MedNexus
2025年 · 第07卷第04期
MedNexus
Currently, factor IX replacement is the mainstay treatment for hemophilia B in clinical practice. However, since the disease cannot be cured, lifelong treatment and frequent infusion is required. In recent years, hemophilia B gene therapy has achieved significant advancements, with 3 adeno-associated virus (AAV) vector-based gene therapy products receiving market authorization. Among these, BBM-H901 (Dalnacogene Ponparvovec Injection) has just been approved in China. AAV vector-based gene therapy is characterized by irreversible treatment effects and potential long-term efficacy. However, cases of suboptimal efficacy have been observed in early clinical trials. Eligibility for AAV vector gene therapy primarily depends on factors including patient diagnosis subtype, age, inhibitor status, AAV capsid antibody titer, and patient/family preferences. Given that AAV vector-based gene therapy for hemophilia has become an accessible frontier treatment, Thrombosis and Hemostasis Group and Hemophilia Treatment Center Collaborative Network of China jointly formulated this guidance. It aims to standardize operational procedures and follow-up recommendations to ensure patients receive standardized management when adopting this novel therapeutic approach.
Single-cell multiomics technologies have significantly advanced our understanding of cellular heterogeneity and biological complexity. The joint profiling of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) within the same cell offers a powerful approach for enabling direct linkage between gene expression and chromatin accessibility at single-cell resolution. This integration significantly enhances sensitivity and specificity in identifying rare cell populations and elucidating epigenetic regulatory mechanisms. In this study, we present a robust, high-throughput droplet-based microfluidic protocol that enables simultaneous profiling of RNA and chromatin accessibility from individual cells. The streamlined workflow incorporates key steps, including cell pretreatment, nuclei isolation, gel bead-in-emulsion (GEM) generation, and the construction of scRNA-seq and scATAC-seq libraries. The protocol supports parallel processing of tens of thousands of cells in a single experiment, offering exceptional scalability and reproducibility. The multimodal nature of this approach allows for integrative analysis of multiple features from the same cell, making it an invaluable tool for dissecting complex biological systems.
Clustered regularly interspaced short palindromic repeats (CRISPR) screens represent a transformative force in biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications. Traditional screening approaches predominantly hinge on cell fitness or established markers, which inherently constrain their abilities for unbiased biological discovery. By contrast, single-cell CRISPR screening technologies, which combine pooled CRISPR screens with an array of sophisticated single-cell omics platforms, permit comprehensive profiling of the transcriptome and epigenome following individual genetic manipulations within complex cellular ecosystems. Over the past decade, a panoply of single-cell CRISPR platforms has emerged, each tailored to address specific experimental challenges. Iterative refinements in protocols have bolstered precision, scalability, and reproducibility, thereby enormously advancing functional genomics and translational research. However, technical obstacles such as perturbation efficiency, scalability, and data integration persist, necessitating cross-disciplinary collaboration and innovation. As single-cell CRISPR platforms evolve to incorporate spatial resolution, multi-omics integration, and AI-guided design, they are poised to bridge the gap between genetic perturbation and system-level interpretation. Here, we summarize recent advances in single-cell CRISPR technologies, outline their applications, and provide a comparative framework to guide platform selection (Perturb-seq, CROP-seq, ECCITE-seq, Direct-seq, and Mosaic-seq).
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a curative therapy for hematological malignancies, with T-cell immune reconstitution playing a pivotal role in determining clinical outcomes. This review comprehensively illustrates the processes and influencing factors of T-cell recovery post-HSCT, highlighting the dual pathways of reconstitution: thymus-independent peripheral expansion and thymus-dependent central regeneration. Key factors such as recipient and donor age, human leukocyte antigen disparity, conditioning regimens, immunosuppressive therapies, cytomegalovirus reactivation, and graft-versus-host disease (GVHD) significantly impact T-cell reconstitution dynamics and functional recovery. Furthermore, the article discusses the critical balance between graft-versus-leukemia (GVL) effects and GVHD, emphasizing how T-cell exhaustion, inhibitory receptor overexpression, and clonal dynamics contribute to relapse. Emerging technologies, including single-cell multiomics, spatially resolved proteomics, T cell receptor repertoire analysis, and artificial intelligence-driven modeling, are explored for their potential to deepen mechanistic understanding and enable personalized therapeutic strategies. Ultimately, enhancing T-cell reconstitution through optimized transplantation protocols and targeted interventions is essential for reducing complications and improving long-term survival.
The demand for homologous blood transfusions has reached an unprecedented level, driven by a declining donor population and the ever-increasing need for blood products. While significant advancements have been made in medical equipment and techniques, a critical gap remains in developing an effective alternative to conventional blood transfusion. Medical research to find a proper blood substitute involves many previous experiments. The search for a blood substitute has been ongoing for patients for whom human blood is unavailable, with a few products showing promise in this field. Recent advancements in medical innovation have begun to address this challenge, notably through the development of artificial oxygen carriers (AOCs). These laboratory-synthesized alternatives to traditional blood transfusions offer a means of bypassing the need for human blood, particularly packed red blood cell (pRBC) transfusions. While AOCs fulfill the singular, critical role of in vivo oxygen delivery, the term is frequently used interchangeably with the broader concept of artificial blood. Various AOC products are currently in different stages of clinical development. Most Notable examples include Perftoran, which has been approved in Russia, Kazakhstan, Ukraine, the Kyrgyz Republic, and Mexico and has been administered to over 35,000 patients. Another significant product, Hemopure, has received clinical use approval in South Africa and Russia and has obtained expanded use approval from the United States Food and Drug Administration (USFDA). This article examines the landscape of AOCs, including their preparation methods, available products, regulatory approval status, current applications, limitations, and potential for future use in medical practice. This review article offers an overview of the different types of AOCs currently available, focusing on their clinical development for human use.
The yolk sac drives vertebrate embryonic hematopoiesis through primitive hematopoiesis and endothelial-to-hematopoietic transition (EHT) waves. However, dynamic cellular and molecular changes during EHT of the yolk sac remain to be elucidated. We built a comprehensive atlas of early endothelial and hematopoietic development in the yolk sac by integrating single-cell transcriptomic data from mouse embryos (E6.75–E11.0). Focusing on the yolk sac (E7.5–E9.5), we established a refined atlas capturing key cell populations of EHT in the yolk sac. This enabled the identification of distinct hemogenic endothelial cell (HEC) subpopulations and revealed 2 fundamentally distinct waves of yolk sac hemogenesis via EHT that differed in temporal emergence, cellular origin, molecular signature, and lineage bias. The first EHT wave, emerging around E8.0, originated from primordial endothelial cells and exhibited a bias toward the generation of erythromyeloid progenitors. In contrast, the second EHT wave, emerging around E8.5, originated from maturing yolk sac endothelial cells, expressed key intraembryonic HEC markers (Hlf, Nupr1, Gfi1), and showed a hematopoietic stem and progenitor cell fate bias. Furthermore, molecular dynamics analysis of the pseudotrajectory during the 2 waves of EHT in mouse yolk sacs revealed different dynamic changes in several pathways, particularly the ribosome and metabolic pathways. The yolk sac endothelial and hematopoietic atlas is accessible from an interactive web server (https://lllab.shinyapps.io/ysshinyapp/). Collectively, this study provides novel insights into the multi-wave nature of yolk sac hematopoiesis, clarifies the fundamental principles of yolk sac EHT at a single-cell resolution, and offers potential guidance for in vitro blood cell regeneration strategies.
Long non-coding RNAs (lncRNAs) play a crucial role in normal and dysregulated hematopoiesis. However, the functional repertoire of lncRNAs across various hematopoietic cell types remains elusive. In this study, we constructed a comprehensive single-cell lncRNA atlas containing 207,113 cells, spanning hematopoietic stem and progenitor cells (HSPCs) to differentiated blood cells, by integrating nine single-cell RNA sequencing (scRNA-seq) datasets derived from 30 healthy donors. The hematopoietic hierarchy based on lncRNA expression was highly consistent with that based on protein-coding genes. We identified 3,463 lineage-specific lncRNAs in HSPCs, neutrophils, monocytes, B cells, and T/natural killer cells; 23 of 30 selected lncRNAs were experimentally validated. Importantly, upregulated lncRNAs in pediatric patients with B cell acute lymphoblastic leukemia, T cell acute lymphoblastic leukemia, and acute myeloid leukemia were primarily associated with oxygen response and immune regulation, indicating the potential contribution of lncRNAs to leukemogenesis. In conclusion, our results portray the landscape of lncRNAs in the hematopoietic system, revealing the functional significance of lncRNAs in both normal and abnormal hematopoiesis and providing potential therapeutic targets for the clinical treatment of leukemia.
FLT3-ITD, NPM1, and DNMT3A mutations are common in acute myeloid leukemia (AML). However, the prognostic role of FLT3-ITD combined with NPM1 and/or DNMT3A mutations after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. In this retrospective study, 100 AML patients were selected from a cohort of 1292 who underwent allo-HSCT between 2014 and 2024. Patients were stratified by co-mutation profiles to compare prognosis, identify predictors of survival and relapse, and assess the efficacy of maintenance therapy. With a median follow-up after allo-HSCT of 16.1 months (interquartile range 8.1–26.2), 2-year overall survival (OS) rates were 65.1%, 68.3%, and 67.1%; leukemia-free survival (LFS) rates were 61.6%, 68.7%, and 63.2%; and cumulative incidence of relapse (CIR) rates were 16.9%, 12.5%, and 15.8%, respectively. No significant differences were observed among the groups. In multivariate analysis with FLT3 inhibitor as a time-dependent covariate, FLT3-ITD measurable residual disease (MRD) positivity prior to allo-HSCT was independently associated with inferior OS (hazard ratio [HR] = 3.51, 95% CI 1.34–9.17), LFS (HR = 3.05, 95% CI 1.26–7.35), and CIR (HR = 4.78, 95% CI 1.55–14.81). In contrast, posttransplant maintenance therapy with FLT3 inhibitors independently conferred a favorable impact on OS (HR = 0.15, 95% CI 0.03–0.66), LFS (HR = 0.24, 95% CI 0.07–0.83), CIR (HR = 0.10, 95% CI 0.01–0.66), and nonrelapse mortality (NRM) (HR = 0.25, 95% CI 0.07–0.89). In conclusion, FLT3-ITD-based double or triple mutations showed comparable posttransplant outcomes. FLT3-ITD MRD status and early maintenance therapy were key prognostic and therapeutic factors.
Gene therapy using lentiviral vectors offers a promising alternative treatment for β-thalassemia, a common monogenic blood disorder. Although the BB305 vector has demonstrated long-term clinical success—enabling most β-thalassemia patients to become transfusion-independent and substantially reducing vaso-occlusive events in sickle cell disease—there remains a significant opportunity to enhance its efficacy and lower production costs. Here, we present G2B, a novel hemoglobin beta gene (HBB) lentiviral vector derived from BB305-like constructs (BB305L), engineered by shortening the locus control region (LCR) and incorporating a mutated HBG2 promoter. Compared to BB305L, G2B achieves a ~4-fold increase in lentiviral titer and a 60% boost in β-globin expression. In a transplantation model using Hbbth4/Hbb+ hematopoietic stem/progenitor cells, G2B demonstrates robust therapeutic efficacy without compromising integration safety. These findings suggest that G2B may offer a more potent, efficient, and potentially cost-effective gene therapy option for β-thalassemia and other hemoglobinopathies.
Keloids, characterized by excessive collagen deposition and recurrence, pose significant therapeutic challenges due to limited mechanistic understanding. Mesenchymal stem cells (MSCs) exhibit potential for keloid management, but their precise mechanisms remain unclear. This study investigated how MSCs modulate extracellular matrix (ECM) remodeling in keloid pathogenesis. Using a co-culture system of human umbilical cord MSCs (UC-MSCs) and immortalized keloid fibroblasts (HDIKFs), we demonstrated that UC-MSCs significantly suppressed HDIKF proliferation (via CCK8 assay) and migration (via wound healing assay). Interestingly, UC-MSCs did not alter keloid xenograft growth in vivo. Mechanistically, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) revealed selective downregulation of matrix metalloproteinases 9 (MMP9) in HDIKFs co-cultured with UC-MSCs, while MMP1, MMP2, and MMP3 remained unaffected. This suppression was linked to inhibition of the transforming growth factor-β1/SMAD (TGF-β1/SMAD) pathway, evidenced by reduced hypoxia‐inducible factor-1α (HIF-1α) and SMAD2 expression, alongside upregulated interleukin-10 receptor alpha (IL-10RA). Additionally, UC-MSCs did not alter collagen I/III (COL I/III) ratios or phosphatidylinositol‐3‐kinase (PI3K)/protein kinase B (AKT) signaling. These findings highlight that MSCs attenuate keloid fibroblast activity through TGF-β1/SMAD-driven MMP9 suppression and IL-10RA enhancement, offering novel insights into MSC-based strategies for ECM homeostasis. This study underscores MMP9 as a therapeutic target and provides a foundation for refining MSC efficacy in keloid treatment.
Chronic graft-versus-host disease (cGVHD) is a major complication of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). In our previous studies, we speculated that cGVHD might be closely related to immune-mediated encephalopathy lesions; however, its involvement in the central nervous system (CNS) is exceedingly rare. We performed a retrospective nested case-control study of patients who underwent haplo-HSCT between January 2013 and June 2022 at our institution. In total, 87 patients with cGVHD who had their cerebrospinal fluid (CSF) tested were enrolled, of whom 43 showed evidence of CNS-cGVHD. In these patients, acute GVHD, CSF proteins, and oligoclonal bands were identified as risk factors for cGVHD onset. We combined these variables to establish a nomogram for the cumulative incidence of cGVHD. We found no statistically significant differences in overall survival, leukemia-free survival, or cumulative incidence of relapse between the cGVHD and control groups. However, a significant difference in non-relapse mortality (NRM) was observed. Multivariate analysis of 43 patients diagnosed with CNS-cGVHD showed that underlying diseases and CSF proteins were independently associated with CNS-cGVHD occurrence. In conclusion, the CSF profile may be associated with the onset of cGVHD and higher NRM in cGVHD following haplo-HSCT.
中枢神经系统（CNS）受累发生在约3%的急性髓性白血病（AML）病例中，并且与不良预后相关。
红细胞生成是造血的一个主要分支，负责产生在全身氧运输中起重要作用的特化红细胞（RBC）。
最近，2025年诺贝尔生理学或医学奖联合授予Mary E.Brunkow、Fred Ramsdell和Shimon Sakaguchi，以表彰他们在外周免疫耐受方面的突破性发现，外周免疫耐受是一个确保免疫系统靶向病原体而不攻击身体自身细胞的微妙过程。他们研究的核心是调节性T细胞（Tregs），它充当免疫系统的“刹车”或保安。
免疫性血小板减少性紫癜(ITP)是一种自身免疫性疾病，其特征是自身抗体介导的血小板破坏和血小板生成受损，导致血小板计数时的出血风险<100 × 109/L。在成人中，ITP通常变成慢性和复发性，需要多线治疗，包括皮质类固醇、静脉注射免疫球蛋白、脾切除术、利妥昔单抗、血小板生成素受体激动剂（TPO-RAs）和福坦替尼。然而，一部分患者仍然是复发/难治（r/r），残留的产生自身抗体的浆细胞驱动持续的疾病。标准免疫抑制，如利妥昔单抗的情况，靶向CD20+B淋巴细胞，并可能保留长寿命浆细胞（LLPCs）。如果LLPCs存活，自身抗体持续存在。Daratumumab结合在浆母细胞和浆细胞（包括LLPC）上表达的CD38；并有效地耗尽它们，去除自身抗体来源。
异基因造血干细胞移植是恶性血液病和某些非恶性血液病最重要的治疗方法之一。
在《超分辨率成像知情scRNA测序分析揭示GDF15在使衰老造血干细胞恢复活力中的关键作用》一文中，
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