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UNLABELLED Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model,then epigenetic modulation might enrich the CSC population,thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis,primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB),a potent DNA methyltransferase-1 inhibitor,and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal,superior tumor-initiating capacity in serial transplantations,and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes,although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g.,EGFR,SRC,and MYC). The common CSC signature was associated with malignant progression,which is enriched in poorly differentiated tumors,and was highly predictive of prognosis in liver and other cancers. CONCLUSION Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs. View Publication

In human B-acute lymphoblastic leukemia (B-ALL),RAG1-induced genomic alterations are important for disease progression. However,given that biallelic loss of the RAG1 locus is observed in a subset of cases,RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf(-/-)Rag1(-/-) mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model,we identified a new,Rag1-independent leukemia-initiating mechanism originating from a Sca1(+)CD19(+) precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM,a similar CD34(+)CD19(+) population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. View Publication

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study,we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2,lipoprotein lipase,and adipsin increased during adipocytogenesis of hMSCs. Simultaneously,the expression of canonical WNT2,10B,13,and 14 decreased,whereas non-canonical WNT4 and 11 increased,and WNT5A was unchanged. A small molecule WNT mimetic,SB-216763,increased accumulation of β-catenin protein,inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast,knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells. View Publication

Pluripotent stem cells,both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC),can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However,the tumorigenic potential of these cells remains a great concern,as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice,most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal,cardiac,or endothelial cells prior to human transplantation,drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study,we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells,and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer,whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall,our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation,and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy. View Publication

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect on hPSC vitality and expansion after dissociation in human pluripotent stem cells. View Publication

Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay,we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives,but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response,revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK,a key NHEJ component,by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast,NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus,DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells. View Publication

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A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast,we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34,lineage markers,CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells),endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo,CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials,characterized by the expression of c-Kit and CXCR4 as well as EphB4,EphB2,and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life. 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

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis and limited therapeutic options. Leukemic stem cells (LSCs),which drive disease progression and confer resistance to therapy,pose a significant challenge to conventional treatment strategies. In this study,we identified and characterized the inhibitory mechanisms of TH37,a small molecule derived from traditional Chinese medicine,which selectively targets AML blasts and LSCs. Our analyses identified peroxiredoxin 1 (PRDX1),an enzyme that catalyzes the breakdown of hydrogen peroxide (a reactive oxygen species),as the primary molecular target of TH37. We demonstrated that TH37 directly interacts with PRDX1,inhibiting its enzymatic activity and thereby elevating intracellular reactive oxygen species levels in AML cells. PRDX1 was found to be overexpressed in AML,and its expression correlated with poor prognosis and the activation of AML- and cancer-associated pathways. Targeting PRDX1,either through lentiviral short-hairpin RNA-mediated silencing or TH37 treatment,induced apoptosis,reduced colony formation,and impaired the engraftment and growth of AML cells in immunodeficient mouse models. Furthermore,TH37 synergized with conventional chemotherapeutic agent to significantly reduce the viability and colony-forming capacity of AML cells. These findings demonstrate the critical role of PRDX1 in AML pathogenesis and highlight its potential as a key therapeutic target to improve clinical outcomes for AML patients. View Publication

Primary human endothelial cells represent an essential tool to model endothelial dysfunction and to screen interventions for its treatment. Here,we developed a protocol for the synchronous isolation of primary human saphenous vein endothelial cells (HSaVEC),human internal thoracic artery endothelial cells (HITAEC),and human microvascular endothelial cells (HMVEC) from SV and ITA utilized as conduits during coronary artery bypass graft surgery and from subcutaneous adipose tissue excised while providing an access to the heart. Treatment by collagenase type IV and magnetic separation with anti-CD31-antibody-coated beads ensured relatively high efficiency of the isolation (≈60% for HSaVEC,≈50% for HITAEC,and ≈20% for HMVEC) and high purity (≥99%) of isolated ECs within ≈2 weeks (HSaVEC),≈2–3 weeks (HITAEC),and ≈3–4 weeks (HMVEC). A colorimetric assay of cell viability and proliferation,as well as real-time bioimpedance monitoring using the xCELLigence instrument,demonstrated high proliferative activity in HSaVEC,HITAEC,and HMVEC,whilst the in vitro tube formation assay indicated their angiogenic potential. The isolation of HSaVEC,HITAEC,and HMVEC from patients undergoing coronary artery bypass graft surgery is a promising option to investigate endothelial heterogeneity,to interrogate endothelial responses to various stresses,and to pinpoint the optimal approaches for restoring endothelial homeostasis,thereby reproducing them within the bedside-to-bench-to-bedside concept. View Publication

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder,with no effective treatment currently available. Recently,non-pharmacological therapy,especially gamma frequency stimulation has shown promising therapeutic effects in Alzheimer’s disease (AD) mouse models. Electric field (EF) is a non-invasive biophysical approach for neuronal protection. However,whether EF is beneficial in AD neuropathology remains unknown. In this study,we exposed the P301S tauopathy mouse model to EF at gamma frequency on the head. We demonstrated that EF treatment significantly improved the cognitive impairments in the P301S mice. This was accompanied by reduced tau pathologies,suppressed microglial activation,neuroinflammation and oxidative stress in the tauopathy mouse brain. Moreover,EF treatment induced cell-specific responses in neural cells,with neurons being more susceptible,followed by microglia and oligodendrocytes. EF also had favorable effects on synaptic protein in neurons,inflammatory response and complement signaling in microglia,and myelination in oligodendrocytes. This study provides strong evidence that EF at gamma frequency may have great potential to be a novel therapeutic intervention for P301S by attenuating neuropathology and offering neuroprotection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13195-025-01859-8. View Publication