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3D constructs composed of differentiated immortalized primary normal human bronchial epithelial (NHBE) cells (CL-1548) were repeatedly exposed at the air-liquid interface to non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day,5days/week,8 repetitions in total) and e-cigarette vapor (50 puffs a day,5 days/week,8 repetitions in total) to build up a permanent burden on the cells. Samples were taken after 4,6 and 8 times of repeated smoke exposure and the cultures were investigated using histopathological methods Compared to the clean air-exposed cultures (process control) and incubator control,the aerosol-exposed cultures showed a reduction of ciliated,mucus-producing and club cells. At the end of the exposure phase,we even found metaplastic areas positive for CK13 antibody in the cultures exposed to mainstream cigarette smoke and e-liquid vapor,commonly seen in squamous cells as a marker for non-cornified squamous epithelium. The control cultures (incubator cells) showed no comparable phenotypical changes. In conclusion,our in vitro model presents a valuable tool to study the induction of phenotypical changes after exposure to hazardous airborne material. View Publication

Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose,lactate,extracellular pH and cell cycle phases were quantified. Without daily feeding,FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast,RGA cultured cells grew to confluence over 3 days. Surprisingly,glucose was not exhausted under any condition. However,extracellular pH reached 6.8 after 72 h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle,while adding an additional 10 mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification,a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification,while still maintaining pluripotency and allowing longer feeding cycles. View Publication

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation,however,remain to be fully elucidated. Here,we demonstrate that Sox8,a member of the SRY-related HMG box transcription factor family,is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression,which is the hallmark of functionally mature M cells. Furthermore,genetic deletion of Sox8 causes a marked decrease in the number of mature M cells,resulting in reduced antigen uptake in Peyer's patches. Consequently,juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses. View Publication

A growing body of clinical literature has described neurodevelopmental delays in infants with chronic prenatal opioid exposure and withdrawal. Despite this,the mechanism of how opioids impact the developing brain remains unknown. Here,we developed an in vitro model of prenatal morphine exposure and withdrawal using healthy human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in monolayer. To optimize our model,we identified that a longer neural induction and regional patterning period increases expression of canonical opioid receptors mu and kappa in midbrain neural progenitors compared to a shorter protocol (OPRM1,two-tailed t-test,p =? 0.004; OPRK1,p =? 0.0003). Next,we showed that the midbrain neural progenitors derived from a longer iPSC neural induction also have scant toll-like receptor 4 (TLR4) expression,a key player in neonatal opioid withdrawal syndrome pathophysiology. During morphine withdrawal,differentiating neural progenitors experience cyclic adenosine monophosphate overshoot compared to cell exposed to vehicle (p =? 0.0496) and morphine exposure conditions (p,=? 0.0136,1-way ANOVA). Finally,we showed that morphine exposure and withdrawal alters proportions of differentiated progenitor cell fates (2-way ANOVA,F =? 16.05,p View Publication

During gastrulation,the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT),which is marked by cadherin switching,where E-Cadherin is downregulated,and N-Cadherin is upregulated. To understand the relationships between differentiation,EMT,and cadherin switching,we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols,as well as variants in which signaling through key pathways including Activin,BMP,and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus,cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently,E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall,cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation. View Publication

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are remarkably effective for treating EGFR-mutant non-small cell lung cancer (NSCLC). However,patients inevitably develop acquired drug resistance,resulting in recurrence or metastasis. It is important to identify novel effective therapeutic targets to reverse acquired TKI resistance. Bioinformatics analysis revealed that nicotinamide N-methyltransferase (NNMT) was upregulated in EGFR-TKI resistant cells and tissues via EGR1-mediated transcriptional activation. High NNMT levels were correlated with poor prognosis in EGFR-mutated NSCLC patients,which could promote resistance to EGFR-TKIs in vitro and in vivo. Mechanistically,NNMT catalyzed the conversion of nicotinamide to 1-methyl nicotinamide by depleting S-adenosyl methionine (the methyl group donor),leading to a reduction in H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3) and subsequent epigenetic activation of EGR1 and ALDH3A1. In addition,ALDH3A1 activation increased lactic acid levels,which further promoted NNMT expression via p300-mediated histone H3K18 lactylation on its promoter. Thus,NNMT mediates the formation of a double positive feedback loop via EGR1 and lactate,EGR1/NNMT/EGR1 and NNMT/ALDH3A1/lactate/NNMT. Moreover,the combination of a small-molecule inhibitor for NNMT (NNMTi) and osimertinib exhibited promising potential for the treatment of TKI resistance in an NSCLC osimertinib-resistant xenograft model. The combined contribution of these two positive feedback loops promotes EGFR-TKI resistance in NSCLC. Our findings provide new insight into the role of histone methylation and histone lactylation in TKI resistance. The pivotal NNMT-mediated positive feedback loop may serve as a powerful therapeutic target for overcoming EGFR-TKI resistance in NSCLC. The online version contains supplementary material available at 10.1186/s12943-025-02285-y. View Publication

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging,however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS),DNA damage,senescence,and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC,as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically,CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress,as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC),improved their niche supporting activity,and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production,driving an HSC aging phenotype. Thus,modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function. View Publication

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these,miR-1305,which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells,increased cell apoptosis and sped up G1/S transition,while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast,knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency,thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency,cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells,we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model,donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly,highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer,accumulated in vivo at advanced proliferative cycles,and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted,apoptosis-resistant phenotype,including: 1) reduced apoptosis after staurosporine addition,serum starvation,or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection,we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4,IL-10,perforin,or Fas ligand; 2) could not be reversed by IL-2,IL-7,or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis,persist in vivo,and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells. View Publication

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf(-/-)) mice die at day 14.5 of gestation from severe anemia. In this study,we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf(-/-) embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis,transcriptional profiling was performed with RNA from wild-type and Eklf(-/-) early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation,with the critical regulator of the cell cycle,E2f2,at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf(-/-) early erythroid progenitor cells,which showed a delay in the G(1)-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier,EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation. View Publication

Killer cell lectin-like receptor F1 (KLRF1) is an activating C-type lectin-like receptor expressed on human NK cells and subsets of T cells. In this study,we show that activation-induced C-type lectin (AICL) is a unique KLRF1 ligand expressed on tumor cell lines of hematopoietic and non-hematopoietic origins. We screened a panel of human tumor cell lines using the KLRF1 reporter cells and found that several tumor lines expressed KLRF1 ligands. We characterized a putative KLRF1 ligand expressed on the U937 cell line. The molecular mass for the deglycosylated ligand was 28 kDa under non-reducing condition and 17 kDa under reducing condition,suggesting that the KLRF1 ligand is a homodimer. By expression cloning from a U937 cDNA library,we identified AICL as a KLRF1 ligand. We generated mAbs against AICL to identify the KLRF1 ligands on non-hematopoietic tumor lines. The anti-AICL mAbs stained the tumor lines that express the KLRF1 ligands and importantly the interaction of KLRF1 with the KLRF1 ligand on non-hematopoietic tumors was completely blocked by the two anti-AICL mAbs. Moreover,NK cell degranulation triggered by AICL-expressing targets was partially inhibited by the anti-AICL mAb. Finally,we demonstrate that AICL is expressed in human primary liver cancers. These results suggest that AICL is expressed on tumor cells of non-hematopoietic origins and raise the possibility that AICL may contribute to NK cell surveillance of tumor cells. View Publication

BACKGROUND Granulocyte colony-stimulating factor (G-CSF) is a pro-inflammatory cytokine that stimulates myeloid stem cell maturation,proliferation,and migration into circulation. Despite being a known growth factor,the impact of G-CSF on solid tumours has not been well examined. G-CSF receptor (G-CSFR) is expressed by some tumours,and thus the aim of this study was to examine the expression and impact of G-CSF and G-CSFR on gastrointestinal tumours. METHODS In this study,G-CSF expression was examined in human gastric and colon tumours and by tumour-derived stromal myofibroblasts and carcinoma cells. G-CSFR expression was examined on carcinoma cells isolated from human tissues. The effects of G-CSF on gastric and colon carcinoma cell proliferation,migration,and signalling were examined. RESULTS G-CSFR was highly expressed in 90% of human gastric and colon carcinomas. G-CSF was also found to be highly produced by stromal myofibroblasts and carcinoma cells. Exposure of carcinoma cells to G-CSF led to increased proliferation and migration,and expansion of a sub-population of carcinoma cells expressing stem-like markers. These processes were dependent on ERK1/2 and RSK1 phosphorylation. CONCLUSIONS These data suggest that the G-CSF/R axis promotes gastric and colorectal cancer development and suggest they are potential tumour targets. View Publication