONCLUSION Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus,PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer. View Publication

BACKGROUND We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4,Sox2,c-Myc,and Klf4). In this study,we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. METHODS Mature microRNAs (mir-302a-d,369-3p and 5p,and mir-200c,as needed) were introduced into colon cancer cells (DLD-1,RKO,and HCT116) using lipofection. RESULTS The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog,Oct3/4,SOX2,and Klf4,as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers,indicating that reprogramming of the cancer cells was partially successful. Moreover,we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo,accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil,possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1,RKO,and HCT116 cells exhibited reduced c-Myc expression,in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. CONCLUSIONS Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application,because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression. View Publication