Experimental hematology最新文献_第9页

2019 – ENDOTHELIAL CELL-DERIVED THROMBOSPONDIN-1 NEGATIVELY AFFECTS HEMATOPOIETIC FUNCTION DURING AGING 2019 -内皮细胞源性血栓反应蛋白-1在衰老过程中对造血功能产生负面影响

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104932

Arianna Smith, Cody Carter, Pradeep Ramalingam, Jason Butler

The pool of candidates for treatment of hematologic malignancies via bone marrow (BM) transplants has greatly expanded due to advancements in conditioning strategies, but these carry an increased risk of failure and complications as patients age. Myelosuppressive treatment can cause irreversible damage to the BM niche, hindering the recovery of the hematopoietic system. BM endothelial cell (EC) signaling is indispensable for supporting hematopoietic stem cell (HSC) self-renewal and differentiation, and age-related loss of vascular integrity negatively affects HSC’s ability to recover postmyelosuppression. Using our previously defined premature vascular aging model, we identified thrombospondin-1 (Thbs1) as a progerontic factor that, when globally knocked out (KO’ed), preserves HSC function in aged mice. We created a Thbs1-GFP reporter mouse, unveiling ECs, megakaryocytes, osteoblasts, and myeloid and stromal cells to be Thbs1-producing cells in the BM niche. Cell-specific deletion of Thbs1 from these cells, followed by competitive stem cell transplantations, revealed that only KO of EC-derived Thbs1 preserves HSC functionality during aging, as seen in the global Thbs1 KO. Furthermore, myelosuppression on our Thbs1-GFP reporter showed dynamic Thbs1 expression throughout recovery within BM cells, indicating that cell-specific Thbs1 plays a role during recovery. Myelosuppressed Thbs1 global KO mice exhibited earlier and more robust multilineage hematologic reconstitution, notably in the clinical marker of neutrophil recovery, which is recapitulated in the EC-specific Thbs1 KO model. Collectively, we show that EC-derived Thbs1 plays a crucial role in the aging of the BM niche and recovery postmyelosuppressive treatment, validating it as a therapeutic target to support the regeneration of the hematopoietic system following myelosuppression in aged patients facing malignancy.

由于调节策略的进步，通过骨髓移植治疗血液系统恶性肿瘤的候选药物池已经大大扩大，但随着患者年龄的增长，这些移植失败和并发症的风险增加。骨髓抑制治疗可对骨髓生态位造成不可逆的损伤，阻碍造血系统的恢复。骨髓内皮细胞（EC）信号传导对于支持造血干细胞（HSC）自我更新和分化是不可或缺的，而与年龄相关的血管完整性丧失会对HSC恢复骨髓抑制后的能力产生负面影响。使用我们之前定义的过早血管衰老模型，我们确定了血栓反应蛋白-1 （Thbs1）作为一种衰老因子，当整体敲除（KO 'ed）时，可以保留老年小鼠的HSC功能。我们创建了Thbs1-GFP报告小鼠，揭示了内皮细胞、巨核细胞、成骨细胞、骨髓细胞和基质细胞是骨髓生态位中产生thbs1的细胞。从这些细胞中特异性删除Thbs1，然后进行竞争性干细胞移植，结果表明，只有ec来源的Thbs1 KO在衰老过程中保留了HSC的功能，这在全球Thbs1 KO中可见。此外，对我们的Thbs1- gfp报告细胞的骨髓抑制显示Thbs1在整个恢复过程中在BM细胞内的动态表达，表明细胞特异性Thbs1在恢复过程中发挥作用。骨髓抑制的Thbs1全球KO小鼠表现出更早和更强大的多系血液学重建，特别是中性粒细胞恢复的临床标志物，这在ec特异性Thbs1 KO模型中得到了概括。总的来说，我们发现ec衍生的Thbs1在骨髓生态位的老化和骨髓抑制治疗后的恢复中起着至关重要的作用，验证了它作为一种治疗靶点来支持老年恶性肿瘤患者骨髓抑制后造血系统的再生。

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引用次数: 0

2023 – HEMATOPOIETIC STEM CELLS UNDERGO BIDIRECTIONAL FATE TRANSITIONS IN VIVO 2023 -造血干细胞在体内经历双向命运转变

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104936

Tsuyoshi Fukushima , Trine Kristiansen , Lai Ping Wong , Yosuke Tanaka , Satoshi Yamazaki , Ruslan Sadreyev , David Scadden

Transitions between subsets of differentiating hematopoietic cells are widely regarded as unidirectional in vivo. Here, we introduced a clonal phylogenetic tracer (CP-tracer) that sequentially tags cells with genetic barcodes, enabling high-resolution analysis of ∼100,000 subclones derived from ∼400 individual hematopoietic stem cells (HSC). Using this approach, we uncovered previously uncharacterized functional subsets, including B-biased multipotent progenitors (B-MPPs) within immunophenotypic HSC. Unexpectedly, we also identified bidirectional fate transitions between myeloid-biased hematopoietic stem cells (My-HSCs) and lineage-balanced hematopoietic stem cells (balanced-HSCs). Contrary to the prevailing view that the more self-renewing My-HSCs unidirectionally transition to balanced-HSCs, phylogenetic tracing reveals that less self-renewing balanced-HSCs can also revert to My-HSCs, with the transition favoring My-HSC accumulation over time. This plasticity persists through serial transplantation, demonstrating durable lineage reprogramming. Further, balanced-HSCs contribute to mature blood cell lineages through two distinct intermediates—My-HSCs and lymphoid-biased HSCs (Ly-HSCs)—with lymphoid competence here shown to be dependent on the homeobox gene, Hhex. Hhex enables Ly-HSC differentiation, but its expression declines with age. Our findings reveal unanticipated hematopoietic stem cell plasticity and establish Hhex as a molecular determinant of myeloid-lymphoid balance.

在体内，分化的造血细胞亚群之间的过渡被广泛认为是单向的。在这里，我们引入了一种克隆系统发育示踪剂（cp示踪剂），该示踪剂用遗传条形码顺序标记细胞，能够对来自~ 400个单个造血干细胞（HSC）的~ 100,000个亚克隆进行高分辨率分析。使用这种方法，我们发现了以前未表征的功能亚群，包括免疫表型HSC中的b偏向多能祖细胞（b - mpp）。出乎意料的是，我们还发现了骨髓偏向性造血干细胞（my - hsc）和谱系平衡型造血干细胞（balanced- hsc）之间的双向命运转变。与普遍观点相反，更多的自我更新的My-HSC单向转变为平衡- hsc，系统发育追踪显示，较少的自我更新的平衡- hsc也可以恢复为My-HSC，随着时间的推移，这种转变有利于My-HSC的积累。这种可塑性通过序列移植持续存在，证明了持久的谱系重编程。此外，平衡型造血干细胞通过两种不同的中间体——my - hsc和淋巴细胞偏倚型造血干细胞（ly - hsc）——促进成熟的血细胞谱系，淋巴细胞能力在这里被证明依赖于同源盒基因Hhex。Hhex使Ly-HSC分化，但其表达随年龄增长而下降。我们的研究结果揭示了意想不到的造血干细胞可塑性，并确立了Hhex作为髓淋巴平衡的分子决定因素。

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引用次数: 0

3002 – INTEGRATION OF CRISPR-CAS9 SCREENS AND MULTI-OMICS PROFILING REVEALS CHD7-ANGPT1 AS A NOVEL MULTIDRUG RESISTANCE AXIS IN AML 3002 - crispr-cas9筛选和多组学分析的整合揭示了chd7-angpt1是aml中新的多药耐药轴

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104942

Mingming Niu , Hong Wang , Long Shen , Dandan Yang , Yang Yang , Tingting Zhang

Acute myeloid leukemia (AML) is a devastating hematologic malignancy and one of the most prevalent forms of leukemia in adults. Despite recent advancements and approval of novel targeted therapies, drug resistance remains a formidable clinical challenge. In this study, we conducted an unbiased clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) knockout screen in AML cells to uncover novel mediators of resistance to the clinically approved FMS-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib. This screen identified chromodomain helicase DNA-binding protein 7 (CHD7) as a new regulator of drug resistance. Strikingly, CHD7 loss not only conferred resistance to FLT3 inhibitors but also extended resistance to a broad range of therapeutics, including venetoclax and daunorubicin (DNR). Mechanistic investigations integrating transcriptomic and proteomic data revealed that CHD7 deletion upregulates angiopoietin-1 (ANGPT1), which drives resistance by activating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. Significantly, genetic knockdown of ANGPT1 or pharmacologic inhibition of its receptor, tyrosine kinase endothelial receptor 2 (TIE2), partially restored drug sensitivity in CHD7-deficient cells. Together, these findings identify the CHD7-ANGPT1 axis as a novel mechanism of multidrug resistance in AML. Preclinical studies further suggest that combining targeted therapies with TIE2 inhibitors offers a promising strategy to overcome drug resistance in AML.

急性髓性白血病（AML）是一种毁灭性的血液系统恶性肿瘤，是成人中最常见的白血病形式之一。尽管最近新的靶向治疗取得了进展和批准，但耐药性仍然是一个巨大的临床挑战。在这项研究中，我们在AML细胞中进行了一项无偏聚类规则间隔短重复序列（CRISPR-Cas9）敲除筛选，以发现对临床批准的fms样酪氨酸激酶3 （FLT3）抑制剂吉特替尼耐药的新介质。筛选发现染色质结构域解旋酶dna结合蛋白7 （CHD7）是一种新的耐药调节因子。引人注目的是，CHD7的缺失不仅赋予了对FLT3抑制剂的耐药性，而且扩大了对包括venetoclax和柔红霉素（DNR）在内的广泛治疗药物的耐药性。结合转录组学和蛋白质组学数据的机制研究显示，CHD7缺失上调血管生成素-1 (ANGPT1)，通过激活磷脂酰肌醇3-激酶/蛋白激酶B （PI3K/AKT）和丝裂原激活的蛋白激酶/细胞外信号调节激酶（MAPK/ERK）信号通路驱动耐药性。值得注意的是，基因敲低ANGPT1或药理抑制其受体酪氨酸激酶内皮受体2 (TIE2)，可以部分恢复chd7缺陷细胞的药物敏感性。总之，这些发现确定了CHD7-ANGPT1轴是AML多药耐药的新机制。临床前研究进一步表明，联合靶向治疗与TIE2抑制剂为克服AML耐药提供了一种有希望的策略。

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引用次数: 0

2014 – TGFΒ1 REGULATES LEUKEMIA STEM CELL MARKER CD123 AND INDUCES CHEMOTHERAPY RESISTANCE 2014 - tgfΒ1调控白血病干细胞标志物cd123，诱导化疗耐药

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104927

Sepideh Azizi Taramsary , Denis Tvorogov , Joanna Woodcock , Hossein Anani , Angel Lopez , Dan Thomas

One of the key challenges to combat chemoresistance in acute myeloid leukemia (AML) lies in the percentage of persistent leukemic stem cells (LSCs) that exhibit aberrant expression of interleukin 3 receptor (IL-3Rα; CD123) and resemble granulocyte-macrophage progenitors. Transforming growth factor β (TGF-β) is an established antiproliferative cytokine that plays a pivotal role in regulating cell cycle inhibition and quiescence, features that have been linked to LSCs.

In this study, we uncovered a novel role for TGF-β1 signaling in the regulation of the LSC marker, IL-3Rα/CD123 (∼fourfold increase), via the TβRI/II-SMAD2-SMAD4 axis in AML. Similar to the LSC phenotype, we observed prolonged exposure to TGF-β1 maintained elevated IL-3Rα expression and promoted cellular quiescence by increasing CDKN1A/p21 expression and decreasing cellular division and proliferation (threefold decrease). Interestingly, we showed that TGF-β1 conferred a survival advantage following cytarabine treatment (∼30%), whereas cytarabine alone induced robust apoptosis. Consistently, CFU assays further showed a 20-fold increase in colony numbers in cells cotreated with TGF-β1 and cytarabine, relative to cytarabine alone.

Clinically, we uncovered overactivation of the TGF-β1 canonical pathway, SMAD2, and SMAD4 in FLT3-ITD and RUNX1-mutated leukemic cells. We observed compelling evidence that FLT3-ITD hijacks the TGF-β1 pathway through inducing constitutive SMAD2 phosphorylation and increased expression of TGF-β-responsive genes (TGFB1, SMAD2, and CDKN1A), as well as IL-3Rα.

Together, these findings elucidate a novel regulatory axis by which TGF-β1 sustains LSC-like properties, including maintaining IL-3Rα upregulation, promoting quiescence, and enforcing chemoresistance. This work has significant translational implications, highlighting the TGF-β1–SMAD2/4 pathway as a potential therapeutic target to eradicate LSC in FLT3-ITD and RUNX1-mutated AML with elevated CD123 expression.

对抗急性髓系白血病（AML）化疗耐药的关键挑战之一在于表现出白细胞介素3受体（IL-3Rα; CD123）和类似粒细胞-巨噬细胞祖细胞异常表达的持续性白血病干细胞（LSCs）的百分比。转化生长因子β (TGF-β)是一种已建立的抗增殖细胞因子，在调节细胞周期抑制和静止中起关键作用，这些特征与LSCs有关。在这项研究中，我们发现TGF-β1信号在AML中通过t -β ri /II-SMAD2-SMAD4轴调控LSC标志物IL-3Rα/CD123（增加4倍）中的新作用。与LSC表型相似，我们观察到长时间暴露于TGF-β1中，通过增加CDKN1A/p21表达，减少细胞分裂和增殖（减少三倍），维持IL-3Rα表达升高，促进细胞静止。有趣的是，我们发现TGF-β1在阿糖胞苷治疗后（约30%）具有生存优势，而阿糖胞苷单独治疗可诱导强烈的细胞凋亡。与此一致的是，CFU实验进一步显示TGF-β1和阿糖胞苷共处理的细胞的菌落数量比单独处理阿糖胞苷的细胞增加了20倍。在临床上，我们发现TGF-β1典型通路、SMAD2和SMAD4在FLT3-ITD和runx1突变的白血病细胞中过度激活。我们观察到令人信服的证据，FLT3-ITD通过诱导SMAD2组成性磷酸化和TGF-β响应基因（TGFB1、SMAD2和CDKN1A）以及IL-3Rα的表达增加，劫持了TGF-β1途径。总之，这些发现阐明了一个新的调控轴，TGF-β1通过该轴维持lsc样特性，包括维持IL-3Rα上调、促进静止和加强化学耐药。这项工作具有重要的翻译意义，强调TGF -β1-SMAD2/4途径是根除FLT3-ITD和runx1突变的AML中CD123表达升高的LSC的潜在治疗靶点。

{"title":"2014 – TGFΒ1 REGULATES LEUKEMIA STEM CELL MARKER CD123 AND INDUCES CHEMOTHERAPY RESISTANCE","authors":"Sepideh Azizi Taramsary , Denis Tvorogov , Joanna Woodcock , Hossein Anani , Angel Lopez , Dan Thomas","doi":"10.1016/j.exphem.2025.104927","DOIUrl":"10.1016/j.exphem.2025.104927","url":null,"abstract":"<div><div>One of the key challenges to combat chemoresistance in acute myeloid leukemia (AML) lies in the percentage of persistent leukemic stem cells (LSCs) that exhibit aberrant expression of interleukin 3 receptor (IL-3Rα; CD123) and resemble granulocyte-macrophage progenitors. Transforming growth factor β (TGF-β) is an established antiproliferative cytokine that plays a pivotal role in regulating cell cycle inhibition and quiescence, features that have been linked to LSCs.</div><div>In this study, we uncovered a novel role for TGF-β1 signaling in the regulation of the LSC marker, IL-3Rα/CD123 (∼fourfold increase), via the TβRI/II-SMAD2-SMAD4 axis in AML. Similar to the LSC phenotype, we observed prolonged exposure to TGF-β1 maintained elevated IL-3Rα expression and promoted cellular quiescence by increasing CDKN1A/p21 expression and decreasing cellular division and proliferation (threefold decrease). Interestingly, we showed that TGF-β1 conferred a survival advantage following cytarabine treatment (∼30%), whereas cytarabine alone induced robust apoptosis. Consistently, CFU assays further showed a 20-fold increase in colony numbers in cells cotreated with TGF-β1 and cytarabine, relative to cytarabine alone.</div><div>Clinically, we uncovered overactivation of the TGF-β1 canonical pathway, SMAD2, and SMAD4 in FLT3-ITD and RUNX1-mutated leukemic cells. We observed compelling evidence that FLT3-ITD hijacks the TGF-β1 pathway through inducing constitutive SMAD2 phosphorylation and increased expression of TGF-β-responsive genes (TGFB1, SMAD2, and CDKN1A), as well as IL-3Rα.</div><div>Together, these findings elucidate a novel regulatory axis by which TGF-β1 sustains LSC-like properties, including maintaining IL-3Rα upregulation, promoting quiescence, and enforcing chemoresistance. This work has significant translational implications, highlighting the TGF-β1–SMAD2/4 pathway as a potential therapeutic target to eradicate LSC in FLT3-ITD and RUNX1-mutated AML with elevated CD123 expression.</div></div>","PeriodicalId":12202,"journal":{"name":"Experimental hematology","volume":"151 ","pages":"Article 104927"},"PeriodicalIF":2.1,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145620054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

2021 – LOCAL BONE REMODELING SHAPES HEMATOPOIETIC STEM CELL (HSC)- MACROPHAGE INTERACTIONS AND COMPARTMENTALIZED HSC EXPANSION UNDER INFLAMMATORY STRESS 2021 -局部骨重塑塑造造血干细胞(hsc)-炎症应激下巨噬细胞相互作用和区隔hsc扩张

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104934

Cih-Li Hong , Ryan Adapathya , Kevin Lee , Joseph Collins , Montgomery Whalen , Laura Calvi , Leonard Zon , Shu-Chi Yeh

Inflammation induces hematopoietic stem cell (HSC) expansion with compromised fitness, a culprit of hematopoietic aging. Elucidating mechanisms to maintain HSC fitness under inflammatory stress is crucial to preserve hematopoietic integrity.

Using intravital imaging and HSC reporter mice (Mds1GFP/+; Flt3Cre), we revealed distinct stages of bone turnover across marrow cavities. HSCs within a subset of marrow cavities that lack bone resorption stayed nonmotile and solitary despite stress from cyclophosphamide/G-CSF (Nature, 2020), lipopolysaccharide (LPS, 35 μg/mouse)-induced acute inflammation, and aging. Harvesting cavity-resident HSCs under image guidance, we further revealed the superior colony-formation capacity of HSCs from nonresorptive cavities both at the steady state and under inflammation. These results suggest that local bone remodeling influences the functionality of compartmentalized HSCs.

Notably, via transcriptomic analyses and in vivo tracking, marrow macrophages in resorptive (RE) cavities were found to be proinflammatory and frequently retrieve cellular cargos from HSCs, followed by incidences of HSC division. Further in vivo staining suggested surface calreticulin expression and elevation of MHC class I on HSCs, resembling the “grooming” behavior reported in zebrafish models from the Zon group (Science, 2022, 2024). Such interaction increases with LPS challenge and aging, and continues to occur predominantly in the RE cavities. Inhibiting bone resorption with zoledronic acid (1.2 μg/mouse) reduced grooming and restored HSC fitness under LPS challenge. Taken together, our findings present novel evidence of macrophage-HSC interactions in murine bone marrow that regulate HSC clonality and unveil previously unrecognized spatial heterogeneity of the HSC niche that may be targeted to intervene in hematopoietic decline under inflammatory stress.

炎症诱导造血干细胞（HSC）扩张，降低适应性，这是造血老化的罪魁祸首。阐明炎症应激下维持HSC健康的机制对于保持造血系统的完整性至关重要。通过活体成像和HSC报告小鼠（Mds1GFP/+; Flt3Cre），我们揭示了骨髓腔骨转换的不同阶段。尽管受到环磷酰胺/G-CSF （Nature, 2020）、脂多糖（LPS， 35 μg/小鼠）诱导的急性炎症和衰老的胁迫，缺乏骨吸收的骨髓腔亚群中的hsc仍保持不动和孤立。在图像引导下收集腔内居住的hsc，我们进一步揭示了稳态和炎症状态下来自非吸收性腔的hsc的优越集落形成能力。这些结果表明，局部骨重塑影响区室化hsc的功能。值得注意的是，通过转录组学分析和体内跟踪，发现吸收（RE）腔中的骨髓巨噬细胞具有促炎作用，经常从HSC中提取细胞货物，随后发生HSC分裂。进一步的体内染色表明，hsc表面钙调蛋白表达和MHC I类升高，类似于Zon组斑马鱼模型中报道的“梳理”行为（Science, 2022, 2024）。这种相互作用随着LPS的挑战和衰老而增加，并且继续主要发生在RE腔中。用唑来膦酸（1.2 μg/只）抑制骨吸收可减少修饰，恢复脂多糖刺激下HSC的适应性。综上所述，我们的研究结果提供了小鼠骨髓中巨噬细胞-HSC相互作用调节HSC克隆的新证据，并揭示了以前未被认识到的HSC生态位的空间异质性，这可能是炎症应激下干预造血功能下降的目标。

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引用次数: 0

3027 – IDENTIFYING PIONEER FACTORS DURING HEMATOPOIETIC STEM CELL EMERGENCE 3027 -鉴定造血干细胞出现过程中的先锋因子

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104968

Ewan Egan, Ragini Medhi, Telma Ventura, Abdenour Soufi, Antonella Fidanza, Katrin Ottersbach

Hematopoietic stem cell (HSC) transplantations are currently the most commonly used regenerative therapy in the clinic, being employed to treat numerous blood cancers and genetic disorders. However, their therapeutic potential is limited by a lack of supply and complications arising from graft-vs-host disease. An in vitro sourced supply of HSCs would circumvent these problems, but this first requires a better understanding of their developmental origins to faithfully recapitulate their generation in the dish. At present, it is understood that HSCs arise from an endothelial-to-hematopoietic transition (EHT) within the embryonic dorsal aorta, yet how this cell fate is determined remains unclear. Increasing evidence demonstrates that cell lineages, during embryonic development, are prespecified through the priming of associated genes by pioneer factors. Thus, we sought to investigate whether pioneer factors play a similar role in the specification of the hematopoietic lineage. To do this, we developed a cytokine-based differentiation system that mimics the EHT in vitro from human pluripotent stem cells. Using this model, we designed a sorting strategy that captures various cell types along the developmental trajectory toward hematopoietic cells. These isolated cells were then subject to functional assays and multiomic analyses such as RNA-Seq and ATAC-Seq. Studying the changes in gene expression and chromatin accessibility allowed us to identify when the hematopoietic fate is specified and will indicate which pioneer factors are responsible. So far, we have identified genes associated with cytoskeleton assembly and lipid metabolism to be key in this specification. Consequently, these findings can provide us with insight into how HSCs emerge as well as more widely highlight the role of pioneer factors during cell fate acquisition in development.

造血干细胞（HSC）移植是目前临床上最常用的再生疗法，用于治疗多种血癌和遗传性疾病。然而，它们的治疗潜力受到供应不足和移植物抗宿主病引起的并发症的限制。体外造血干细胞的供应可以避免这些问题，但这首先需要更好地了解它们的发育起源，以便在培养皿中忠实地再现它们的生成过程。目前，人们了解造血干细胞是由胚胎背主动脉内的内皮向造血转化（EHT）产生的，但这种细胞命运是如何决定的仍不清楚。越来越多的证据表明，在胚胎发育过程中，细胞系是通过先驱因子启动相关基因而预先指定的。因此，我们试图调查先锋因子是否在造血谱系的规范中发挥类似的作用。为了做到这一点，我们开发了一个基于细胞因子的分化系统，模拟体外的人类多能干细胞的EHT。利用这个模型，我们设计了一种分选策略，可以捕获沿着造血细胞发育轨迹的各种细胞类型。然后对这些分离的细胞进行功能分析和多组学分析，如RNA-Seq和ATAC-Seq。研究基因表达和染色质可及性的变化使我们能够确定何时指定造血命运，并将表明哪些先驱因素是负责任的。到目前为止，我们已经确定了与细胞骨架组装和脂质代谢相关的基因是这一规范的关键。因此，这些发现可以为我们提供关于造血干细胞如何出现的见解，以及更广泛地强调先锋因子在细胞命运获得过程中的作用。

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引用次数: 0

3030 – NICHE INTERACTIONS DURING DEVELOPMENT DOWNREGULATE INFLAMMATORY PATHWAYS IN HEMATOPOIETIC STEM AND PROGENITOR CELLS 3030 -发育过程中的生态位相互作用下调造血干细胞和祖细胞的炎症通路

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104971

Nicole Woodhead , Octavia Santis-Larrain , Sobhika Agarwala , Wantong Li , Rodolfo Calderon , Kylie Sweeny , Clyde A. Campbell , Raquel Espin-Palazon , Bradley W. Blaser , Owen J. Tamplin

In the mammalian embryo, the fetal liver supports hematopoietic stem and progenitor cell (HSPC) maturation and transition to quiescence. However, the genetic regulatory networks involved are poorly understood. To dissect these networks, we used viable integrin α4 (itga4) mutant zebrafish where HSPCs fail to lodge in the caudal hematopoietic tissue (CHT), the mammalian fetal liver equivalent. Bulk RNA-seq of sorted wild type (WT) and itga4 mutant Runx:mCherry+ HSPCs from 5 days post fertilization (dpf) larvae found 286 upregulated and 49 downregulated transcripts in itga4 mutant versus WT (q < 0.05). Gene set enrichment analysis revealed upregulated inflammatory pathways in itga4 mutant HSPCs. Bulk ATAC-seq found 84,236 peaks unique to WT and 16,875 peaks unique to the itga4 mutant (q < 0.05). Motif analysis identified ETS and AP-1 as potential HSPC maturation regulatory factors. Additional single cell RNA-seq of sorted Runx:mCherry+ HSPCs at five dpf confirmed inflammatory pathway enrichment and revealed upregulated cell cycle pathways in itga4 mutant HSPCs versus WT. To validate inflammatory signaling changes, we compared itga4 or control morpholino injected into NFκB:d2eRFP;cd41:GFP+ reporters, and NFκB activity was increased 1.5-fold in itga4 morphant HSPCs versus WT at the beginning of the CHT stage (3 dpf). Live imaging in established zebrafish fluorescent cell cycle indicator (zFUCCI); Runx:mCherry+ lines revealed a significantly higher proportion of itga4 mutant HSPCs in the S/G2/M phase at 3 dpf compared with WT (16.46% vs. 6.045%, p < 0.0001). To understand the loss of itga4 in HSPCs through development and into adulthood, we compared whole kidney marrow (WKM) of young (6-month-old) WT, itga4 mutant, and vcam1b mutant, and aged (24-month-old) WT adults via split-pool barcoding scRNA-seq, flow cytometric lineage analysis, and cell cycle analysis. Together, our results show that itga4-dependent niche interactions are required for HSPC stability through development and into adulthood.

在哺乳动物胚胎中，胎儿肝脏支持造血干细胞和祖细胞（HSPC）的成熟和向静止的过渡。然而，人们对其中的基因调控网络知之甚少。为了解剖这些网络，我们使用了活的整合素α4 （itga4）突变斑马鱼，其中HSPCs无法在哺乳动物胎儿肝脏的尾端造血组织（CHT）中植入。对野生型（WT）和itga4突变体Runx:mCherry+受精后5天（dpf）幼虫的HSPCs进行大量rna测序发现，与WT相比，itga4突变体有286个转录本上调，49个转录本下调（q < 0.05）。基因集富集分析显示itga4突变型HSPCs中炎症通路上调。Bulk ATAC-seq发现WT特有的峰84,236个，itga4突变体特有的峰16,875个（q < 0.05）。基序分析发现ETS和AP-1是潜在的HSPC成熟调节因子。在5个dpf位点对Runx:mCherry+ HSPCs进行排序后的单细胞rna测序，证实了itga4突变HSPCs中炎症通路的富集，并揭示了itga4突变HSPCs与WT相比细胞周期通路的上调。为了验证炎症信号的变化，我们比较了注射到NFκB:d2eRFP中的itga4或对照morpholino；cd41:GFP+报告者和NFκB活性在itga4型HSPCs中与WT相比，在CHT阶段开始时增加了1.5倍（3 dpf）。建立的斑马鱼荧光细胞周期指示器（zFUCCI）的实时成像Runx:mCherry+系显示，与WT相比，在3 dpf时，itga4突变体HSPCs在S/G2/M期的比例显著更高（16.46% vs. 6.045%, p < 0.0001）。为了了解itga4在HSPCs发育和成年过程中的丢失，我们通过分裂池条形码scRNA-seq、流式细胞术谱系分析和细胞周期分析，比较了幼年（6个月）WT、itga4突变体和vcam1b突变体和老年（24个月）WT成人的全肾骨髓（WKM）。总之，我们的研究结果表明，依赖itga4的生态位相互作用是HSPC从发育到成年稳定所必需的。

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引用次数: 0

3019 – STAGE-DEPENDENT CHANGES IN SIGNALING REQUIREMENTS AND SPATIAL LOCALIZATION DURING HEMATOPOIETIC STEM CELL DEVELOPMENT 3019 -造血干细胞发育过程中信号需求和空间定位的阶段依赖性变化

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104960

Saori Morino-Koga , Mariko Tsuruda , Xueyu Zhao , Shogo Oshiro , Tomomasa Yokomizo , Mariko Yamane , Shunsuke Tanigawa , Koichiro Miike , Shingo Usuki , Kei-ichiro Yasunaga , Ryuichi Nishinakamura , Toshio Suda , Minetaro Ogawa

The development of hematopoietic stem cells (HSCs) originates from endothelial cells in the aorta-gonad-mesonephros (AGM) region during midgestation. Specific endothelial cells, known as hemogenic endothelial cells (HECs), differentiate into HSC precursors (pre-HSCs), which subsequently migrate to the fetal liver and mature into HSCs. Although several signaling molecules essential for HSC development have been identified, previous studies have predominantly relied on serum-containing culture conditions, which complicate the dissection of underlying molecular mechanisms. In this study, we aimed to identify key signaling molecules required for HSC induction using a serum-free in vitro culture system. To achieve this, we conducted single-cell RNA-sequencing (scRNA-seq) analysis of mouse embryos to identify candidate signaling molecules involved in HSC development and to evaluate their functional roles under serum-free conditions. Our results revealed that HSC development proceeds through a stepwise process orchestrated by multiple signaling pathways. Stem cell factor (SCF) and endothelial cell-derived factors were found to be crucial for the differentiation of HECs into pre-HSCs. After migration to the fetal liver, the maturation of pre-HSCs into HSCs was dependent on both SCF and thrombopoietin (TPO) signaling. Notably, the addition of these signaling molecules in vitro enabled the efficient differentiation of HECs and pre-HSCs into transplantable HSCs. Furthermore, we confirmed the presence of these signaling molecules in the HSC developmental environment. Taken together, our findings contributed to the establishment of a serum-free in vitro culture system that recapitulates the development of HSCs from pluripotent stem cells.

造血干细胞（hsc）起源于妊娠中期主动脉-性腺-中肾（AGM）区域的内皮细胞。特异性内皮细胞，被称为造血内皮细胞（hec），分化为造血干细胞前体（前造血干细胞），随后迁移到胎儿肝脏并成熟为造血干细胞。虽然已经确定了几个对HSC发育至关重要的信号分子，但先前的研究主要依赖于含血清的培养条件，这使得对潜在分子机制的解剖复杂化。在本研究中，我们旨在通过无血清体外培养系统鉴定诱导HSC所需的关键信号分子。为此，我们对小鼠胚胎进行了单细胞rna测序（scRNA-seq）分析，以确定参与HSC发育的候选信号分子，并评估其在无血清条件下的功能作用。我们的研究结果表明，HSC的发展是通过多种信号通路协调的逐步过程进行的。干细胞因子（SCF）和内皮细胞衍生因子在hec向前造血干细胞分化过程中起着至关重要的作用。在迁移到胎儿肝脏后，前造血干细胞向造血干细胞的成熟依赖于SCF和血小板生成素（TPO）信号。值得注意的是，在体外添加这些信号分子可以使hec和pre- hsc有效地分化为可移植的hsc。此外，我们证实了这些信号分子在HSC发育环境中的存在。综上所述，我们的发现有助于建立一个无血清的体外培养系统，该系统概括了多能干细胞向造血干细胞的发展。

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引用次数: 0

3007 – HSPCS DIFFERENTIATE TO A DIVERSE SET OF LYMPHOID LINEAGE IMMUNE CELLS DURING DEVELOPMENT 3007 - HSPCS在发育过程中分化为多种淋巴系免疫细胞

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104947

Anastasia Nizhnik, Bianca Ulloa, Kevyn Jackson, Deyou Zheng, Teresa Bowman

During embryogenesis, hematopoietic stem cells (HSCs) and HSC-independent progenitor cells form contemporaneously and produce overlapping and distinct immune cell types. The full repertoire of immune cells generated during development and their cellular origin across vertebrates is not completely elucidated. Here, we did temporal lineage tracing of the emerging hematopoietic system during zebrafish larval development to delineate the differentiation potential of HSCs and HSC-independent progenitors in vivo. We used an inducible Cre-Lox lineage tracing system to label the emerging hematopoietic system at three early timepoints in development and traced the heritable fluorescent mCherry signal. To identify the mature cell types emanating from HSCs and HSC-independent progenitors, we did single-cell RNA sequencing of the mCherry-expressing cells as a time course through development. We observed a wave of lymphoid lineage differentiation derived from the earliest induced labeling, enriched for HSC-independent progenitors. The wave of differentiated cells included a greater diversity of lymphoid immune cell types than previously recognized, such as populations with innate lymphoid cell (ILC) identities. The ILC-like cells were dependent on the transcription factor Runx1 and on interleukin 2 receptor γ, but not on the function of the T- and B-cell-specific Rag1 recombinase. Larval ILC-like cells were detected using in situ hybridization in both lymphoid and mucosal organs and were responsive to a viral mimicry-induced stimulation, indicating their functionality in early vertebrate life. The work revealed the developmental origins and early functionality of a more diverse set of lymphoid cells during zebrafish development, and illustrates the utility of the zebrafish model to investigate the origins and larval activities of ILCs.

在胚胎发生过程中，造血干细胞（hsc）和不依赖hsc的祖细胞同时形成，并产生重叠和不同的免疫细胞类型。在脊椎动物发育过程中产生的免疫细胞的全部功能及其细胞起源尚未完全阐明。在这里，我们对斑马鱼幼虫发育过程中新兴的造血系统进行了时间谱系追踪，以描绘体内造血干细胞和不依赖造血干细胞的祖细胞的分化潜力。我们使用可诱导的Cre-Lox谱系追踪系统在发育的三个早期时间点标记新兴的造血系统，并追踪可遗传的荧光mCherry信号。为了鉴定从造血干细胞和不依赖造血干细胞的祖细胞中产生的成熟细胞类型，我们对表达mccherry的细胞进行了单细胞RNA测序。我们观察到一波淋巴细胞谱系分化源于最早的诱导标记，丰富了hsc独立祖细胞。这波分化细胞包括比以前认识到的更多样化的淋巴免疫细胞类型，例如具有先天淋巴样细胞（ILC）身份的群体。ilc样细胞依赖于转录因子Runx1和白细胞介素2受体γ，但不依赖于T和b细胞特异性Rag1重组酶的功能。利用原位杂交技术在淋巴和粘膜器官中检测到ilc样细胞幼虫，它们对病毒模拟诱导的刺激有反应，表明它们在早期脊椎动物生命中的功能。这项工作揭示了斑马鱼发育过程中更多样化的淋巴样细胞的发育起源和早期功能，并说明了斑马鱼模型在研究ilc的起源和幼虫活性方面的实用性。

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引用次数: 0

3008 – INTEGRATED IN SILICO AND IN VITRO APPROACHES MATCH DEVELOPMENTAL CELL-OF-ORIGIN TO THERAPEUTIC VULNERABILITIES IN INFANT LEUKEMIA 3008 -集成在硅和体外的方法匹配发育细胞的起源，以治疗婴儿白血病的脆弱性

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 Epub Date: 2025-12-01 DOI: 10.1016/j.exphem.2025.104948

Denise Ragusa , Ylenia Cicirò , Emily Johnson , Ayona Johns , Sina Kanannejad , Remisha Gurung , Alice Giustacchini , Sabrina Tosi , Cristina Pina

Pediatric leukemias remain challenging to model and to treat. A subset originates in utero and has age-specific genetic abnormalities and/or an embryonic cell-of-origin (CoO), which result in unique molecular features. Using patient transcriptomics and a spatio-temporally accurate three-dimensional (3D) gastruloid model of embryonic hematopoiesis (haemGx), we previously modeled an acute myeloid leukemia (AML) subtype exclusive to infants—t(7;12)(q36;p13), which overexpresses motor neuron and pancreas homeobox 1 (MNX1)—and pinpointed the CoO at the endothelial-to-hematopoietic transition (EHT). An independent t(7;12) cell line we engineered recapitulated similar molecular and cellular composition. Here, we harnessed t(7;12) unique transcriptional features to model therapeutic vulnerabilities in silico. By correlating its characteristic signatures with gene expression and drug sensitivity data from public databases, we identified candidate compounds selectively targeting t(7;12) AML. We tested these drugs on our t(7;12)-engineered cell line and found that HSP90 inhibition selectively eliminates t(7;12)-expanded progenitors with clonogenic potential, whereas decreasing the expression of MNX1 and t(7;12)-defining genes. The targeted progenitors exhibit EHT programs, highlighting a selective activity against cells with features reminiscent of t(7;12) developmental origin, which are also observed in patients. Furthermore, the antileukemic effects of heat shock protein 90 (HSP90) inhibition were recapitulated in the haemGx model; within the EHT susceptibility window to MNX1 overexpression we previously defined. Overall, we successfully integrated computational and multimodal in vitro approaches to extract ontogeny placement of a putative t(7;12) AML CoO, revealing a drug vulnerability that is clinically valuable and allows mechanistic dissection of embryonic trajectories in infant AML.

儿童白血病的建模和治疗仍然具有挑战性。一个亚群起源于子宫，具有年龄特异性遗传异常和/或胚胎起源细胞（CoO），这导致独特的分子特征。利用患者转录组学和一个时空精确的胚胎造血（haemGx）三维（3D）胃原质模型，我们先前模拟了一种婴幼儿特有的急性髓性白血病（AML）亚型-t (7;12)(q36;p13)，该亚型过度表达运动神经元和胰腺同源盒1 (MNX1)，并确定了内皮细胞向造血过渡（EHT）的CoO。我们设计的一个独立的t（7;12）细胞系再现了类似的分子和细胞组成。在这里，我们利用t（7;12）独特的转录特征来模拟硅中的治疗脆弱性。通过将其特征特征与公共数据库中的基因表达和药物敏感性数据相关联，我们确定了选择性靶向t(7;12) AML的候选化合物。我们在我们的t（7;12）工程细胞系上测试了这些药物，发现HSP90抑制选择性地消除了具有克隆潜能的t（7;12）扩增祖细胞，同时降低了MNX1和t（7;12）定义基因的表达。靶向祖细胞表现出EHT程序，突出了对具有t（7;12）发育起源特征的细胞的选择性活性，这也在患者中观察到。此外，抑制热休克蛋白90 （HSP90）的抗白血病作用在haemGx模型中得到了重现；在我们之前定义的对MNX1过表达的EHT敏感性窗口内。总体而言，我们成功地整合了计算和多模式体外方法来提取假定的t(7;12) AML CoO的个体发生位置，揭示了具有临床价值的药物脆弱性，并允许对婴儿AML的胚胎轨迹进行机械解剖。

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引用次数: 0