Background Hyperlipidemia is prevalent in primary immune thrombocytopenia (ITP) and may exacerbate bleeding severity and compromise treatment responses. Statins possess lipid-lowering and anti-inflammatory properties, but their influence on corticosteroid therapy in ITP patients with hyperlipidemia remains unclear. Objectives This study aimed to compare the efficacy and safety of initial corticosteroid therapy between patients with or without concomitant statin use for pre-existing hyperlipidemia in a cohort of newly diagnosed ITP patients. Design A multicenter retrospective propensity score-matched study. Methods Newly diagnosed ITP patients with pre-existing hyperlipidemia from 9 medical centers were analyzed. Patients were divided into 2 groups based on statin use (statin vs . non-statin). Propensity score matching (PSM) was employed to balance baseline characteristics. Treatment outcomes, including response rates, duration of response (DOR), and safety, were compared. Results After PSM, 104 patients (52 per group) were included. Baseline characteristics were comparable. Initial response rates were similar between the statin and non-statin groups (67.3% vs. 65.4%, P = 0.836), with the 95% confidence interval (CI) for the between-group risk difference ranging from -15.83% to 19.51%. The median time to response was numerically shorter in the statin group, although this difference did not reach statistical significance (4 [2-8] days vs. 5 [2-12] days, P = 0.054). Notably, the sustained response (SR) rate at 6 months was significantly higher in the statin group compared to the non-statin group (50.0% vs. 30.8%, P = 0.046), with the 95% CI for the between-group risk difference ranging from 0.41% to 36.25%. Kaplan-Meier analysis also demonstrated a significant longer duration of response (DOR) in the statin group ( P = 0.044). No serious adverse events were observed in either group during follow-up. Conclusion In this retrospective, propensity score-matched cohort, concomitant statin use was associated with higher 6-month SR rates and longer DOR in newly diagnosed ITP patients with pre-existing hyperlipidemia, without an apparent increase in serious adverse events. These observational findings warrant further validation in larger, prospective cohorts.

Hematopoietic stem and progenitor cells (HSPCs) give rise to the blood system and maintain hematopoiesis throughout the human lifespan. Here, we report a transcriptional census of human bone-marrow-derived HSPCs from the neonate, infant, child, adult, and aging stages, showing two subpopulations of multipotent progenitors separated by CD52 expression. From birth to the adult stage, stem and multipotent progenitors shared similar transcriptional alterations, and erythroid potential was enhanced after the infant stage. By integrating transcriptome, chromatin accessibility, and functional data, we further showed that aging hematopoietic stem cells (HSCs) exhibited a bias toward megakaryocytic differentiation. Finally, in comparison with the HSCs from the cord blood, neonate bone-marrow-derived HSCs were more quiescent and had higher long-term regeneration capability and durable self-renewal. Taken together, this work provides an integral transcriptome landscape of HSPCs and identifies their dynamics in post-natal steady-state hemopoiesis, thereby helping explore hematopoiesis in development and diseases.

BackgroundSevere and persistent cancer treatment-induced thrombocytopenia (CTIT) remains a major obstacle to continuation of antineoplastic therapy and faces an increased risk of bleeding. Standard therapy has yet to be established in this setting.ObjectivesThis study aimed to evaluate the efficacy of avatrombopag switching from other thrombopoietic agents and baseline factors associated with non-response or early response in severe and persistent CTIT.MethodsIn this multicentre real-world cohort study, we included 78 patients with solid tumors or non-myeloid hematologic malignancies who showed no response or loss of response to prior thrombopoietic agents and had grade 4 thrombocytopenia for at least 4 weeks. Platelet response and relevant influencing factors, transfusion dependence, interruptions to antineoplastic therapy, and safety were assessed.ResultsMedian baseline platelet count was 10 × 109/L and increased to 102 × 109/L after switching to avatrombopag (p < 0.001). Platelet response rates increased over time, from 28.2% at week 4 to 43.6% at week 8 and 52.6% at week 12. The overall difference across these time points was significant (p < 0.001). After switching therapy, platelet transfusion dependence decreased significantly from 69.2% to 19.2% (p < 0.001), along with interruptions to antineoplastic therapy decreased from 88.5 to 50.0% (p < 0.001). The median time to platelet response was 11.7 weeks. Hemoglobin>123 g/L at baseline might be associated with early platelet response. Major bleeding at baseline might independently predicted poor platelet response across different time points. No grade ≥3 adverse events were observed.ConclusionSwitching to avatrombopag was associated with platelet recovery, reduced transfusion dependence, fewer interruptions to antineoplastic therapy, and an acceptable safety profile for severe and persistent CTIT.

ABSTRACT Extramedullary disease (EMD) in newly diagnosed multiple myeloma (NDMM) is aggressive and associated with worse survival. This study aimed to comprehensively evaluate the clinical outcomes and factors affecting prognosis in NDMM patients with EMD receiving novel agents‐based induction therapy. EMD presented in 94 cases (21.2%) among 443 screened NDMM patients. Eighty‐five EMD patients with complete clinical and follow‐up data were subdivided into extramedullary bone‐related disease (EMB, n = 55) and extramedullary extraosseous disease (EME, n = 30). EMD patients did not present a significantly high tumor burden or worse disease stage than non‐EMD patients. With novel agents‐based induction therapy, the overall response (OR) rate in the EMD group was significantly lower than in the non‐EMD group (88.2% vs 96.8%, p = 0.005), but was comparable between EMB and EME (90.9% vs 83.3%, p = 0.494). The number of patients achieving complete remission (CR) or stringent CR (sCR) in the EME group was significantly less than that in the EMB or non‐EMD group. After a median follow‐up of 49 months, EME patients showed significantly inferior progression‐free survival (PFS) compared with non‐EMD and EMB patients (median PFS: 20 vs 39 vs 32 months, p = 0.000). Exploratory subgroup analyses suggested that patients with EME and high‐risk cytogenetic abnormalities (HRCAs) experienced the most unfavorable survival outcomes, although these findings require validation in larger cohorts. Patients achieving sCR/CR demonstrated significantly prolonged PFS and overall survival (OS). Autologous stem cell transplantation (ASCT) was associated with improved survival. Novel agents‐based induction therapy achieved favorable responses and encouraging survival outcomes in patients with EMD, particularly among those achieving deep remissions. EME patients appear to represent a clinically aggressive disease phenotype whose adverse prognosis is closely intertwined with high‐risk clinical and cytogenetic characteristics rather than being entirely independent. Larger prospective multicenter studies are warranted to validate these findings and optimize treatment strategies for patients with EMD.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) cures hematological malignancies but severely impairs long-term immune reconstitution by inducing T cell senescence. Previous studies have linked anti-tumor therapy to immune aging; however, it remains unclear whether haploidentical HSCT (haplo-HSCT) independently drives progressive premature T cell senescence beyond pre-transplant chemotherapy, and how the recipient microenvironment shapes donor T cell aging patterns. This study aimed to determine the independent effect of haplo-HSCT on time-dependent T cell senescence and to elucidate the bidirectional regulatory role of recipient age-associated microenvironments on phenotypic and molecular aging signatures of donor-derived T cells. A total of 34 subjects were enrolled, including 24 haplo-HSCT recipients stratified by post-transplant duration (<12 months, n = 12; ≥12 months, n = 12) and 10 patients receiving chemotherapy alone. Eighteen age-reciprocal donor-recipient pairs were analyzed. Multiparameter flow cytometry, multiplex cytokine assay, SASP PCR array, epigenetic clock profiling, and telomere length measurement were performed to comprehensively characterize T cell senescence features. Haplo-HSCT induced robust, time-dependent senescence in both CD4⁺ and CD8⁺ T cells, accompanied by substantial T cell subset remodeling. This included a reduced CD4/CD8 ratio, contracted naive and central memory T cell compartments, and expansion of senescence-prone terminal effector memory T cells, which was particularly pronounced in the CD8⁺ compartment. Terminal senescent CD27⁻CD28⁻CD57⁺ T cells (both CD4⁺ and CD8⁺) from long-term recipients exhibited significantly upregulated canonical senescence molecules p16INK4a and γH2AX, providing solid molecular evidence for transplant-associated T cell senescence. In contrast, chemotherapy alone failed to induce comparable terminal T cell senescence phenotypes. Haplo-HSCT further established persistent peripheral inflammatory imbalance and remodeled the prosenescent SASP transcriptional profile of circulating T cells. Notably, recipient age exerted bidirectional modulation of donor T cell epigenetic aging, telomere homeostasis, and SASP remodeling. Haplo-HSCT serves as an independent, time-dependent driver of premature donor T cell senescence, distinct from chemotherapy-induced immune alteration. The recipient age-related microenvironment is a critical regulator of donor T cell epigenetic aging, telomere maintenance and SASP reprogramming. This study systematically delineates multi-layered signatures of post-transplant T cell senescence and provides a theoretical foundation for developing age-adapted strategies to improve long-term immune reconstitution after haplo-HSCT.

ABSTRACT:CD4+CD25+Foxp3+ regulatory T cells (Tregs) are pivotal negative regulators of the adaptive immune system. Abnormalities in the number and/or function of Tregs contribute to the pathogenesis of primary immune thrombocytopenia (ITP). Strategies aimed at modulating Tregs offer potential therapeutic opportunities for ITP management. In this study, we demonstrated that inhibition of cyclin-dependent kinase 8 (CDK8) and CDK19 activity by the small-molecule inhibitor AS2863619 (AS) robustly promoted the conversion of CD4+CD25- effector T cells (Teffs) into CD4+CD25+Foxp3+ Tregs, endowing the converted Tregs with lineage stability and potent suppressive capacity. Mechanistically, AS rapidly augmented STAT5 phosphorylation and subsequent Foxp3 induction. STAT5 blockade completely abrogated this effect, confirming that the Treg-promoting activity of AS was critically dependent on STAT5 signaling. In parallel, AS suppressed STAT3 phosphorylation under interleukin-6-driven conditions, thereby attenuating T helper 17 (Th17) polarization. These mechanistic findings were supported by global transcriptomic analysis, which revealed a profound transcriptional shift by broadly suppressing gene programs of Teff differentiation and function while simultaneously upregulating a robust signature characteristic of stable Tregs. Crucially, unbiased upstream analysis of these changes pinpointed STAT5, STAT3, and FOXP3 as the core transcription factors mediating the drug's effect. Functional metabolic analysis further revealed that AS mediated metabolic reprogramming in T cells by suppressing glycolysis, thereby providing the necessary metabolic adaptations for Treg conversion. In a murine model of active ITP, CDK8/CDK19 inhibition elevated Treg frequencies and ameliorated thrombocytopenia in a STAT5-dependent manner. Collectively, our study highlighted the therapeutic potential of CDK8/CDK19 inhibition in restoring immune homeostasis and managing ITP.

Corticosteroid resistance is a major obstacle to the management of patients with primary immune thrombocytopenia (ITP), as the factors contributing to the variability in treatment response remain largely unknown. We analyzed a broad set of plasma protein biomarkers from patients who are corticosteroid-sensitive (CSp; n = 30), patients who are corticosteroid resistant (CRp; n = 26), and healthy controls (HCs; n = 25) using a 92-plex immunoassay from Olink Proteomics Technology. A total of 54 inflammation-related proteins demonstrated significant differences among the three groups. Twenty-seven biomarkers showed statistical differences between CSp and CRp. Machine learning-based feature selection identified four potential biomarkers, which were closely related to corticosteroid resistance: CXCL10, IL-1α, glial cell line-derived neurotrophic factor (GDNF), and CCL11. A Nomograph model was developed based on these 4 biomarkers, demonstrating remarkable discriminative ability, with an area under the curve (AUC) of 0.920 (95% confidence interval: 0.830-1.000) in this exploratory cohort. Although requiring external validation in larger studies, the identification of the four potential biomarkers suggested their value in predicting corticosteroid resistance in patients with newly diagnosed ITP, and might guide the initial choice of treatment.

ABSTRACT:Immune thrombocytopenia (ITP) is characterized by the overproduction of antiplatelet autoantibodies. Although B-cell depletion therapies show promise in ITP, their high relapse rates suggest a potential de novo breakdown of tolerance during an early stage of B-cell development. Here, we investigated how central B-cell tolerance mechanisms affect autoantibody production in ITP. Paired single-cell RNA/B-cell receptor (BCR) sequencing and bulk BCR sequencing revealed reduced V-J genomic distances in immunoglobulin kappa-chain (IGK) genes within bone marrow and peripheral B cells from patients with ITP, along with decreased expression of recombination-activating gene in the immature B cells, suggesting insufficient receptor editing. Single-cell antibody cloning demonstrated increased autoreactive and polyreactive naïve B cells in ITP, indicating defective central B-cell tolerance. Through an in vivo study, we established a causal link between receptor editing defects and antiplatelet antibody production, validating the immature B-cell stage as the key phase of dysregulation. These findings suggest that insufficient receptor editing of immature B cells triggers central B-cell tolerance deficiency and autoantibody accumulation in ITP.

While platelets are well-documented contributors to tumorigenesis, their role in multiple myeloma (MM) progression and risk stratification remains underexplored. To assess platelet function in MM and verify the prognostic value of platelet-related genes(PRGs) in patients with multiple myeloma, further providing new ideas for the development of MM. We combined the clinical assessment of platelet activation in MM patients with functional co-culture experiments using MM cell lines (RPMI8226, MM.1 S) to investigate platelet-driven tumor progression. Additionally, an integrated analysis of bulk (GSE124310) and single-cell transcriptomic datasets (GSE6477, TCGA-MM data, GSE4581, GSE24080,and GSE136337) was performed to identify platelet-related prognostic genes (PRGs). Through single-cell RNA sequencing, we identified aberrant erythroid-megakaryocyte components in multiple myeloma and further demonstrated dysfunctional platelet activity that promotes tumor cell proliferation and suppresses apoptosis. Using comprehensive bioinformatic screening across 116 algorithms, we identified a combined forward stepwise Cox and Ridge regression model as optimal and established a 13-gene platelet-related prognostic signature. The genetic risk model effectively stratified MM patients into distinct prognostic groups, with high-risk patients exhibiting poorer outcomes in both training and validation cohorts. Finally, we integrated the genetic risk model and clinically relevant information and visualized it with dynamic Nomogram plots, and the ROC and DCA curves demonstrated that the integrated model had better predictive ability. Our study establishes a significant association between platelet activity and disease progression in MM. The platelet-related prognostic signature we developed is correlated with patient outcomes and may have utility in risk stratification.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction and impaired regulatory T-cell (Treg) function, yet its molecular basis remains poorly defined. Here, we show that CD4+ naive T cells from patients with ITP exhibit diminished T-cell receptor (TCR) signaling and impaired in vitro Treg induction relative to healthy controls. RNA-sequencing revealed widespread alternative splicing dysregulation, most notably exon 8 skipping in LCK, a kinase central to TCR signaling and Treg differentiation. Using an LCK minigene combined with RNA pull-down mass spectrometry and RNA immunoprecipitation assays, we identified the splicing factor SRSF1 as a direct upstream regulator of LCK exon 8 skipping. Notably, SRSF1 expression was reduced in CD4+ naive T cells from ITP patients, and its overexpression restored in vitro Treg induction capacity. Antisense oligonucleotide (ASO)-mediated blockade of SRSF1 binding to LCK enhanced exon 8 skipping and attenuated TCR activation in Jurkat cells. Although murine Lck lacks the human-specific recursive splicing sites required for exon 8 exclusion, adoptive transfer of CD4+ naive T cells expressing the exon 8-skipped murine Lck into CD61-knockout mice significantly reduced Treg proportions and platelet counts in an active ITP model. Mechanistically, Jurkat cells engineered to express only the exon 8-skipped LCK variant showed markedly reduced binding to ZAP70 and CD3ζ, which may partly account for the attenuated TCR signaling and downstream FOXP3 induction. Together, these findings define a novel SRSF1-LCK splicing axis that may regulate Treg development in ITP.

About 25% of patients with myelodysplastic syndromes (MDS) have combined autoimmune diseases (AIDs). However, the relationships between MDS and AIDs, especially a causal relationship and the underlying shared pathophysiological mechanisms, remain largely unknown. We aimed to evaluate the association between MDS and AIDs using a multicenter retrospective study, Mendelian randomization (MR), and bioinformatics analysis. About 26.6% of patients with MDS from all centers presented with AIDs. Compared to MDS patients without AIDs, MDS with AIDs was less likely to progress to acute myeloid leukemia (AML) (6.6% vs. 15.1%, p = 0.037), and the pre-existing AIDs could be used as an independent protective factor of survival (HR: 0.504, p = 0.048). Bidirectional MR results showed that MDS could cause the risk of systemic lupus erythematosus (SLE, OR: 1.09, p = 0.015), although with no significant causal relationship in other AIDs. The effect of MDS on SLE may be partially mediated by naïve CD4+ T-cells (median proportion 6.9%) and CD45RA-CD4+ T memory cells (median proportion 9.0%). Furthermore, two hub genes (IFI27 and VSIG4) were identified by machine learning and curve analysis as potential diagnostic markers for MDS with SLE. Our study suggested that the impact and mechanisms of AIDs in MDS need to be taken seriously, which could provide more accurate treatment guidance.

Postacute coronavirus disease 2019 (COVID-19) syndrome (PACS), or long COVID, encompasses a range of symptoms persisting beyond the acute phase of severe acute respiratory syndrome coronavirus 2 infection. Although acute-phase coagulation disturbances in COVID-19 are well documented, these abnormalities during recovery and their association with PACS remain inadequately explored. Our study aimed to investigate the long-term changes in coagulation function and inflammatory markers in patients with PACS, elucidating their pathological mechanisms and providing insights for patient management. This retrospective cohort study included 3783 adult inpatients in Jinan, China, divided into COVID-19-positive and -negative groups, with 363 patients with COVID-19 further diagnosed with PACS. Coagulation and inflammatory markers were collected at baseline and during 1-year follow-up, and changes over time were analyzed using generalized estimating equations. Most inflammatory markers and some coagulation parameters showed significant recovery, including lymphocyte counts and fibrinogen. However, several parameters remained abnormal even at 7 to 12 months after infection. Of note, D-dimer (Z = 5.692, P < .001, abnormal rate 65.79%) and erythrocyte sedimentation rate (Z = 2.749, P = .006, abnormal rate 57.32%) remained elevated above the normal upper limit. Additionally, certain coagulation parameters, particularly prothrombin time (β = -0.10 [95% confidence interval, -0.88 to 0.69]; P = .81, prolonged rate 17.29%) and platelet counts, did not normalize by 7 to 12 months. Our findings in survivors of severe COVID-19 pneumonia support the concept of PACS as a chronic thromboinflammatory syndrome characterized by sustained coagulation abnormalities. The prolonged elevation of D-dimer and incomplete recovery of coagulation parameters highlight the need for long-term monitoring and personalized management strategies to mitigate thrombotic risks in survivors of COVID-19.

Our previous clinical studies show that all-trans retinoic acid (ATRA) induces a sustained response in patients with immune thrombocytopenia (ITP). However, its mechanisms of action remain unclear. In this study, we observed disorganized cytoskeleton and impaired proplatelet formation (PPF) in megakaryocytes from patients with ITP. Metabolite profiling revealed reduced sphingosine 1-phosphate (S1P) levels in ITP. Decreased sphingosine kinase 2 (SPHK2) expression was responsible for the low S1P levels in ITP. In addition, S1P was essential for activating S1P receptor 1 and Rac1, which regulate cytoskeletal reorganization and PPF. Furthermore, hypoxia-inducible factor-1α (HIF-1α) was shown to mediate SPHK2 and S1P production. Decreased HIF-1α expression in megakaryocytes from patients with ITP contributed to impaired PPF. We subsequently found that ATRA up-regulated HIF-1α and corrected impaired PPF in vitro and in vivo. These findings reveal that ATRA targets the HIF-1α/SPHK2/S1P pathway to improve PPF dysfunction, offering mechanistic insights into its clinical efficacy in ITP.

Advances in label-free optical detection technologies hold significant scientific and clinical value, such as for the diagnosis of leukemia diseases, yet single-modality techniques often miss diverse biophysical features of cancer cells. We present a novel label-free multimodal imaging system for intelligent identification of patient-derived leukemia cells by simultaneously obtaining the light scattering imaging (LSI) and quantitative phase imaging (QPI) modalities. LSI captures defocused scattering patterns sensitive to subwavelength morphological features, while QPI provides quantitative biophysical maps of intracellular content including refractive index (RI), together enabling comprehensive single-cell characterization unattainable by either modality alone. The system simultaneously determines particle size with sub-micron resolution and RI, enabling the independent extraction of these complementary features. In our technological validation, multimodal fusion achieved 98.2% accuracy for distinguishing chronic lymphocytic leukemia (CLL) from chronic myeloid leukemia (CML) cells, outperforming single modalities by at least 1.8%. Furthermore, it is interesting to note that LSI exceeded QPI by 2.1% in classifying CLL from normal cells (NCs), while QPI surpassed LSI by approximately 12% for CML from NCs, indicating that neither modality consistently outperformed the other across diverse sample types. Notably, multimodal integration consistently yielded the highest classification accuracy, outperforming either single-modality approach. These findings demonstrate that in the absence of a priori knowledge regarding which cellular features are most diagnostically relevant for unknown cancer samples, simultaneous acquisition of complementary scattering and phase information provides a robust and broadly applicable strategy for cytopathological characterization, providing a powerful tool for potential diagnosis of hematological malignancies.

BACKGROUND:Evans syndrome is a rare autoimmune disease characterized by simultaneous or sequential primary immune thrombocytopenia and autoimmune hemolytic anemia. Despite the low incidence of Evans syndrome after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, its progression may threaten public health. This review offers an up-to-date summary of the works on the association between coronavirus disease 2019 (COVID-19) and Evans syndrome to explore the pathogenic mechanisms, epidemiological characteristics, clinical presentations, diagnostic markers, and treatment strategies. MATERIAL AND METHODS:We searched PubMed and Web of Science to identify articles that explored the relationship between COVID-19 and Evans syndrome. We collected and organized all reported cases of Evans syndrome following COVID-19 or SARS-CoV-2 vaccination over the past 4 years and also expanded the search to examine other cases of post-infection Evans syndrome. RESULTS:Thirteen cases were included with an average age of 42 years of whom 12 survived and one died. Two cases were associated with pregnancy and four with vaccination, two involved epileptic seizures, and three had a history of autoimmune disease. DISCUSSION:Patients with Evans syndrome and exposure to SARS-CoV-2 have a potential risk of bleeding. This risk should prompt close monitoring of bleeding biomarker dynamics and early initiation of hemostatic treatments, including platelet transfusion, corticosteroids, thrombopoietin receptor agonists, intravenous immunoglobulin and rituximab.

Background: CD8+ T cells participate in the pathogenesis of primary immune thrombocytopenia (ITP). Natural killer cell granule protein 7 (NKG7) is essential for natural killer cell and CD8+ T cell cytotoxicity. The function of NKG7 in CD8+ T cells in ITP remains unclear. Objectives: We investigated the expression and roles of NKG7 in CD8+ T cells in ITP. Methods: We analyzed NKG7 and CD107a expression and CD8+ T cell-mediated platelet apoptosis in patients with ITP and controls. NKG7 knockdown was performed using small interfering RNA, and the extracellular signal-regulated kinases 1 and 2 pathway was analyzed by western blot analysis. Results: NKG7 was significantly increased in CD8+ T cells and positively correlated with CD107a and CD8+ T cell-induced platelet apoptosis in ITP. Based on NKG7 levels, patients with ITP were divided into NKG7 high-expression and low-expression groups. Patients with high expression of NKG7 had significantly higher levels of CD107a and CD8+ T cell-induced platelet apoptosis than controls, whereas no difference was found between patients with low NKG7 expression and controls. Platelet counts in patients with high NKG7 expression were significantly lower than those in patients with low NKG7 expression. We knocked down NKG7 in CD8+ T cells from patients with ITP and found decreased CD107a expression and less platelet apoptosis in vitro. We further found that NKG7 affected the cytotoxicity of CD8+ T cells through the extracellular signal-regulated kinase 1 and 2 pathway. Conclusion: NKG7 plays an important role in CD8+ T cell-mediated cytotoxicity might be a potential therapeutic target for ITP.

Introduction Hematopoietic stem cells (HSCs) form the basis of the hematopoietic system, with essential functions including long-term self-renewal and the generation of all mature blood cell lineages in vivo. Their development relies on the endothelial-to-hematopoietic transition (EHT) of endothelial cells in the aorta-gonad-mesonephros (AGM) region. Mechanical forces, particularly fluid shear stress, have been shown to play a crucial role in HSCs emergence during embryogenesis. Piezo1, a key mechanosensitive ion channel protein in endothelial cells, is known to regulate various physiological processes; however, its specific role in EHT remains unclear. This study aims to elucidate the function of Piezo1 in HSCs generation during EHT, investigate the in vitro hematopoietic function and in vivo transplantation reconstitution potential of hemogenic endothelial cells (HECs) after Piezo1 knockout, and explore the mechanisms by which Piezo1 responds to embryonic hemodynamic changes to regulate EHT in the murine AGM region. Methods We generated an endothelial cell-specific Piezo1 knockout mouse model. Flow cytometry was used to detect fetal liver hematopoietic activity from E12.5 to E14.5 mouse embryos. Meanwhile, the AGM region of E10.5 mice was dissected to quantify the number and proportion of hemogenic endothelial cells and hematopoietic progenitor cells, and the colony-forming ability of AGM-derived cells was further evaluated. Subsequently, we sorted HECs from the AGM region using flow cytometry for in vitro colony-forming assays and OP9 co-culture experiments to examine the impact of Piezo1 knockout on their hematopoietic potential. Concurrently, in vivo transplantation experiments were conducted using mouse AGM-derived cells, and donor chimerism was measured at different time points post-transplantation to evaluate the effect of Piezo1 knockout on hematopoietic reconstitution capacity of HECs. To explore the underlying mechanisms, we performed RNA sequencing (RNA-seq) on sorted HECs from Piezo1 knockout mice, followed by validation of differentially regulated pathways identified by sequencing and conducted rescue experiments using in vitro assays such as OP-9 co-culture. Results Using an endothelial cell-specific Piezo1 knockout mouse model, we found that Piezo1 deletion inhibited fetal liver hematopoietic activity in E12.5 to E14.5 mouse embryos and reduced the proportions of HSCs and hematopoietic stem/progenitor cells (HSPCs) in the fetal liver. We revealed that these effects were not caused by vascular developmental abnormalities in the fetal liver induced by Piezo1 knockout. Subsequently, analysis of the E10.5 AGM region further revealed that Piezo1 knockout decreased the numbers of HECs and hematopoietic progenitor cells, and significantly impaired the colony-forming capacity of AGM-derived cells in vitro. Flow cytometry–based isolation of HECs from the AGM region, followed by colony-forming assays and OP9 co-culture, confirmed that Piezo1 deficiency compromises the in vitro hematopoietic potential of HECs. Moreover, transplantation experiments showed that AGM-derived cells from Piezo1-deficient mice exhibited a reduced chimerism rate after transplantation, indicating impaired hematopoietic reconstitution capacity. Mechanistically, RNA-seq analysis of isolated HECs from Piezo1 knockout mice revealed downregulation of multiple pathways associated with extracellular matrix remodeling and intercellular junctions. Among these, the Rap1–PI3K signaling pathway was significantly suppressed in Piezo1-deficient HECs, and activation of Rap1 partially rescued the in vitro hematopoietic capacity of HECs. Conclusions Based on these findings, we conclude that Piezo1-mediated mechanical effects act as a key factor in the EHT process, influencing the emergence of functional HSCs during mouse embryogenesis. This study elucidates the specific role and underlying mechanism of Piezo1 in EHT regulation, providing new insights for future understanding of the mechanical mechanism of EHT in embryonic hematopoietic development.

Introduction Aplastic anemia (AA), a rare disorder characterized by bone marrow failure and pancytopenia, poses exceptionally high risks when it occurs during pregnancy. This condition endangers both the mother and fetus, significantly increasing the likelihood of maternal complications such as hemorrhage and infection, as well as adverse perinatal outcomes such as preterm birth and fetal growth restriction. Consequently, pregnancy with AA demands careful management. However, tools to predict these adverse outcomes in affected pregnant women are currently lacking. Here, we applied a machine learning approach to develop and validate a prediction model for adverse pregnancy outcomes in patients with AA, with the goal of guiding early clinical decision-making and improving their overall health outcomes. Methods This study was registered at Clinicaltrials.gov: NCT07101770. We collected data from 310 pregnant women with AA admitted between January 2000 and December 2024 to 15 tertiary hospitals in China. Adverse pregnancy outcomes included at least one of placental abruption, amniotic fluid embolism, postpartum hemorrhage, postpartum infection, maternal mortality, stillbirths, preterm birth, low birthweight, fetal growth restriction, neonatal intensive care unit admission, or neonatal mortality (BJOG, 2014). Feature selection was performed through least absolute shrinkage and selection operator (LASSO) regression. The reliability of the models was evaluated using the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, accuracy, F1 score, calibration plots, and decision curve analysis (DCA). The SHapley Additive exPlanation (SHAP) method was used to rank the feature importance and explain the final model. Results Among the 310 patients with AA (median age, 30.2 [27.6-33.9]), 201 from 7 specialized tertiary hospitals composed the derivation cohort (training set), whereas an independent cohort of 109 patients from 8 distinct academic medical centers formed the external validation set. To ensure robust model development, the training set underwent a stratified random split, yielding a model-building subset (136 patients, 67.7%) and a hold-out internal validation subset (65 patients, 32.3%), preserving the distribution of adverse outcomes, including postpartum hemorrhage, placental abruption, fetal growth restriction, and preterm delivery. In this study, anemia was present in 280 patients (90.3%). Overall, 195 patients (62.9%) experienced adverse pregnancy outcomes. Notably, among the subgroup with severe aplastic anemia (SAA, n=8), the rate of adverse pregnancy outcomes rose significantly to 75.0% (6/8). These findings underscored the high-risk nature of this cohort, particularly those with SAA, highlighting the critical need for accurate prediction tools to guide targeted antenatal interventions. The data for the variables evaluated in this study, including demographic and clinical characteristics, laboratory results, and treatment, were obtained from patient electronic medical records. Using multivariable LASSO regression, we selected the top five features for model construction: age, hemoglobin level, platelet count, neutrophil count, and the percentage of lymphocytes. Seven state-of-the-art machine learning algorithms were rigorously trained and tuned. The RF model emerged as optimal, demonstrating good discriminative ability both in internal validation (AUC: 0.765, 95% CI: 0.737–0.851) and, crucially, in external validation (AUC: 0.743, 95% CI: 0.723–0.814), confirming its generalizability across heterogeneous health care settings. Furthermore, calibration plots revealed agreement between the predicted probabilities and observed event rates, indicating reliability across risk strata. DCA indicated that the clinical implementation of the prognostic model could benefit pregnant women with AA. Conclusions To our knowledge, it's the world's largest cohort of pregnant women with AA to date. We demonstrated that the model could predict the risk of adverse pregnancy outcomes in patients with AA. The model will help clinicians identify pregnant women at high risk early and provide a basis for individualized patient treatment plans.