Experimental hematology最新文献_第6页

3029 – MODELING EFFICIENT HSC DEVELOPMENT FROM HUMAN PLURIPOTENT STEM CELLS 3029 -模拟人类多能干细胞高效发展HSC

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104970

Vladimir Manchev, Jamie Kwan, Marion Kennedy, Quynh Nguyen, Nicole Zhao, Brenda Cohen, Gordon Keller

Hematopoietic stem cell (HSC) transplantation is a proven treatment for hematologic/immunologic disorders, including leukemias, immunodeficiencies, and autoimmune diseases. The on-demand production of HSCs from pluripotent stem cells (PSCs) for such therapies is one of the greatest promises of regenerative medicine and, as recent work has shown, is now possible. In this study, we have built on these and other findings to establish a protocol that promotes the reliable and efficient generation of long-term engrafting HSCs from both human embryonic and induced PSCs. Three key differentiation steps were refined and optimized in this work. For the first step, mesoderm induction, the WNT, FGF, ACTIVIN/NODAL, and BMP pathways were manipulated in a staged fashion to yield populations consisting of greater than 90% APJ+ and RALDH2+ cells from all hPSC lines tested. The next step, specification of the hemato-vascular fate, was optimized with precise levels of VEGF/BMP signaling to generate populations of > 90% CD34+ cells. Finally, the differentiation of the CD34+ hemato-vascular cells to CD34+ hematopoietic progenitors was carried out under conditions designed to maintain engraftment potential. Day 15 CD34+ cells generated in this way demonstrate multilineage engraftment (10%–60%) 20 weeks after transplantation into NSG W41 mice. Notably, most mice (> 75%) transplanted with between 5 × 10⁵ and 2 × 10⁶ cells show multilineage engraftment. To characterize the temporal patterns of repopulation, we sampled bone marrow of the recipient animals at different times and found that a graft of > 5% present relatively early is predictive of long-term engraftment (at 20 weeks). The ability to assess engraftment early will significantly decrease the iteration time for further improving the efficiency of HSC production, an essential next step for translation to therapeutic applications.

造血干细胞（HSC）移植是一种被证实的治疗血液/免疫疾病的方法，包括白血病、免疫缺陷和自身免疫性疾病。从多能干细胞（PSCs）中按需生产造血干细胞用于此类治疗是再生医学最伟大的承诺之一，正如最近的工作所表明的那样，现在是可能的。在这项研究中，我们建立在这些和其他发现的基础上，建立了一个方案，促进可靠和有效地从人类胚胎和诱导的造血干细胞中产生长期移植的造血干细胞。本文对三个关键的鉴别步骤进行了细化和优化。第一步，中胚层诱导、WNT、FGF、ACTIVIN/NODAL和BMP通路被分阶段操纵，以产生由所有测试的hPSC系中超过90%的APJ+和RALDH2+细胞组成的群体。下一步，指定血管命运，通过精确水平的VEGF/BMP信号来优化生成>； 90% CD34+细胞群。最后，在维持移植潜能的条件下，将CD34+血管细胞分化为CD34+造血祖细胞。第15天，以这种方式产生的CD34+细胞在移植到NSG W41小鼠体内20周后显示出多系植入（10%-60%）。值得注意的是，大多数移植了5个 × 105和2个 × 106细胞的小鼠（> 75%）表现出多系移植。为了描述再种群的时间模式，我们在不同时间对受体动物的骨髓进行了采样，发现相对较早出现的5%的移植物预示着长期移植（20周）。早期评估移植的能力将显著减少迭代时间，进一步提高HSC生产效率，这是转化为治疗应用的重要下一步。

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引用次数: 0

1005 – MAPPING THE DEVELOPMENTAL PATHWAYS TO HUMAN HEMATOPOIETIC AND LEUKEMIC STEM CELLS 1005 -绘制人类造血和白血病干细胞的发育途径

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104885

Hanna Mikkola

No Abstract Submitted

未提交摘要

引用次数: 0

1001 – HOW CELL STAGE-SPECIFIC TRANSCRIPTION FACTORS AND OUTSIDE SIGNALS REGULATE HEMATOPOIETIC STEM CELL DEVELOPMENT AND DIFFERENTIATION – A JOURNEY THROUGH TIME 细胞阶段特异性转录因子和外部信号如何调节造血干细胞的发育和分化-穿越时间的旅程

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104881

Constanze Bonifer

The ability of cells to respond to external stimuli is one of the characteristics of life. Multicellular organisms have developed a huge machinery that interprets the cellular environment and instigates an appropriate cellular response by changing gene expression, cell differentiation state, metabolism, proliferation state, and motility. Decades of research have studied the pathways transmitting the various signals within the cell. However, although we know most of the molecular players, we still do not fully understand how extrinsic signals are interpreted and integrated within the genome and lead to genome-wide responses within a specific chromatin environment.

In this lecture, I touch upon this question by summarizing our work, which used both mouse and human embryonic stem (ES) cell differentiation into blood as models to explain how signaling-responsive cis-regulatory elements interact with signaling-responsive and constitutively expressed transcription factors and drive differentiation and growth. I describe how differential enhancer activation drives the changes in gene regulatory networks during blood cell development. We show that inducible transcription factors that transmit signaling processes to the genome are essential for changing gene expression within a given gene regulatory network, how they interact with cell-type-specific factors within a pre-existing chromatin landscape, and how they integrate multiple signaling pathways at specific enhancer elements to both maintain and alter cellular identities. In addition, we show that the signaling-dependent programming of chromatin and the opening-up of blood-specific developmental pathways start long before the onset of blood-specific gene expression, providing a mechanistic explanation for the finding that a precisely timed relay of signals is essential for executing correct blood cell development. Finally, I will show data demonstrating that signaling processes and signaling-responsive transcription factors are at the heart of acute myeloid leukemia development.

细胞对外界刺激作出反应的能力是生命的特征之一。多细胞生物已经发展出一个巨大的机制来解释细胞环境，并通过改变基因表达、细胞分化状态、代谢、增殖状态和运动来引发适当的细胞反应。几十年来，人们一直在研究细胞内传递各种信号的途径。然而，尽管我们知道大多数的分子参与者，我们仍然不完全了解外在信号是如何在基因组中被解释和整合的，并导致特定染色质环境下的全基因组反应。在本次讲座中，我将通过总结我们的工作来探讨这个问题，我们使用小鼠和人类胚胎干细胞分化为血液作为模型来解释信号响应性顺式调控元件如何与信号响应性和组成性表达的转录因子相互作用，并驱动分化和生长。我描述了差异增强子激活如何驱动血细胞发育过程中基因调控网络的变化。我们表明，在给定的基因调控网络中，向基因组传递信号过程的诱导转录因子对于改变基因表达至关重要，它们如何与预先存在的染色质景观中的细胞类型特异性因子相互作用，以及它们如何整合特定增强子元件的多种信号通路以维持和改变细胞身份。此外，我们表明，染色质的信号依赖性编程和血液特异性发育途径的开放早在血液特异性基因表达开始之前就开始了，这为精确定时的信号传递对于执行正确的血细胞发育至关重要的发现提供了机制解释。最后，我将展示的数据表明，信号传导过程和信号响应转录因子是急性髓性白血病发展的核心。

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引用次数: 0

3023 – DECODING THE ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION IN HUMAN IPSC-DERIVED HEMATOPOIETIC ORGANOIDS: TEMPORAL DYNAMICS, ENDOTHELIAL DIVERSITY, AND TRANSCRIPTIONAL DRIVERS 3023 -解码人类ipsc衍生的造血器官从内皮到造血的转变：时间动力学、内皮多样性和转录驱动因素

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104964

Oyishee Ahmad , Huan Zhang , Lucas Kuijpers , Marien van der Stel , Arthur Svendsen , Eszter Varga , Emile van den Akker

Blood transfusions and hematopoietic stem and progenitor cell (HSPC) transplantation can be improved by generating patient-specific, engraftable HSCs and transfusable blood cells in vitro. During fetal development, functional HSCs arise from hemogenic endothelium (HE) via endothelial-to-hematopoietic transition (EHT). HE originates in CXCR4⁺ arterial endothelium in the dorsal aorta of the aorta-gonad mesonephros. We developed an induced pluripotent stem cell-derived hematopoietic organoid (HeO) that produces definitive enucleated erythrocytes. We hypothesize that hematopoiesis in this model occurs via EHT. Here, we characterized HE cell (HEC) dynamics and EHT, examined endothelial heterogeneity during EHT, and identified intrinsic EHT regulators.

HECs were defined as CD144⁺CD309⁺CD34⁺CD31⁺CD43⁻CD73⁻. Flow cytometry of dissociated HeOs showed tightly regulated HEC emergence (days 19–21; 3%), followed by a surge in CD43⁺ hematopoietic cells. Single-cell RNA sequencing of CD309⁺CD34⁺ endothelial cells at peak HE identified six clusters. Comparison with in vivo EHT data sets revealed shared marker profiles for arterial endothelium (CXCR4⁺, GJA⁺), pre-HE (CXCR4⁺GJA⁺IL33⁺), HE (KCNK17⁺CD44⁺RUNX1⁺), and HSPCs (SPN⁺PTPRC⁺SPINK2⁺) in our arterial and hematopoietic-endothelial clusters. We found known and novel EHT regulators, including upregulated NOTCH signaling in the arterial cluster and hematopoietic regulators (RUNX1, GFI1/GFI1B) and translation genes in the hematopoietic-endothelial cluster. Culturing purified HECs or CD309⁺CD34⁺ populations confirmed EHT progression using flow cytometry and microscopy.

These findings define EHT timing and HE emergence, show endothelial heterogeneity, and confirm, via transcriptomics, cells undergoing EHT in the HeOs. Together, the data support improved HSPC yield from our HeO and validate its use as a developmental hematopoietic model.

输血和造血干细胞和祖细胞（HSPC）移植可以通过在体外产生患者特异性的、可移植的造血干细胞和可输注的血细胞来改善。在胎儿发育过程中，造血干细胞由造血内皮（HE）通过内皮-造血转化（EHT）产生。HE起源于主动脉-性腺中肾背主动脉的CXCR4 +动脉内皮。我们开发了一种诱导多能干细胞衍生的造血器官（HeO），可以产生最终的去核红细胞。我们假设该模型中的造血是通过EHT发生的。在这里，我们表征了HE细胞（HEC）动力学和EHT，检查了EHT过程中的内皮异质性，并确定了EHT的内在调节因子。hec定义为CD144 + CD309 + CD34 + CD31 + CD43⁻CD73⁻。游离HeOs的流式细胞术显示HEC出现受到严格调节（第19-21天；3%），随后CD43 +的造血细胞激增。CD309 + CD34 +内皮细胞HE峰的单细胞RNA测序鉴定出6个簇。与体内EHT数据集的比较显示，在我们的动脉和造血内皮簇中，动脉内皮（CXCR4 +、GJA +）、预HE （CXCR4 + GJA + IL33 +）、HE （KCNK17 + CD44 + RUNX1 +）和HSPCs （SPN + PTPRC + SPINK2 +）具有共同的标志物特征。我们发现了已知的和新的EHT调节因子，包括动脉簇和造血调控因子（RUNX1, GFI1/GFI1B）中上调的NOTCH信号，以及造血内皮簇中的翻译基因。培养纯化的hec +或CD309 + CD34 +群体使用流式细胞术和显微镜证实EHT进展。这些发现确定了EHT的时间和HE的出现，显示了内皮的异质性，并通过转录组学证实了HeOs中发生EHT的细胞。总之，这些数据支持HeO提高HSPC产量，并验证其作为发育造血模型的用途。

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引用次数: 0

3009 – BLOOD-RESIDENT MACROPHAGES (BMΦ) SAFEGUARD BLOOD AND VESSEL INTEGRITY 3009 -血液巨噬细胞（bmΦ）保护血管的完整性

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104949

Bart Weijts , Jeroen Demmers , Catherine Robin

Tissue-resident macrophages are highly heterogeneous phagocytic cells essential for immune defense, tissue development, and the onset and progression of diseases. They originate from yolk sac progenitors during early embryonic development, colonizing most tissues, where they persist ubiquitously throughout life. However, no dedicated macrophage population is known to function within the vast network of blood vessels, despite the importance of maintaining a healthy vascular and blood system. Using the zebrafish model and high-resolution fluorescent microscopy, we identified a population of macrophages that exclusively functions within the blood vessels, which we term blood-resident macrophages (bMΦ). bMΦ actively surveys the blood to eliminate foreign particles and unfit cells and acts as the first responder to endothelial damage. bMΦ emerge directly from the main axial blood vessels through an endothelial-to-macrophage transition (EMacT). This process is independent of Runx1, which is known for its role in the endothelial-to-hematopoietic transition leading to hematopoietic stem cell production, and of Csf1r, a gene crucial for normal myeloid differentiation. Similar to tissue-resident macrophages, bMΦ persist into adulthood, where they also clear foreign particles from the bloodstream. Importantly, our preliminary data indicate that bMΦ are conserved in mice, where they fulfil a similar role to their zebrafish counterparts. Thus, we unveiled the existence of a previously unrecognized population of macrophages that function within the confines of the vasculature to safeguard blood from foreign threats and maintain vascular integrity. This discovery not only expands our understanding of immune surveillance mechanisms but also offers potential avenues for the treatment of bloodborne diseases and vascular injuries.

组织常驻巨噬细胞是高度异质性的吞噬细胞，对免疫防御、组织发育和疾病的发生和进展至关重要。它们起源于胚胎早期发育的卵黄囊祖细胞，在大多数组织中定植，并在生命中无处不在。然而，尽管巨噬细胞对维持健康的血管和血液系统很重要，但目前还没有专门的巨噬细胞群在庞大的血管网络中发挥作用。利用斑马鱼模型和高分辨率荧光显微镜，我们发现了一种专门在血管内起作用的巨噬细胞，我们称之为血液常驻巨噬细胞（bMΦ）。bMΦ主动检查血液以消除外来颗粒和不适合的细胞，并作为内皮损伤的第一反应者。bMΦ通过内皮细胞向巨噬细胞转化（EMacT）直接从主轴血管产生。这一过程独立于Runx1和Csf1r，前者在内皮细胞向造血细胞的转化过程中发挥作用，导致造血干细胞的产生，而后者是正常髓细胞分化的关键基因。与组织内的巨噬细胞类似，bMΦ会持续存在到成年，在成年后它们也会清除血液中的外来颗粒。重要的是，我们的初步数据表明bMΦ在小鼠中是保守的，它们在斑马鱼中发挥着类似的作用。因此，我们揭示了一种以前未被认识的巨噬细胞群体的存在，它们在脉管系统范围内发挥作用，保护血液免受外来威胁并维持血管完整性。这一发现不仅扩大了我们对免疫监视机制的理解，而且为治疗血源性疾病和血管损伤提供了潜在的途径。

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引用次数: 0

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 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

2002 – EXPEDITED MONOCYTE-INDEPENDENT MACROPHAGE DIFFERENTIATION FROM A DEVELOPMENTALLY RESTRICTED HUMAN DEFINITIVE HEMATOPOIETIC STEM CELL 2002 -加速单核细胞非依赖性巨噬细胞从发育受限的人类最终造血干细胞分化

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104915

Grant Rowe , Katie Frenis , Brianna Badalamenti , Ohnaz Mamigonian , Chen Wang , Dahai Wang , Sara Fierstein , Parker Cote , Hesper Khong , Hojun Li , Edroaldo Lummertz da Rocha , Vijay Sankaran

The impact of hematopoietic development on human macrophage diversity is incompletely understood but is broadly relevant to inflammatory diseases. We performed M-CSF-driven differentiation assays using human fetal or adult hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). Strikingly, we found that fetal HSCs/MPPs rapidly produce macrophages within 4–5 days compared with adult HSCs/MPPs, which require 10–14 days. To understand this developmental divergence in macrophage specification, we performed single-cell RNA sequencing of human fetal and adult definitive HSCs, monoblasts, monocytes, and macrophages. In this dataset, we identified proinflammatory monocyte-derived macrophages (MMacs) with clear canonical differentiation trajectories from HSCs via monoblasts and monocytes at both ages. However, we observed a fetal-specific anti-inflammatory tissue-resident-like macrophage (TRM) population that appeared to bypass canonical intermediates to emerge via an alternative pathway. Informed by this dataset, we identified and functionally validated a transient M-CSFR+ population of definitive human fetal HSCs primed for expedited noncanonical TRM differentiation. We then hypothesized that the rapid differentiation from these primed HSCs enables retention of HSC-like self-renewal programs in TRMs. We confirmed that, compared with MMacs, fetal TRMs undergo self-renewal that can be amplified by inhibition of the aryl hydrocarbon receptor (AHR). Finally, we demonstrate that selective TRM expansion via AHR antagonism mitigates inflammation in vivo in a model of eczema. Overall, we identified a developmentally restricted, TRM-primed, definitive HSC population. We postulate that developmental differences in macrophage programming at the level of HSCs underlie human macrophage heterogeneity, a finding that can be exploited in the treatment of inflammatory diseases.

造血发育对人类巨噬细胞多样性的影响尚不完全清楚，但与炎症性疾病广泛相关。我们使用人胎儿或成人造血干细胞（hsc）和多能祖细胞（mpp）进行了m - csf驱动的分化实验。引人注目的是，我们发现胎儿hsc /MPPs在4-5天内迅速产生巨噬细胞，而成人hsc /MPPs则需要10-14天。为了理解这种巨噬细胞发育分化，我们对人类胎儿和成人终代造血干细胞、单核细胞、单核细胞和巨噬细胞进行了单细胞RNA测序。在这个数据集中，我们发现促炎单核细胞来源的巨噬细胞（MMacs）在两个年龄段都具有从造血干细胞到单核细胞和单核细胞的明确的典型分化轨迹。然而，我们观察到胎儿特异性抗炎组织驻留样巨噬细胞（TRM）群体似乎绕过典型中间体，通过另一种途径出现。根据该数据集，我们鉴定并功能验证了一个短暂的M-CSFR+人类胎儿造血干细胞群体，为加速非典型TRM分化做了准备。然后，我们假设从这些引物hsc的快速分化使trm中保留hsc样自我更新程序。我们证实，与MMacs相比，胎儿TRMs可以通过抑制芳烃受体（AHR）进行自我更新。最后，我们在湿疹模型中证明，通过AHR拮抗选择性TRM扩张可以减轻体内炎症。总的来说，我们确定了一个发育受限的、trm引发的、确定的HSC人群。我们假设巨噬细胞编程在造血干细胞水平上的发育差异是人类巨噬细胞异质性的基础，这一发现可以用于炎症性疾病的治疗。

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引用次数: 0

1028 – MESENCHYMAL STROMAL CELL SENESCENCE AUGMENTS HEMATOPOIETIC CELL FITNESS IN CLONAL HEMATOPOIESIS 在克隆造血中，间充质间质细胞的衰老增强了造血细胞的适应性

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104908

Jennifer Trowbridge , Jayna Mistry , Kira Young , Anna Navarro Figueredo , Patricia Colom Diaz , Maria Telpoukhovskaia , Inés Fernández Maestre , Sheng Cai , Katharina Götze , Ross Levine

Noncell-autonomous mechanisms impact tumor growth and metastasis, including remodeling of the tissue microenvironment. The extent to which remodeling of the microenvironment promotes premalignant clonal fitness remains unknown. Here, using single-cell RNA sequencing of the bone marrow microenvironment in a mouse model of DNMT3A-mutant clonal hematopoiesis, we identify subsets of mesenchymal stromal cells (MSCs) in a molecular state of cellular senescence. Using in vivo and ex vivo approaches, we found that MSC senescence is induced by Dnmt3a-mutant hematopoietic cells in a cell contact-independent manner through the cytokines interleukin 6 (IL-6) and tumor necrosis factor (TNF). Mechanistically, these cytokines activate a STAT3/ROS/p53-driven pathway selectively in MSCs and not in endothelial cells, underscoring the cell context specificity of senescence induction. Depletion of senescent MSCs using genetic or pharmacologic methods reduces the fitness of Dnmt3a-mutant hematopoietic stem and progenitor cells and delays development of myeloproliferation, which is a precursor to myeloid malignancy. Together, our work identifies that remodeling of the tissue microenvironment modifies premalignant clonal fitness and suggests disruption of the cross-talk between premalignant cells and their niche as a cancer prevention strategy.

非细胞自主机制影响肿瘤的生长和转移，包括组织微环境的重塑。微环境重塑在多大程度上促进癌前克隆适应度仍不清楚。在这里，我们使用dnmt3a突变克隆造血小鼠模型骨髓微环境的单细胞RNA测序，鉴定了细胞衰老分子状态下的间充质基质细胞（MSCs）亚群。通过体内和离体方法，我们发现dnmt3a突变的造血细胞通过细胞接触无关的方式通过白细胞介素6 （IL-6）和肿瘤坏死因子（TNF）诱导间充质干细胞衰老。从机制上讲，这些细胞因子在MSCs中选择性地激活STAT3/ROS/p53驱动通路，而不是在内皮细胞中，强调了细胞背景诱导衰老的特异性。使用遗传或药理学方法消耗衰老的MSCs会降低dnmt3a突变的造血干细胞和祖细胞的适应度，并延迟骨髓增殖的发展，而骨髓增殖是髓系恶性肿瘤的前兆。总之，我们的工作确定了组织微环境的重塑改变了癌前细胞克隆适应度，并建议破坏癌前细胞及其生态位之间的串扰作为一种癌症预防策略。

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引用次数: 0

1024 – A GENETIC LABELING STRATEGY FOR COMPETITIVE XENOTRANSPLANTATION OF HUMAN HSCS 人类造血干细胞竞争性异种移植的基因标记策略

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104904

Satoshi Yamazaki

Xenotransplantation into immunodeficient mice remains the gold standard for assessing the stemness and multilineage engraftment potential of human hematopoietic stem cells (HSCs) following ex vivo expansion. However, current approaches rely primarily on single-condition transplants, limiting the ability to directly compare the functional properties of HSCs cultured under different conditions. In the murine system, competitive transplantation assays using congenic markers (e.g., CD45.1 and CD45.2) enable side-by-side evaluation of HSC engraftment, but comparable strategies for human HSCs are lacking.

Here, we report the development of a CRISPR/Cas9-based genetic labeling system that enables competitive xenotransplantation assays for human HSCs. We targeted the PTPRC gene, which encodes the pan-hematopoietic marker CD45, in cord blood-derived CD34⁺ cells to introduce distinguishable surface epitopes. This approach allows robust, nonviral, and heritable labeling of expanded human HSCs, thereby enabling the simultaneous tracking of cells cultured under different ex vivo conditions in a single recipient.

This platform provides a quantitative and efficient method to evaluate the stemness and functional potential of human HSCs and will accelerate the development of optimized ex vivo expansion protocols and regenerative therapies.

异种移植到免疫缺陷小鼠中仍然是评估人造血干细胞（hsc）体外扩增后的干性和多系移植潜力的金标准。然而，目前的方法主要依赖于单条件移植，限制了直接比较不同条件下培养的造血干细胞功能特性的能力。在小鼠系统中，使用同源标记（例如CD45.1和CD45.2）的竞争性移植试验能够对HSC移植进行并行评估，但缺乏用于人类HSC的可比策略。在这里，我们报告了一种基于CRISPR/ cas9的遗传标记系统的发展，该系统能够对人类造血干细胞进行竞争性异种移植检测。我们在脐带血来源的CD34 +细胞中靶向编码泛造血标记CD45的PTPRC基因，以引入可区分的表面表位。这种方法允许对扩增的人造血干细胞进行稳健的、非病毒的和可遗传的标记，从而能够在单个受体中同时跟踪在不同体外条件下培养的细胞。该平台提供了一种定量和高效的方法来评估人类造血干细胞的干性和功能潜力，并将加速优化体外扩增方案和再生疗法的发展。

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引用次数: 0

3037 – A SUBSET OF MEGAKARYOCYTES REGULATES DEVELOPMENT OF HEMATOPOIETIC STEM CELL PRECURSORS 3037 -巨核细胞的一个亚群调节造血干细胞前体的发育

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104978

Wenlang Lan , Zhuan Li , Jinping Li , Zehua Ye

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocytes emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we used platelet factor 4 (PF4)-Cre; RosatdTomato+ cells to report the presence of megakaryocytes in the HSC developmental niche. Further, we used the PF4-Cre; Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in an OP9-DL1 coculture system. Single-cell RNA sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with the highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

了解胚胎发育过程中促进造血干细胞（HSC）规范的调控机制对体外造血干细胞的生成具有重要意义。巨核细胞从卵黄囊中出现并产生血小板，血小板参与多种生物过程，如预防出血。然而，巨核细胞是否调节胚胎主动脉-性腺-中肾（AGM）区域的HSC发育尚不清楚。在这里，我们使用血小板因子4 (PF4)-Cre；RosatdTomato+细胞报告巨核细胞在HSC发育生态位中的存在。此外，我们使用了PF4-Cre；Rosa-DTA （DTA）耗尽模型揭示巨核细胞在小鼠胚胎中控制HSC规格。巨核细胞缺乏阻断了造血干细胞前的生成和成熟，并改变了造血干细胞在AGM中的活性。此外，巨核细胞在OP9-DL1共培养系统中促进内皮细胞向造血细胞的转变。单细胞RNA测序鉴定细胞表面标记物CD226阳性的巨核细胞是通过分泌TNFSF14促进内皮细胞造血命运的最高潜力亚群。因此，TNFSF14治疗可以挽救巨核细胞缺失共培养中的造血细胞功能。综上所述，巨核细胞促进造血前细胞的产生和成熟，在胚胎造血过程中作为一个关键的微环境控制因子。

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引用次数: 0