Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

Background

Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.

Results

By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.

Conclusion

The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.     

锂对造血干细胞和神经干细胞作用影响的研究进展

锂在现代精神病学中使用超过65年,其构成了双相情感障碍（BD）长期治疗的基础。锂的许多生物学特性已经被证实,包括抗病毒、血液系统和神经系统保护作用。本文系统综述了锂对造血干细胞（HSCs）、神经干细胞（NSCs）以及诱导多能干细胞（iPSCs）作用影响的研究进展及其目前已证实的分子机制。自20世纪70年代以来,锂对保持HSCs和生长因子高水平的作用已被报道。锂可以改善HSCs的归巢能力、形成菌落的能力和自我更新的能力。关于锂对神经发生影响的研究表明,锂可促进海马齿状回的干细胞增殖,并导致施旺氏细胞有丝分裂活性增强。锂被证实与神经保护和神经营养作用相关,具体作用反映在锂可改善突触的可塑性,促进细胞存活,抑制细胞凋亡等。在临床研究中发现,锂离子的治疗可增加大脑灰质的成分,尤其作用在额叶、海马和杏仁核等位置。锂对干细胞的作用涉及多条介质和信号通路,其中最重要的介质和信号通路被认为是糖原合成酶激酶-3（GSK-3）和Wnt/β-catenin通路,另外包括调节cAMP、蛋白激酶B、磷脂酰肌醇3-激酶（pi3k）和肌醇单磷酸酶（IMP）水平的信号通路等也与锂作用有紧密的联系。锂在现阶段被利用于治疗BD和降低痴呆症患病风险的临床实验中,并对神经退行性疾病发挥有益作用。除此之外,为了研究的发病机制和锂离子在其中的作用机制,从BD患者中获得的iPSCs也被广泛应用。     

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a “biomimetic niche.” The CD34⁺ cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2–5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34⁺ cells from umbilical cord blood in the 3D system increased 3.3–4.8 folds when compared with the initial CD34⁺ cells. CD34⁺/CD38⁻ cells accounted for 82–90% of CD34⁺ cells. After 5 weeks, CD34⁺/CD38⁻ cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10³ vs. 1.0 ± 0.5 × 10⁴, p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10³ vs. 2.5 ± 0.7 × 10², p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6–9.3 folds vs. 1.0–1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2–7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.     

From gut to glutes: The critical role of niche signals in the maintenance and renewal of adult stem cells

Stem cell behavior is tightly regulated by spatiotemporal signaling from the niche, which is a four-dimensional microenvironment that can instruct stem cells to remain quiescent, self-renew, proliferate, or differentiate. In this review, we discuss recent advances in understanding the signaling cues provided by the stem cell niche in two contrasting adult tissues, the rapidly cycling intestinal epithelium and the slowly renewing skeletal muscle. Drawing comparisons between these two systems, we discuss the effects of niche-derived growth factors and signaling molecules, metabolic cues, the extracellular matrix and biomechanical cues, and immune signals on stem cells. We also discuss the influence of the niche in defining stem cell identity and function in both normal and pathophysiologic states.     

Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition

The expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) with the support of microencapsulated osteoblasts under hypoxia environment was investigated. The expansion of HSPCs was evaluated through the total number of UCB mononuclear cells (MNCs) produced, their repopulating potential with the colony-forming unit assay (CFU-Cs) and CD34⁺ phenotypic analysis with flow cytometry. At the end of 7 days of culture, the UCB-MNCs, CFU-Cs and CD34⁺ cells had achieved 18.7 ± 1.6, 11.6 ± 0.9 and 23.4 ± 2-fold expansions, respectively, in the test groups. These were significantly different from those in control groups. Microencapsulated osteoblasts under hypoxia conditions had therefore a significant effect on the expansion potential of HSPCs in vitro.     

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as “nonhematopoietic” sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.     

Floating culture promotes the maintenance of hematopoietic stem cells

In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine.     

Environmental control of lineage plasticity and stem cell memory

Tissue-resident stem cells (SCs) are critical players in the maintenance of tissue homeostasis. SCs reside in complex and uniquely anatomically organized microenvironments (SC niches), that carefully control SC lineage outputs depending on localized tissue needs. Upon environmental perturbations and tissue stressors, SCs respond and restore the tissue to homeostasis, as well as protect it from secondary assaults. Critical to this function are two key processes, SC lineage plasticity and SC memory. In this review, we delineate the multifactorial determinants and key principles underlining these two remarkable SC behaviors. Understanding lineage plasticity and SC memory will be critical not only to design new regenerative therapies but also to determine how these processes are altered in a multitude of pathologies such as cancer and chronic tissue damage.     

亲缘供者外周血红细胞参数对造血干细胞采集影响的经验分析

目的探讨亲缘供者外周血红细胞参数对COBE Spectra血细胞分离机的自动外周血干细胞采集程序（AutoPBSC程序）与单个核细胞采集程序（MNC程序）的影响及经验分析。 方法选取河北燕达陆道培医院2019年6月至2021年2月小红细胞亲缘供者31例45次采集为小红细胞组,选取同期非小红细胞亲缘供者51例60次采集为非小红细胞组,分别应用AutoPBSC程序和MNC程序,比较两组采集情况及采集产品相关指标。采用独立样本t检验和Mann-Whitney U检验分析2组计量资料的差异。 结果与小红细胞AutoPBSC程序组比较,小红细胞MNC程序组血小板（PLT）降低率[（25.88±15.83）﹪比（36.64±10.22）﹪]、采集效率[32.65﹪（23.60﹪,73.82﹪）比63.74﹪ （59.83﹪,68.55﹪）]、采集物体积[（158.83±34.39）比（222.91±63.9）mL]、MNC总数[（218.04±117.57）×10⁸/L比（350.24±127.64）×10⁸/L]、CD34⁺细胞总数[113.83×10⁶/L （79.25×10⁶/L,154.10×10⁶/L）比233.26×10⁶/L （177.18×10⁶/L,392.51×10⁶/L）]、MNC计数[（4.04±2.61）×10⁸/kg比（5.54±2.22）×10⁸/ kg]、CD34⁺计数[1.84×10⁶/kg （1.16×10⁶/kg,4.41×10⁶/kg）比3.64×10⁶/kg （2.49×10⁶/kg,6.37×10⁶/kg）]均升高,差异有统计学意义（P < 0.05）;与非小红细胞AutoPBSC程序组比较,非小红细胞组MNC程序组采集物体积[（162.83±51.74）比（242.56±43.25）mL]升高,差异有统计学意义（P < 0.05）。 结论对造血干细胞移植供者红细胞体积偏小时,应用MNC程序采集外周血造血干细胞,比应用AutoPBSC程序更有优势。     

Communications between bone cells and hematopoietic stem cells

The skeletal system, while characterized by a hard tissue component, is in fact an extraordinarily dynamic system, with disparate functions ranging from structural support, movement and locomotion and soft-organ protection, to the maintenance of calcium homeostasis. Amongst these functions, it has long been known that mammalian bones house definitive hematopoiesis. In fact, several data demonstrate that the bone microenvironment provides essential regulatory cues to the hematopoietic system. In particular, interactions between the bone-forming cells, or osteoblasts, and the most primitive Hematopoietic Stem Cells (HSC) have recently been defined. This review will focus mainly on the role of osteoblasts as HSC regulatory cells, discussing the signaling mechanisms and molecules currently thought to be involved in their modulation of HSC behavior. We will then review additional cellular components of the HSC niche, including endothelial cells and osteoclasts. Finally, we will discuss the potential clinical implications of our emerging understanding of the complex HSC microenvironment.     

记忆性T细胞的形成、维持和功能

免疫记忆是指机体在对某一抗原产生特异性识别及应答的同时,记住该抗原,当再次遭遇同一抗原时,能发生快速和强烈的免疫应答。树突状细胞吞噬病原微生物后,通过主要组织相容性复合体分子提呈抗原短肽段,与T细胞相互作用。在T细胞抗原受体信号和共刺激信号的协同作用下,抗原特异性T细胞增殖,收缩,小部分细胞作为记忆细胞长期存活。免疫记忆T细胞在表型特征和功能上都存在多样性。深入研究机体记忆性T细胞的特征,不仅能指导新型疫苗的设计,而且可望帮助治疗疾病。     

Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy

Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin-treated mice, insulin- and TNF-α co-expressing bone marrow-derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high-fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co-expression of insulin and TNF-α in c-kit⁺Sca-1⁺lineage⁻ (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.     

白细胞介素4降低脐带间充质干细胞对造血干细胞分化潜能的维持能力

目的探讨白细胞介素4（IL-4）对脐带间充质干细胞（UC-MSC）维持造血干细胞分化的影响。 方法将UC-MSC与脐带血CD34⁺造血干细胞按照造血支持能力常用的方案共培养,实验分为对照组和IL-4组,IL-4处理组加入IL-4（20 ng/ml）培养14 d。收集细胞并计数,使用流式细胞仪检测表达CD34的细胞比例。取3×10³个细胞,加入到半固体培养基,培养14 d后,通过倒置显微镜观察比较各种集落的形成,并使用流式细胞仪分析其中巨噬细胞和粒细胞表面特异性蛋白CD11b、CD14和CD15的表达。对两独立样本进行t检验统计学分析。 结果加入IL-4后,共培养体系中细胞数量（1.31±0.05）×10⁵个/孔与对照组（2.80±0.28）×10^5?个/孔相比下降,差异有统计学意义（t = 7.31,P < 0.05）,并且流式细胞分析显示其中的CD34⁺细胞比例也有降低。3×10³个IL-4组得到的细胞形成巨噬细胞集落形成单位的能力（9.33±1.53）?个/孔较对照组（17.67±0.58）个/孔有明显下降,差异有统计学意义（t = 8.84,P?< 0.001）;形成粒-巨噬集落形成单位的能力（15.67±3.22）个/孔较对照组（29.33±4.04）?个/?孔有明显下降,差异有统计学意义（t = 4.58,P < 0.05）;形成总集落单位的能力（39.33±9.07）个/?孔较对照组（62.67±6.66）?个/?孔也有下降,差异有统计学意义（t = 3.59,P < 0.05）。IL-4组得到的细胞分化出的细胞总数（3.67±1.71）×10⁵个/孔与对照组（9.50± 3.13）×10⁵个/孔相比也明显下降,差异有统计学意义（t = 2.83,P < 0.05）,而流式细胞术分析发现分化成的细胞中CD14⁺细胞比例也下降。 结论IL-4可以降低UC-MSC对造血干细胞分化潜能的维持能力,提示在Ⅱ型辅助T细胞相关体液免疫疾病中使用间充质干细胞治疗时,也需要兼顾机体造血相关功能。     

Adult stem cells in neural repair: Current options,limitations and perspectives

Stem cells represent a promising step for the future of regenerative medicine. As they are able to differentiate into any cell type, tissue or organ, these cells are great candidates for treatments against the worst diseasesthat defy doctors and researchers around the world. Stem cells can be divided into three main groups:(1) embryonic stem cells;(2) fetal stem cells; and(3) adult stem cells. In terms of their capacity for proliferation, stem cells are also classified as totipotent, pluripotent or multipotent. Adult stem cells, also known as somatic cells, are found in various regions of the adult organism, such as bone marrow, skin, eyes, viscera and brain. They can differentiate into unipotent cells of the residing tissue, generally for the purpose of repair. These cells represent an excellent choice in regenerative medicine, every patient can be a donor of adult stem cells to provide a more customized and efficient therapy against various diseases, in other words, they allow the opportunity of autologous transplantation. But in order to start clinical trials and achieve great results, we need to understand how these cells interact with the host tissue, how they can manipulate or be manipulated by the microenvironment where they will be transplanted and for how long they can maintain their multipotent state to provide a full regeneration.     

IL-33转基因小鼠骨髓造血干／祖细胞向髓系细胞分化的能力增强

目的：利用IL-33转基因小鼠研究IL-33对造血干/祖细胞的增殖和分化影响。方法利用流式细胞仪分析IL-33转基因小鼠及同窝野生对照小鼠的外周血、脾脏、骨髓细胞的免疫表型及造血干细胞分化不同阶段细胞的数量变化;利用体外成克隆实验和细胞周期分析研究IL-33对于造血干细胞增殖能力的影响。结果与野生型小鼠相比,IL-33转基因小鼠B细胞和T细胞在外周血中都明显降低,粒细胞在外周血和骨髓中都有明显增加;IL-33转基因小鼠的骨髓造血干细胞和多能祖细胞数量减少,共同淋系祖细胞数量减少,共同髓系祖细胞和粒单系祖细胞数量增加;IL-33转基因小鼠的造血干细胞处于S-G2-M的细胞增多;体外单克隆实验发现IL-33转基因小鼠造血干细胞形成的集落数增加。结论 IL-33转基因小鼠造血干细胞增殖能力增强,更易向髓系细胞分化。