Experimental hematology最新文献_第8页

3032 – MAPPING VARIABILITY IN PLURIPOTENT STEM CELL-DERIVED HEMOGENIC ENDOTHELIAL CELLS USING CELLULAR INDEXING OF TRANSCRIPTOMES AND EPITOPES SEQUENCING 3032 -利用转录组和表位测序的细胞索引来定位多能干细胞来源的造血内皮细胞的变异

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104973

Beth Castle, Thristan Taberna, Nora Kotkas, Ross Jones, Peter Zandstra

Induced pluripotent stem cells (iPSCs) are a renewable source for the generation of blood cells in vitro. The current gold standard for production of iPSC-derived blood cells consists of a multi-day aggregate differentiation to generate hemogenic endothelium (HE), the precursor to multipotent blood progenitors. Blood lineage competence can be dictated by the HE, but we do not yet understand how that occurs and how to best identify HE that generates a given type of blood. Further, high variability in HE produced limits the ability to use this protocol for research and clinical use. Efforts commonly rely on flow cytometry to measure surface markers for staging and identifying HE phenotypes. However, despite numerous markers reported in the literature, variability in their expression and kinetics across iPSC lines and blood differentiation protocols remains a major challenge in the field. To characterize and classify HE, we performed cellular indexing of transcriptomes and epitope sequencing (CITE-seq) on iPSC-derived HE samples. To capture common forms of variability, we captured HE populations from a time course spanning arterialization to blood induction, small and large aggregates, different blood induction protocols, and two iPSC lines. The mapping of cell surface markers to transcriptomes enabled a precise understanding of what stage a marker represents and whether this is maintained across protocols and cell lines. Pseudotime mapping with gene regulatory network analysis using IQCELL allowed time-resolved mapping of changes in transcriptional networks and their correlation with surface marker profiles. Predicted lineage competence of the HE was determined by differential gene expression and mapping populations to in vivo human blood development. Ongoing efforts are focused on sorting these populations to determine endothelial to hematopoietic transition (EHT) and lineage competence.

诱导多能干细胞（iPSCs）是体外生成血细胞的可再生来源。目前生产ipsc衍生的血细胞的黄金标准包括多日的聚集分化，以产生造血内皮（HE），多能造血祖细胞的前体。血液谱系能力可以由HE决定，但我们还不知道这是如何发生的，也不知道如何最好地识别产生特定血型的HE。此外，HE产生的高度可变性限制了将该方案用于研究和临床应用的能力。努力通常依靠流式细胞术来测量表面标记物的分期和鉴定HE表型。然而，尽管文献中报道了许多标记物，但它们在iPSC系和血液分化方案中的表达和动力学的可变性仍然是该领域的主要挑战。为了对HE进行表征和分类，我们对ipsc衍生的HE样本进行了转录组的细胞索引和表位测序（CITE-seq）。为了捕捉常见的变异形式，我们从动脉化到血液诱导、小聚集体和大聚集体、不同的血液诱导方案和两个iPSC系的时间过程中捕获了HE群体。将细胞表面标记物映射到转录组，可以精确地了解标记物所代表的阶段，以及这是否在不同的方案和细胞系中保持不变。使用IQCELL进行基因调控网络分析的伪时间作图，可以对转录网络的变化及其与表面标记谱的相关性进行时间分辨作图。预测HE的谱系能力是通过差异基因表达和定位人群来确定的。正在进行的工作集中在分类这些人群，以确定内皮到造血过渡（EHT）和谱系能力。

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引用次数: 0

1016 – THE BIOLOGY AND PATHOBIOLOGY OF BONE MARROW-DERIVED FIBROCYTES 1016 -骨髓源性纤维细胞的生物学和病理学

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104896

Shannon Elf, William Heaton

Despite a well-characterized mutational landscape, the molecular and cellular etiology of myelofibrosis (MF) remains poorly understood. Several reports in recent years have demonstrated a crucial role for neoplastic fibrocytes in MF, with patients displaying significant expansion of this cell population in the bone marrow. The fibrocyte is a unique monocyte-derived, proinflammatory, collagen-producing cell type that has been shown to play a key role in wound healing, tissue repair, and inflammation. Fibrocytes possess features of both monocytic lineage cells and fibroblasts, but a lack of knowledge regarding definitive cell surface markers, gene expression profiles, and general function has made it a difficult cell type to study. As such, precisely how fibrocyte differentiation and expansion are regulated, what controls normal and neoplastic fibrocyte fate, and how these cells can be targeted for therapeutic gain in fibrotic diseases all remain elusive.

We have developed an in vitro human fibrocyte system in which we can differentiate primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) from aged healthy donors through the monocyte lineage and into putative fibrocytes. Using this system, we have performed multiomics and single-cell sequencing studies to identify definitive cell surface markers, metabolic features, and gene expression profiles that distinguish fibrocytes from other monocytic lineage cells and fibroblasts. We have further identified unique features of neoplastic fibrocytes possessing different MF driver mutations that may explain why some myeloproliferative neoplasm (MPN) mutations are more frequent in MF than others, including differences in MHC-I cell surface presentation and immune-mediated clearance.

In sum, this study revealed new insights into the molecular mechanisms underlying the biology of normal fibrocytes and the pathobiology of MF fibrocytes, allowing us to identify novel points of therapeutic intervention that have the potential to cure MF and spare healthy cells.

尽管有一个很好的特征突变景观，髓纤维化（MF）的分子和细胞病因仍然知之甚少。近年来的几篇报道表明，肿瘤纤维细胞在MF中起着至关重要的作用，患者骨髓中显示出这种细胞群的显著扩增。纤维细胞是一种独特的单核细胞来源，促炎，胶原生成细胞类型，已被证明在伤口愈合，组织修复和炎症中发挥关键作用。纤维细胞具有单核细胞谱系细胞和成纤维细胞的特征，但缺乏关于明确的细胞表面标记、基因表达谱和一般功能的知识，使其成为一种难以研究的细胞类型。因此，纤维细胞的分化和扩张是如何被调控的，是什么控制着正常和肿瘤纤维细胞的命运，以及这些细胞如何在纤维化疾病中靶向治疗，这些都是难以捉摸的。我们已经开发了一种体外人纤维细胞系统，在该系统中，我们可以通过单核细胞谱系将来自健康老年供者的原代人CD34+造血干细胞和祖细胞（HSPCs）分化为假定的纤维细胞。使用该系统，我们进行了多组学和单细胞测序研究，以确定确定的细胞表面标记，代谢特征和基因表达谱，以区分纤维细胞与其他单核细胞谱系细胞和成纤维细胞。我们进一步确定了具有不同MF驱动突变的肿瘤纤维细胞的独特特征，这可能解释了为什么一些骨髓增生性肿瘤（MPN）突变在MF中比其他突变更频繁，包括MHC-I细胞表面呈现和免疫介导清除的差异。总之，这项研究揭示了正常纤维细胞生物学和MF纤维细胞病理生物学的分子机制的新见解，使我们能够确定新的治疗干预点，这些点有可能治愈MF并保护健康细胞。

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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 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

1006 – HEMATOPOIETIC STEM CELL ACTIVATION FOR BLOOD REGENERATION 1006 -造血干细胞活化用于血液再生

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104886

Sean Morrison

We found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR⁺) stromal cells is required to maintain nerve fibers in adult bone marrow (Nature Cell Biology, 2023). In nerveless bone marrow, steady-state hematopoiesis was normal, but hematopoietic and vascular regeneration were impaired after myeloablation. Nerves promoted regeneration by activating β2 and β3 adrenergic receptor signaling in LepR⁺ cells, increasing their production of multiple hematopoietic and vascular regeneration growth factors.

These results raised the possibility that nonselective β blockers could inhibit engraftment after hematopoietic cell transplants (HCTs). Mice treated with a nonselective β blocker (carvedilol), but not a β1-selective inhibitor (metoprolol), exhibited impaired hematopoietic regeneration after syngeneic or allogeneic HCTs. At two institutions, patients who received nonselective, but not β1-selective, β blockers after allogeneic HCT exhibited delayed platelet engraftment and reduced survival (Cancer Discovery, 2025). This was particularly observed in patients who received posttransplant chemotherapy for graft-versus-host disease (GVHD) prophylaxis, which accentuated the inhibitory effect of carvedilol on engraftment in mice.

In terms of intrinsic mechanisms, hematopoietic stem cells (HSCs) and erythropoiesis are activated during pregnancy and after bleeding by the derepression of retrotransposons, including endogenous retroviruses and long interspersed nuclear elements (LINEs) (Science, 2024). Retrotransposon transcription activates the innate immune sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), which induce interferon and interferon-regulated genes in HSCs, increasing HSC division and erythropoiesis. Inhibition of reverse transcriptase or deficiency of cGAS or STING had little or no effect on hematopoiesis in nonpregnant mice but depleted HSCs and erythroid progenitors in pregnant mice, reducing red blood cell counts. Retrotransposons and interferon-regulated genes were also induced in mouse HSCs after serial bleeding and in human HSCs during pregnancy. Reverse transcriptase inhibitor use was associated with anemia in pregnant, but not nonpregnant, women, suggesting conservation of these mechanisms from mice to humans.

我们发现瘦素受体表达（LepR+）基质细胞产生的神经生长因子（NGF）是维持成人骨髓神经纤维所必需的（Nature Cell Biology, 2023）。在无神经的骨髓中，稳态造血功能正常，但骨髓消融后造血功能和血管再生受损。神经通过激活LepR+细胞中β2和β3肾上腺素能受体信号，增加其多种造血和血管再生生长因子的产生，从而促进再生。这些结果提出了非选择性β受体阻滞剂抑制造血细胞移植（hct）后移植的可能性。用非选择性β受体阻滞剂（卡维地洛）而非β1选择性抑制剂（美托洛尔）治疗的小鼠，在同基因或异体造血干细胞移植后表现出造血再生受损。在两家机构中，同种异体HCT后接受非选择性而非β1选择性β受体阻滞剂的患者表现出血小板植入延迟和生存率降低（Cancer Discovery, 2025）。这在接受移植后化疗以预防移植物抗宿主病（GVHD）的患者中尤其明显，这加强了卡维地洛对小鼠移植的抑制作用。就内在机制而言，造血干细胞（hsc）和红细胞生成在怀孕期间和出血后通过抑制逆转录转座子，包括内源性逆转录病毒和长穿插核元件（LINEs）而激活（Science, 2024）。反转录转座子转录激活先天免疫传感器环GMP-AMP合成酶（cGAS）和干扰素基因刺激因子（STING），诱导造血干细胞中的干扰素和干扰素调节基因，增加造血干细胞分裂和红细胞生成。抑制逆转录酶或缺乏cGAS或STING对未怀孕小鼠的造血功能影响很小或没有影响，但在怀孕小鼠中耗尽hsc和红细胞祖细胞，减少红细胞计数。逆转录转座子和干扰素调节基因也在连续出血后的小鼠造血干细胞和妊娠期间的人造血干细胞中被诱导。逆转录酶抑制剂的使用与怀孕妇女的贫血有关，但与非怀孕妇女无关，这表明这些机制从小鼠到人类都是守恒的。

{"title":"1006 – HEMATOPOIETIC STEM CELL ACTIVATION FOR BLOOD REGENERATION","authors":"Sean Morrison","doi":"10.1016/j.exphem.2025.104886","DOIUrl":"10.1016/j.exphem.2025.104886","url":null,"abstract":"<div><div>We found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR<sup>+</sup>) stromal cells is required to maintain nerve fibers in adult bone marrow (Nature Cell Biology, 2023). In nerveless bone marrow, steady-state hematopoiesis was normal, but hematopoietic and vascular regeneration were impaired after myeloablation. Nerves promoted regeneration by activating β2 and β3 adrenergic receptor signaling in LepR<sup>+</sup> cells, increasing their production of multiple hematopoietic and vascular regeneration growth factors.</div><div>These results raised the possibility that nonselective β blockers could inhibit engraftment after hematopoietic cell transplants (HCTs). Mice treated with a nonselective β blocker (carvedilol), but not a β1-selective inhibitor (metoprolol), exhibited impaired hematopoietic regeneration after syngeneic or allogeneic HCTs. At two institutions, patients who received nonselective, but not β1-selective, β blockers after allogeneic HCT exhibited delayed platelet engraftment and reduced survival (Cancer Discovery, 2025). This was particularly observed in patients who received posttransplant chemotherapy for graft-versus-host disease (GVHD) prophylaxis, which accentuated the inhibitory effect of carvedilol on engraftment in mice.</div><div>In terms of intrinsic mechanisms, hematopoietic stem cells (HSCs) and erythropoiesis are activated during pregnancy and after bleeding by the derepression of retrotransposons, including endogenous retroviruses and long interspersed nuclear elements (LINEs) (Science, 2024). Retrotransposon transcription activates the innate immune sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), which induce interferon and interferon-regulated genes in HSCs, increasing HSC division and erythropoiesis. Inhibition of reverse transcriptase or deficiency of cGAS or STING had little or no effect on hematopoiesis in nonpregnant mice but depleted HSCs and erythroid progenitors in pregnant mice, reducing red blood cell counts. Retrotransposons and interferon-regulated genes were also induced in mouse HSCs after serial bleeding and in human HSCs during pregnancy. Reverse transcriptase inhibitor use was associated with anemia in pregnant, but not nonpregnant, women, suggesting conservation of these mechanisms from mice to humans.</div></div>","PeriodicalId":12202,"journal":{"name":"Experimental hematology","volume":"151 ","pages":"Article 104886"},"PeriodicalIF":2.1,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145620511","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

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 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

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 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 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 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

3012 – VASCULAR ENDOTHELIAL GROWTH FACTOR SIGNALING BLOCKS THE DEVELOPMENT OF HUMAN HEMATOPOIETIC STEM CELLS FROM INDUCED PLURIPOTENT STEM CELLS 3012 -血管内皮生长因子信号传导可阻断人造血干细胞从诱导多能干细胞的发育

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104952

Alexander Maytum, Ritika Saxena, Gulcan Sarila, Hasindu Edirisinghe, Katerina Terolli, Constanze Bonifer, Edouard Stanley, Andrew Elefanty, Elizabeth Ng

The generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has great therapeutic potential for stem cell therapy. In a world first, our laboratory has generated human HSCs from iPSCs, named induced HSCs (iHSCs), which are capable of long-term multilineage engraftment (Ng et al., 2024). The generation of iHSCs depends on cytokines and their receptors transmitting signals to transcription factors that bind to cis-regulatory elements, such as enhancers, in the nucleus. However, how chromatin-level signal interpretation defines gene regulatory networks and cellular identity remains unclear.

Using a murine serum-free embryonic stem (ES) cell differentiation system, we showed that the withdrawal of vascular endothelial growth factor (VEGF) strongly increased the efficiency of the endothelial-to-hematopoietic transition (EHT), yielding higher numbers of HSCs (Edginton-White et al., 2023). When these observations were applied to the human protocol, the removal of VEGF after endothelium formation resulted in a rapid loss of the endothelial marker CXCR4+ and an accelerated EHT with increased numbers of iHSCs. VEGF withdrawal increased both the rate of total engraftment of iHSCs and of multilineage engraftment. To investigate at the molecular level how VEGF blocks iHSC development, we performed single-cell RNA sequencing (scRNA-Seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) analysis on differentiating cells over an extensive time course with or without VEGF, followed by transplantation of iHSCs into mice. With VEGF, RUNX1 targets fail to be upregulated, and conversely, endothelial genes are not downregulated, impacting iHSC commitment. We also found that with VEGF, iHSCs rapidly lose their stem cell phenotype and commit to differentiation. Our data demonstrated that cytokines are true regulators of cell fate changes and need to be applied in a timed fashion to regulate hematopoietic differentiation in vitro.

诱导多能干细胞（iPSCs）生成造血干细胞（hsc）在干细胞治疗中具有巨大的治疗潜力。我们的实验室在世界上首次从多能干细胞中生成了人类造血干细胞，称为诱导造血干细胞（ihsc），能够长期多系移植（Ng et al., 2024）。造血干细胞的产生依赖于细胞因子及其受体向转录因子传递信号，转录因子与细胞核中的顺式调控元件（如增强子）结合。然而，染色质水平的信号解释如何定义基因调控网络和细胞身份仍不清楚。通过小鼠无血清胚胎干（ES）细胞分化系统，我们发现血管内皮生长因子（VEGF）的退出显著提高了内皮向造血转化（EHT）的效率，产生了更多的造血干细胞（edgington - white et al., 2023）。当这些观察结果应用于人类方案时，内皮形成后去除VEGF导致内皮标记物CXCR4+的快速丢失，EHT加速，ihsc数量增加。VEGF停药增加了ihsc的总移植率和多系移植率。为了在分子水平上研究VEGF如何阻断iHSC的发育，我们对有或没有VEGF的分化细胞进行了单细胞RNA测序（scRNA-Seq）和转座酶可及染色质高通量测序（ATAC-Seq）分析，然后将iHSC移植到小鼠体内。对于VEGF， RUNX1靶点不能上调，相反，内皮基因不下调，影响iHSC承诺。我们还发现，在VEGF的作用下，ihsc迅速失去其干细胞表型并致力于分化。我们的数据表明，细胞因子是细胞命运变化的真正调节器，需要在体外以定时方式应用来调节造血分化。

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引用次数: 0

3014 – CELLULAR MORPHOMETRIC ANALYSIS OF MOUSE FETAL LIVER IDENTIFIES HEPATOBLASTIC NICHE FOR HEMATOPOIETIC STEM CELLS 3014 -小鼠胎肝细胞形态计量学分析确定造血干细胞的肝母细胞生态位

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104955

Harsh Agrawal , Atreyi Biswas , Shubham Mehatre , Soham Chatterjee , Siddharth Kurne , Athira Suresh , Gayathri Kartha , Satish Khurana

The fetal liver (FL) is a key hematopoietic niche during embryonic development, allowing the expansion and differentiation of hematopoietic stem cells (HSCs). The spatial distribution of HSCs and the cellular components that constitute their niche within the FL remain poorly understood. Here, we investigated the spatiotemporal dynamics of HSC localization and their microenvironmental interactions at embryonic days (E) 14.5 and E18.5. Using high-resolution immunofluorescence microscopy and three-dimensional image reconstruction, we found that HSCs at E14.5 preferentially associate with DLK1+ hepatoblasts that form a supportive microenvironment for proliferation and expansion. By E18.5, HSCs migrate toward perisinusoidal regions as the influence of hepatoblasts diminishes. Analysis of single-cell RNA-sequencing data from murine FL showed enriched expression of hematopoietic regulators in hepatoblasts at E14.5. CellChat analysis further identified key hematopoietic signaling pathways enriched between hepatoblasts and HSCs at E14.5, suggesting active crosstalk that supports HSC maintenance and expansion. The expression of hematopoietic support factors declined significantly by E17.5, coinciding with the establishment period of BM as the new hematopoietic site. This study demonstrates the dynamic nature of the FL niche, where early-stage hepatoblasts play a pivotal role in HSC support, transitioning to a sinusoidal-dominant niche as development progresses. These insights deepen our understanding of fetal hematopoiesis and the evolving microenvironment that shapes HSC behavior. By elucidating these temporal changes in the FL niche, our findings provide valuable perspectives on HSC biology and potential strategies for enhancing ex vivo HSC expansion for therapeutic purposes.

胎儿肝脏（FL）是胚胎发育过程中一个关键的造血生态位，允许造血干细胞（hsc）的扩张和分化。造血干细胞的空间分布和构成其在FL内生态位的细胞成分仍然知之甚少。在此，我们研究了胚胎期(E) 14.5和E18.5时HSC定位的时空动态及其微环境相互作用。通过高分辨率免疫荧光显微镜和三维图像重建，我们发现E14.5的hsc优先与DLK1+肝母细胞结合，形成支持增殖和扩张的微环境。到E18.5时，随着肝母细胞的影响减弱，造血干细胞向肝周区域迁移。小鼠FL单细胞rna测序数据分析显示，E14.5时肝母细胞中造血调节因子的表达丰富。CellChat分析进一步确定了在E14.5时肝母细胞和HSC之间富集的关键造血信号通路，表明主动串扰支持HSC的维持和扩展。造血支持因子的表达在E17.5时显著下降，与BM作为新的造血部位的建立时间一致。这项研究证明了FL生态位的动态性质，其中早期肝母细胞在HSC支持中起关键作用，随着发育的进展，过渡到正弦主导的生态位。这些见解加深了我们对胎儿造血和塑造HSC行为的不断发展的微环境的理解。通过阐明FL生态位的这些时间变化，我们的研究结果为HSC生物学提供了有价值的视角，并为提高HSC体外扩增的治疗目的提供了潜在的策略。

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引用次数: 0