Experimental hematology最新文献_第4页

3033 – ATLAS OF CYNOMOLGUS MACAQUE HEMATOPOIESIS 3033 -食蟹猴造血图谱

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104974

Ryo Yamamoto

Self-renewal and differentiation are inherent properties of hematopoietic stem cells (HSCs) that are necessary to support hematopoiesis; however, the underlying mechanisms, especially in humans, remain unclear. Here, using the cynomolgus macaque as a surrogate model, we develop a new gating strategy to isolate with high purity transplantable cynomolgus HSCs and generate a single-cell transcriptomic map of cynomolgus HSCs and progenitor cells—covering gestational periods previously not analyzed in humans.

We performed single-cell transcriptomic analysis using 10 × genomics and Smart-seq technologies. This study presents a single-cell transcriptomic analysis of CD34hi hematopoietic stem and progenitor cells (HSPCs) from cynomolgus monkeys across developmental stages—fetal liver, fetal bone marrow, and adult bone marrow. We found that there are dynamic shifts in HSPC composition with development: HSC/MPP frequencies peak in late second trimester and decrease with age, whereas myeloid and lymphoid progenitors increase and erythroid/megakaryocyte lineages decline. Developmental and tissue-specific gene expression differences were identified in HSCs, such as higher mitochondrial activity in fetal HSCs and NF-κB–related genes in aged ABM-HSCs. Furthermore, novel surface markers for transcriptionally purified HSCs were identified, offering improved tools for future HSC isolation. We anticipate that our comprehensive data set will serve as a basis for building an HSC atlas of cynomolgus monkeys that will facilitate a better understanding of conserved and nonconserved properties as well as mechanisms between nonhuman primates (NHPs) and humans, which will be necessary for better translational applications in the future.

自我更新和分化是造血干细胞（hsc）的固有特性，是支持造血所必需的；然而，潜在的机制，特别是在人类中，仍然不清楚。本研究以食蟹猕猴作为替代模型，开发了一种新的门控策略，分离出高纯度可移植的食蟹造血干细胞，并生成了覆盖人类妊娠期的食蟹造血干细胞和祖细胞的单细胞转录组图谱。我们使用10 × 基因组学和Smart-seq技术进行单细胞转录组学分析。本研究对食蟹猴不同发育阶段（胎儿肝脏、胎儿骨髓和成年骨髓）的造血干细胞和祖细胞（HSPCs）进行了单细胞转录组学分析。我们发现，随着发育，HSPC组成存在动态变化：HSC/MPP频率在妊娠中期达到峰值，并随着年龄的增长而下降，而髓系和淋巴系祖细胞增加，红细胞/巨核细胞谱系减少。在造血干细胞中发现了发育和组织特异性基因表达差异，例如胎儿造血干细胞中线粒体活性较高，老年abm -造血干细胞中NF-κ b相关基因较高。此外，还发现了转录纯化HSC的新表面标记，为未来HSC的分离提供了改进的工具。我们预计，我们的综合数据集将作为构建食蟹猴HSC图谱的基础，这将有助于更好地理解非人类灵长类动物（NHPs）与人类之间的保守和非保守特性以及机制，这将是未来更好的翻译应用所必需的。

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引用次数: 0

3034 – IN VITRO INDUCTION OF HEMATOPOIETIC STEM CELLS FROM EARLY HEMOGENIC ENDOTHELIAL CELLS 3034 -早期造血内皮细胞体外诱导造血干细胞

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104975

Mariko Tsuruda , Saori Morino-Koga , Xueyu Zhao , Shingo Usuki , Kei-ichiro Yasunaga , Tomomasa Yokomizo , Ryuichi Nishinakamura , Toshio Suda , Minetaro Ogawa

During embryogenesis, hematopoietic stem cells (HSCs) arise from hemogenic endothelial cells (HECs). HECs enter endothelial-to-hematopoietic transition (EHT) and differentiate into HSCs via pre-HSCs. Previously, we reported that two cytokines and feeder cells were sufficient to induce HSCs from HECs and pre-HSCs isolated from mouse embryos at embryonic day 10.5 (E10.5). However, the signaling requirements at earlier developmental stages remain largely undefined. In this study, we sought to identify the key signaling factors required to induce HSCs from E9.5 HECs in vitro. Transcriptomic profiling of E9.5 mouse embryos detected activation of bone morphogenetic protein (BMP) signaling in HECs. BMP4 treatment in the culture system enhanced the generation of multilineage hematopoietic colonies and long-term repopulating HSCs, as confirmed using colony-forming and transplantation assays. Furthermore, early-stage HECs capable of forming HSCs expressed the BMP4 receptor. These results suggest that BMP4 plays a key role in promoting the emergence of transplantable HSCs from early HECs and provides insight into stage-specific requirements for HSC development. These findings may contribute to establishing conditions for in vitro derivation of HSCs from pluripotent stem cells.

在胚胎发生过程中，造血干细胞（hsc）由造血内皮细胞（HECs）产生。hec进入内皮到造血转化（EHT），并通过造血干细胞前分化为造血干细胞。此前，我们报道了两种细胞因子和饲养细胞足以在胚胎10.5天（E10.5）从小鼠胚胎中分离出hec和前hsc诱导造血干细胞。然而，早期发育阶段的信号需求在很大程度上仍然不明确。在这项研究中，我们试图确定E9.5 hec体外诱导造血干细胞所需的关键信号因子。E9.5小鼠胚胎的转录组学分析检测到hec中骨形态发生蛋白（BMP）信号的激活。培养系统中的BMP4处理增强了多系造血集落的产生和长期再生的造血干细胞，这一点在集落形成和移植试验中得到了证实。此外，能够形成造血干细胞的早期hec表达BMP4受体。这些结果表明，BMP4在促进早期hec可移植HSC的出现中发挥了关键作用，并为HSC发展的阶段特异性需求提供了见解。这些发现可能有助于建立多能干细胞体外衍生造血干细胞的条件。

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引用次数: 0

3035 – HEMOGENIC GASTRULOIDS CAPTURE DYNAMIC SPECIFICATION OF HE AND EHT AND INFORM RULES OF HSCS FORMATION 3035 -造血原胃样蛋白捕获he和ht的动态规格，并告知造血干细胞形成的规则

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104976

Ylenia Cicirò , Denise Ragusa , Ayona Johns , Camilla Cerutti , Cristina Pina

Emergence of definitive hematopoietic stem cells (HSCs) during embryonic development is underpinned by the crucial process of endothelial-to-hematopoietic transition (EHT). Therein, HSCs and progenitors are specified from a specialized subset of endothelial cells termed hemogenic endothelium (HE). These cells reside in the aorta-gonad-mesonephros (AGM) region, namely in the ventral wall of the dorsal aorta, and acquire hematopoietic potential through an incompletely characterized morphogenetic and transcriptional programming process that generates the first self-renewing, multilineage HSCs.

We made use of our previously described mouse embryonic stem cell-based 3D hemogenic gastruloid (haemGx) to dissect the dynamic specification of HE and decipher the morpho-molecular reconfiguration that underpins EHT. The haemGx model recapitulates temporally accurate emergence of CD31+CD34+Kit+ cells with HE characteristics, followed by sequential waves of CD41+ and CD45+ cells, corresponding to hematopoietic progenitors.

We integrated high-content microscopy, flow cytometry, and scRNA-seq, coupled to gene regulatory network analysis, to comprehensively capture the transcriptional dynamics of EHT and model HSCs’ emergence in silico. We further dissected intrinsic and extrinsic contributions to EHT and hematopoietic specification by establishing a novel endothelial culture system derived from haemGx and exploring endothelial and hematopoietic formation with different cellular compositions.

Our integrated approach provides a mechanistically tractable model of HE specification and EHT. Comprehensive inference of the dynamic rules underpinning the earliest events in hematopoietic specification will be pivotal to the design of high-efficiency protocols for in vitro production of HSCs.

胚胎发育过程中确定的造血干细胞（hsc）的出现是由内皮到造血转化（EHT）的关键过程支撑的。其中，造血干细胞和祖细胞是由内皮细胞称为造血内皮（HE）的特殊亚群指定的。这些细胞位于主动脉-性腺-中肾（AGM）区域，即背主动脉腹侧壁，并通过不完全表征的形态发生和转录编程过程获得造血潜能，从而产生第一批自我更新的多系造血干细胞。我们利用先前描述的基于小鼠胚胎干细胞的3D造血原胃样蛋白（haemGx）来解剖HE的动态规范，并破译支撑EHT的形态-分子重构。haemGx模型概括了具有HE特征的CD31+CD34+Kit+细胞的时间准确出现，随后是CD41+和CD45+细胞的顺序波，对应于造血祖细胞。我们整合了高含量显微镜、流式细胞术和scRNA-seq，结合基因调控网络分析，全面捕捉了EHT和模型hsc在硅中的出现的转录动力学。我们通过建立源自haemGx的新型内皮培养系统，并探索不同细胞组成的内皮和造血形成，进一步剖析了EHT和造血规范的内在和外在贡献。我们的集成方法提供了HE规范和EHT的机械可处理模型。全面推断造血规范中支持最早事件的动态规则对于设计体外造血干细胞生产的高效方案至关重要。

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引用次数: 0

3036 – AUTOPHAGY REGULATES THE MATURATION OF HEMATOPOIETIC PRECURSORS IN THE EMBRYO 3036 -自噬调节胚胎中造血前体的成熟

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104977

Yumin Liu , Sifan Luo , Yuehang Chen , Zhuan Li

An understanding of the mechanisms regulating embryonic hematopoietic stem cell (HSC) development would facilitate their regeneration. The aorta gonad-mesonephros (AGM) region is the site for HSC production from hemogenic endothelial cells (HEC) through a process termed endothelial-to-hematopoietic transition (EHT). Although several regulators are involved in this process, the role of autophagy (macroautophagy) in preliver hematopoiesis remains unclear. Here, we investigated how autophagy influences the EHT in the AGM region. First, we showed that different states of autophagy existed in hematopoietic precursors and correlated with hematopoietic potential based on the RFP-EGFP-LC3 (LC3R/G) mouse model. The treatment with autophagy inhibitors resulted in the reduction of hematopoietic progenitors, indicating the possible roles of autophagy in hematopoietic development.

Furthermore, we generated Atg5 conditional knockout (cKO) mice (Vec-Cre; Atg5fl/fl) to disrupt autophagy. By combining LC3R/G with cKO embryos, we detected that the autophagic process was blocked in the hematopoietic-related cells, validating the role of Atg5 in autophagy. Atg5 deficiency impaired hematopoietic stem/progenitor cell (HSPC) function in the AGM region, expanded hematopoietic clusters, and increased immature pre-HSC I production. Trajectory analysis further demonstrated the delay of pre-HSC I maturation, collectively indicating disrupted EHT. Comparison analysis of transcriptomics between the cKO and control group showed increased interactions of Ncl-Ptn and Ncl-Mdk. Our functional and immunostaining data confirmed the altered nucleolin (NCL) distribution and the rescue role played by AS1411 (the aptamer of NCL) in the HEC and pre-HSC I fractions. In summary, we have discovered that autophagy regulates the EHT process and the maturation of pre-HSC from HEC, possibly through NCL distribution.

了解胚胎造血干细胞（HSC）发育的调控机制有助于其再生。主动脉性腺-中肾（AGM）区是造血内皮细胞（HEC）通过内皮-造血转化（EHT）过程产生造血干细胞的部位。虽然有几种调节因子参与这一过程，但自噬（巨噬）在肝前造血中的作用尚不清楚。在这里，我们研究了自噬如何影响AGM区域的EHT。首先，我们基于RFP-EGFP-LC3 （LC3R/G）小鼠模型发现造血前体细胞中存在不同状态的自噬，并与造血潜能相关。使用自噬抑制剂治疗导致造血祖细胞减少，提示自噬在造血发育中的可能作用。此外，我们制造了Atg5条件敲除（cKO）小鼠（Vec-Cre; Atg5fl/fl）来破坏自噬。通过将LC3R/G与cKO胚胎结合，我们检测到造血相关细胞的自噬过程被阻断，验证了Atg5在自噬中的作用。Atg5缺陷损害了AGM区域的造血干细胞/祖细胞（HSPC）功能，扩大了造血集群，增加了未成熟的前hsc I的产生。轨迹分析进一步表明，hsc I前成熟延迟，共同表明EHT中断。cKO与对照组的转录组学比较分析显示，Ncl-Ptn和Ncl-Mdk的相互作用增加。我们的功能和免疫染色数据证实了核仁蛋白（NCL）分布的改变以及AS1411 （NCL适配体）在HEC和预hsc I中发挥的拯救作用。综上所述，我们发现自噬可能通过NCL分布调节HEC的EHT过程和预hsc的成熟。

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引用次数: 0

1019 – DYING TO REGENERATE: ENDOGENOUS MECHANISMS OF TISSUE REPAIR IN THE THYMUS 死亡再生：胸腺组织修复的内源性机制

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104899

Jarrod Dudakov

The thymus, which is the primary site of T cell development, is extremely sensitive to insult but also has a remarkable capacity for endogenous repair. However, even though there is continual thymic involution and regeneration in response to everyday insults like stress and infection, profound thymic damage, such as ionizing radiation, leads to prolonged T cell lymphopenia. One approach to developing therapies to boost thymic function after radiation injury is to understand and exploit the processes underlying endogenous repair.

We have identified that the balance of cell death detection is crucial for triggering endogenous regenerative responses in the thymus. Specifically, immunologically silent apoptosis (which is abundant in thymocytes during steady-state) is suppressive to the regenerative program, but in contrast, after thymic damage, a switch toward immunogenic cell death (ICD) can promote regeneration. Importantly, many of these pathways can be therapeutically targeted to improve thymic recovery after radiation injury. However, induction of ICD (and specifically the cleavage of caspase-1) also leads to the activation of proinflammatory factors such as IL-18 and IL-1β. Although IL-1β did not seem to impact regeneration after injury, IL-18 limited repair by stimulating NK cells and their cytotoxic program, which targets thymic epithelial cells, which are crucial for supporting T cell development. Notably, this proinflammatory axis is not the only limitation to thymus repair, with aging-associated epithelial remodeling also contributing to blunted regenerative responses with age.

Together, these studies not only further defined the cellular and molecular regulators of thymic damage and repair after injury but also demonstrated the critical need for balancing proregenerative and suppressive signals in developing optimal therapies for thymus recovery and T cell reconstitution.

胸腺是T细胞发育的主要部位，对损伤极其敏感，但也具有显著的内源性修复能力。然而，即使胸腺持续退化和再生，以应对日常的伤害，如压力和感染，严重的胸腺损伤，如电离辐射，导致长期的T细胞淋巴减少。研究放射损伤后增强胸腺功能的方法之一是了解和利用内源性修复的潜在过程。我们已经确定，细胞死亡检测的平衡是触发胸腺内源性再生反应的关键。具体来说，免疫沉默细胞凋亡（在稳定状态下大量存在于胸腺细胞中）抑制再生程序，但相反，胸腺损伤后，向免疫原性细胞死亡（ICD）的转变可以促进再生。重要的是，许多这些途径可以靶向治疗，以改善放射损伤后的胸腺恢复。然而，ICD的诱导（特别是caspase-1的裂解）也会导致IL-18和IL-1β等促炎因子的激活。虽然IL-1β似乎不影响损伤后的再生，但IL-18通过刺激NK细胞及其细胞毒性程序限制修复，其目标是胸腺上皮细胞，这对支持T细胞的发育至关重要。值得注意的是，这种促炎轴并不是胸腺修复的唯一限制，衰老相关的上皮重塑也会导致再生反应随着年龄的增长而减弱。总之，这些研究不仅进一步定义了胸腺损伤和损伤后修复的细胞和分子调节因子，而且还表明，在开发胸腺恢复和T细胞重建的最佳疗法时，平衡促再生和抑制信号是至关重要的。

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引用次数: 0

1008 – SELF-RENEWAL ACTIVITY OF HEMATOPOIETIC STEM CELLS FROM BIRTH TO AGED 1008 -造血干细胞从出生到衰老的自我更新活性

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104888

Toshio Suda

We have recently shown that hematopoiesis can occur independently of hematopoietic stem cells (HSCs) during fetal development. We analyzed the formation of HSCs and progenitors from intra-arterial hematopoietic clusters, which contain HSC precursors and express the Hlf transcription factor, using in vivo genetic tracing. A kinetic study revealed the simultaneous formation of HSCs and defined progenitors, followed by prompt hierarchical hematopoietic structure formation in the fetal liver. The transcription factor Evi1^high cells are predominantly localized to intraembryonic arteries and preferentially give rise to HSCs. By genetically manipulating Evi1 expression, we can alter HSC and progenitor output from precursors in vivo.

On the other hand, the aging of HSCs leads to diminished stem cell function, with clonal bias potentially resulting in an inadequate supply of blood cells and the development of hematopoietic disorders. Mitochondria play an essential role in the metabolic processes of cells, and their dysfunction is a hallmark of aging. However, the relationship between mitochondrial mass and quality in old HSCs and their influence on HSC function remains incompletely elucidated. Here, we demonstrated that mitochondria-rich old HSCs, like young HSCs, are healthy and possess high stemness capacity, and the high mitochondrial mass does not necessarily damage mitochondrial accumulation. This insight enriches our comprehension of HSC aging and provides new perspectives on assessing old HSCs, highlighting the significance of mitochondrial dynamics in aging. Taken together, these data suggest that self-renewal of HSCs is separated from the generation of progenitors and functional blood cells.

我们最近的研究表明，在胎儿发育过程中，造血可以独立于造血干细胞（hsc）发生。我们使用体内遗传追踪分析了动脉内造血集群中HSC和祖细胞的形成，这些造血集群包含HSC前体并表达Hlf转录因子。一项动力学研究显示，造血干细胞和确定的祖细胞同时形成，随后在胎儿肝脏中迅速形成分层造血结构。转录因子Evi1high细胞主要定位于胚胎内动脉，并优先产生造血干细胞。通过基因操纵Evi1表达，我们可以改变体内造血干细胞和前体的祖细胞输出。另一方面，造血干细胞的老化导致干细胞功能减弱，克隆偏倚可能导致血细胞供应不足和造血功能障碍的发生。线粒体在细胞代谢过程中起着重要作用，其功能障碍是衰老的标志。然而，老龄HSC中线粒体质量和质量的关系及其对HSC功能的影响尚不完全清楚。在这里，我们证明了富含线粒体的老年hsc和年轻hsc一样，是健康的，具有高干性能力，高线粒体质量并不一定会损害线粒体积累。这一发现丰富了我们对HSC衰老的理解，并为评估老年HSC提供了新的视角，突出了线粒体动力学在衰老中的重要性。综上所述，这些数据表明造血干细胞的自我更新与祖细胞和功能性血细胞的产生是分离的。

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引用次数: 0

IFC Editorial Board IFC编委会

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/S0301-472X(25)00579-X

引用次数: 0

3017 – TRANSCRIPTION FACTOR PROGRAMMING BY TRANSIENT MRNA DELIVERY ENHANCES IPSC-DERIVED CAR T CELL DIFFERENTIATION, MATURATION AND CYTOTOXIC FUNCTION 3017 -转录因子编程通过瞬时mrna传递增强ipsc衍生的car - t细胞分化，成熟和细胞毒性功能

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104958

Brett Napiwocki, Lage von Dissen, Anthony Trombetta, Prateek Thenge, Jason Bell, Branden Moriarity, Beau Webber

Engineered T cells are a promising avenue to treat advanced cancers and autoimmune diseases; however, modification of autologous T cells is limited by disease status or prior therapies, necessitating alternative T-cell sources. Human-induced pluripotent stem cells (iPSCs) represent a scalable source of off-the-shelf T cells for therapy; however, current differentiation protocols rely on engineered murine feeder cells, limiting their translational utility. Here, we reported a novel 2D monolayer differentiation protocol incorporating transient expression of hematopoietic transcription factors ERG, HOXA5, HOXA9, HOXA10, and RUNX1 by mRNA delivery (TF-mRNA) that results in the production of iPSC-hematopoietic progenitor cells (iHPCs) with increased T-cell potential. In both fibroblast iPSC (F-iPSC) and T cell-derived iPSC (T-iPSC) lines, we showed TF-mRNA increases the frequency of CD34+CD45+ iHPCs compared with cells treated with GFP-mRNA. Additionally, removing hematopoietic cytokines (VEGF, SCF, IL-3, IL-6, TPO, and SR-1) from the iHPC differentiation yielded greater CD34+CD45+ expression in the TF-mRNA minimal-cytokine (MC) condition compared with the GFP-mRNA MC condition. Remarkably, the MC condition increased the frequency of CD34+CD117+ progenitors compared with the all-cytokine condition. In feeder-free T-cell differentiation culture, iHPC conversion into CD4+CD8+CD3+TCRαβ+ induced T (iT) cells was higher in the TF-mRNA condition compared with GFP-mRNA. Lastly, iT cells engineered with a CD19 chimeric antigen receptor (CAR) by targeted integration at TRAC or random integration with the TcBuster transposon system displayed effective killing against Raji target cells compared with nonengineered controls. These findings demonstrated the effectiveness of transient TF-mRNA delivery to enhance CAR iT cell production from engineered iPSCs, providing a new approach for the scalable production of CAR iT cells for therapeutic application.

工程T细胞是治疗晚期癌症和自身免疫性疾病的一种很有前途的途径；然而，自体T细胞的修饰受到疾病状态或先前治疗的限制，需要替代T细胞来源。人诱导多能干细胞（iPSCs）是一种可扩展的现成T细胞治疗来源；然而，目前的分化方案依赖于工程小鼠饲养细胞，限制了它们的转化效用。在这里，我们报道了一种新的二维单层分化方案，该方案通过mRNA传递（TF-mRNA）瞬时表达造血转录因子ERG、HOXA5、HOXA9、HOXA10和RUNX1，导致ipsc -造血祖细胞（iHPCs）的产生，并增加t细胞电位。在成纤维细胞iPSC （F-iPSC）和T细胞衍生iPSC （T-iPSC）系中，我们发现与GFP-mRNA处理的细胞相比，TF-mRNA增加了CD34+CD45+ iHPCs的频率。此外，从iHPC分化中去除造血细胞因子（VEGF， SCF, IL-3, IL-6， TPO和SR-1），在TF-mRNA最小细胞因子（MC）条件下，与GFP-mRNA MC条件相比，CD34+CD45+表达更高。值得注意的是，与全细胞因子条件相比，MC条件增加了CD34+CD117+祖细胞的频率。在无饲料的T细胞分化培养中，tgf - mrna条件下iHPC向CD4+CD8+CD3+TCRαβ+诱导的T （iT）细胞的转化高于GFP-mRNA。最后，用CD19嵌合抗原受体（CAR）工程化的iT细胞，通过靶向整合TRAC或随机整合TcBuster转座子系统，与非工程化对照相比，对Raji靶细胞显示出有效的杀伤效果。这些发现证明了瞬时TF-mRNA递送在增强工程iPSCs产生CAR - iT细胞方面的有效性，为大规模生产用于治疗的CAR - iT细胞提供了一种新方法。

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引用次数: 0

2024 – A STEP-WISE AND DETERMINISTIC LEUKEMIA MOUSE MODEL REMINISCENT OF JMML WITH ENDOGENOUS ACQUISITION OF PATIENT-RELEVANT RTK–RAS MUTATIONS 2024 -一个循序渐进和确定性的白血病小鼠模型，让人想起内源性获得患者相关rtk-ras突变的JMML

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104937

Marija Zarocsinceva , Moosa Qureshi , Nicola Wilson , George Giotopoulos , Fernando Calero-Nieto , Iwo Kucinski , Sarah Kinston , David Adams , Brian Huntly , Bertie Gottgens

Leukemia develops through a multistep process in which normal hematopoietic cells are progressively transformed into malignant cells, with relapse frequently driven by therapy-resistant premalignant clones. Although the molecular features of fully developed leukemia are well studied, the early clinically silent premalignant phase and its progression remain less understood. In this study, we established a model that captures the preleukemic stage by introducing a patient-derived CEBPA mutation into the Hoxb8-FL multipotent hematopoietic progenitor cells. Upon transplantation, these cells gave rise to overt leukemia after a prolonged latency of 12 months. Remarkably, all resulting tumors acquired secondary driver mutations in the RTK–RAS pathway genes—including Ptpn11, Kras, Nras, Cbl, or Flt3—with the exact amino acid changes observed in patients with cancer. Single-cell transcriptomic analyses revealed that CEBPA mutant cells retained both myeloid and lymphoid outputs during the preleukemic phase in vivo, and the leukemic transformation did not eliminate differentiated cell populations. This, combined with the strong preference for RTK–RAS pathway mutations, recapitulates key features of juvenile myelomonocytic leukemia (JMML), an aggressive pediatric disease for which robust cellular models have been challenging to develop. Additionally, using CRISPR-Cas9 technology, we generated an extensive transcription factor network map, identifying candidate regulators that may drive the aberrant preleukemic state in CEBPA mutant cells. Overall, we present a clinically relevant and experimentally accessible model of leukemogenesis, providing a valuable platform for studying disease mechanisms and facilitating preclinical drug discovery.

白血病的发展是一个多步骤的过程，在这个过程中，正常的造血细胞逐渐转化为恶性细胞，并且经常由耐药的癌前克隆驱动复发。虽然完全发展的白血病的分子特征已经得到了很好的研究，但早期临床沉默的癌前期及其进展仍然知之甚少。在这项研究中，我们通过将患者来源的CEBPA突变引入Hoxb8-FL多能造血祖细胞，建立了一个捕获白血病前期的模型。移植后，这些细胞在延长12个月的潜伏期后产生明显的白血病。值得注意的是，所有由此产生的肿瘤都获得了RTK-RAS通路基因的继发性驱动突变，包括Ptpn11、Kras、Nras、Cbl或flt3，并且在癌症患者中观察到确切的氨基酸变化。单细胞转录组学分析显示，CEBPA突变细胞在体内白血病前期保留髓样和淋巴样输出，白血病转化不会消除分化的细胞群。这与RTK-RAS通路突变的强烈偏好相结合，概括了青少年髓细胞白血病（JMML）的关键特征，JMML是一种侵袭性儿科疾病，其强大的细胞模型一直具有挑战性。此外，利用CRISPR-Cas9技术，我们生成了广泛的转录因子网络图谱，确定了可能驱动CEBPA突变细胞异常白血病前期状态的候选调节因子。总的来说，我们提出了一个临床相关和实验可获得的白血病发生模型，为研究疾病机制和促进临床前药物发现提供了一个有价值的平台。

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引用次数: 0

2003 – MOLECULAR DISSECTION OF AGING CLARIFIES THE IMPACT OF MDS GENETICS AND THERAPY 2003 -衰老的分子解剖阐明了MDS基因和治疗的影响

IF 2.1 4区医学 Q2 HEMATOLOGY

Experimental hematology

Pub Date : 2025-11-01 DOI: 10.1016/j.exphem.2025.104916

H. Leighton Grimes , Xuan Zhang , Mutian Tang , Guangyuan Li , David Bernardicius , Jennifer Van Oudenhove , Amy DeZern , Gabriel Ghiaur , Stephanie Halene , Nathan Salomonis

Among molecular and cellular hallmarks of aging, changes in genetics, the epigenome, and inflammation are now recognized as key mediators of decreased regenerative potential. Such molecular changes compound the impact of myelodysplastic syndrome (MDS) mutations (e.g., SRSF2, RUNX1) and underlie the selection of mutant clones. To understand the mechanistic basis of MDS, leukemic transformation, and therapy resistance, we applied a multimodal single-cell profiling strategy to serial bone biopsies for patients with MDS (cellular indexing of transcriptomes and epitopes by sequencing [CITE-Seq], long-read isoform, and genotyping). The analysis spanned young and aged normal donors to complement MDS at diagnosis, hypomethylating agent (HMA) treatment, and secondary acute myeloid leukemia (sAML). Isoform, splicing, and clonal impacts were determined using a new bioinformatic toolkit, AltAnalyze-LR. In normal donor material, we observed significant age-related differences in specific populations, including the most primitive hematopoietic stem cells (HSCs), where we found significant deregulation of inflammation and ubiquitin pathway gene expression. These changes were accompanied by broad splicing alterations, impacting known regulators of cell survival, clonal hematopoiesis, and inflammation. Comparison of normal aged marrow versus diagnostic biopsies for patients with MDS defined a highly coordinated gene program in MDS hematopoietic stem/progenitor cell (HSPC), largely associated with downregulation of inflammatory, glucocorticoid, and cytokine signaling and splicing deregulation. We find that HMA therapy drives HSC output toward megakaryocyte-erythroid progenitor (MEP) and megakaryocyte progenitor (MkP) regardless of clinical response and induces treatment and genotype-specific changes in splicing (including bona fide SRSF2-P95 targets). The latter was confirmed in both a mouse model of MDS (Srsf2-P95 Runx1−/−) and an SRSF2-P95 human cell line. The role of alternative isoform regulation in aging, MDS progression, and therapy has been largely underestimated, resulting in profound differences in the cell state impacts of MDS oncoproteins.

在衰老的分子和细胞特征中，遗传学、表观基因组和炎症的变化现在被认为是再生潜力下降的关键介质。这种分子变化复合了骨髓增生异常综合征（MDS）突变（如SRSF2、RUNX1）的影响，并成为突变克隆选择的基础。为了了解MDS、白血病转化和治疗耐药的机制基础，我们应用多模式单细胞谱分析策略对MDS患者进行了系列骨活检（通过测序[CITE-Seq]、长读异构体和基因分型对转录组和表位进行细胞索引）。该分析涵盖了年轻和老年正常供体，在诊断时补充MDS，低甲基化剂（HMA）治疗和继发性急性髓系白血病（sAML）。使用新的生物信息学工具altanalysis - lr来确定异构体、剪接和克隆影响。在正常供体材料中，我们观察到特定人群中显著的年龄相关差异，包括最原始的造血干细胞（hsc），我们发现炎症和泛素通路基因表达显著失调。这些变化伴随着广泛的剪接改变，影响已知的细胞存活、克隆造血和炎症的调节因子。MDS患者的正常衰老骨髓与诊断活检的比较确定了MDS造血干细胞/祖细胞（HSPC）中高度协调的基因程序，主要与炎症、糖皮质激素和细胞因子信号的下调以及剪接的失调有关。我们发现HMA治疗驱动HSC向巨核细胞-红细胞祖细胞（MEP）和巨核细胞祖细胞（MkP）输出，而不考虑临床反应，并诱导剪接的治疗和基因型特异性变化（包括真正的SRSF2-P95靶点）。后者在MDS小鼠模型（Srsf2-P95 Runx1−/−）和Srsf2-P95人细胞系中得到证实。替代异构体调控在衰老、MDS进展和治疗中的作用在很大程度上被低估了，导致MDS癌蛋白对细胞状态影响的深刻差异。

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