造血干细胞体外培养模式的研究进展

造血干细胞(hematopoietic stem cells,HSC)的体外培养可为造血干细胞移植提供大量的造血干细胞,具有重要的临床意义。HSC体外培养方式从二维培养发展到三维培养,从静态培养发展到动态培养,其培养技术及效果日趋成熟,其中以生物反应器为主的动态三维培养具有明显的优势。本文就HSC体外培养的研究进展作一综述。     

脐血来源造血干细胞体外培养扩增技术研究进展

随着造血干细胞(HSC)移植技术的发展,脐血已成为干细胞的主要来源之一。为突破HSC来源及数量不足的制约,HSC体外扩增技术发展迅速,涉及到细胞因子、小分子化合物的应用以及联合培养及三维培养技术的发展和氧环境在干细胞扩增技术的作用以及基因修饰技术。本文就近年上述技术中采用的主要方法进行综述。     

基于单细胞转录组分析的造血微环境研究进展

成体造血干细胞(hematopoietic stem cell,HSC)是一类主要定居于骨髓,具有自我更新和多向分化潜能的成体干细胞。HSC能够源源不断地产生各谱系成熟血细胞以维持造血系统的动态平衡。HSC功能特性的维持不仅依赖细胞本身的内在调控,还受到其所处的造血微环境或造血龛(hematopoietic microenvironment or hematopoietic niche)中多种成分的影响,因此研究造血微环境对揭示稳态造血调控、HSC体外扩增以及血液系统疾病的发生和发展等具有重要意义。以往研究利用体内成像技术和细胞特异的基因敲除技术,发现了造血微环境中多种细胞、细胞因子以及细胞外基质等成分。但作为一个复杂的三维系统,造血微环境的细胞组成、空间区域划分以及在正常造血维持及应激条件下的作用目前仍有争议。近年来,单细胞组学技术的迅速发展为阐明造血微环境的细胞和分子的异质性提供了新方法,为造血微环境的研究翻开了新的篇章。该文就单细胞转录组测序技术在骨髓微环境研究领域的最新进展进行综述。     

造血干细胞衰老体外模型构建及其生物学研究初探

造血干细胞（HSC）衰老与机体衰老密切相关。HSC衰老研究中的关键问题是HSC衰老模型的构建,迄今还未有公认的HSC衰老的体外模型,建立HSC衰老体外模型可为深入研究HSC衰老的生物学机理及调控机制奠定基础。本实验运用免疫磁珠分选法分离纯化小鼠Soft-1^＋ HSC,流式细胞术鉴定分选细胞的纯度达85％,免疫荧光示大量带绿色荧光Sca-1^＋细胞,     

综述: 造血干细胞衰老的研究进展

成体干细胞衰老是组织器官老化的重要原因之一.越来越多的证据显示,免疫系统的衰老起始于造血干细胞(HSC)功能的下降,即造血干细胞的衰老直接影响免疫系统的功能.然而,有关HSC衰老的机理和分子机制仍旧不清楚.在这篇综述中,我们总结了造血干细胞衰老的表型,同时从细胞内在及外在两个方面探讨论了HSC衰老的分子机制.     

造血干细胞衰老的研究进展

成体干细胞衰老是组织器官老化的重要原因之一.越来越多的证据显示,免疫系统的衰老起始于造血干细胞(HSC)功能的下降,即造血干细胞的衰老直接影响免疫系统的功能.然而,有关HSC衰老的机理和分子机制仍旧不清楚.在这篇综述中,我们总结了造血干细胞衰老的表型,同时从细胞内在及外在两个方面探讨论了HSC衰老的分子机制.     

混合培养体系中间充质干细胞与造血干细胞生物学特性

为了体外同时获得符合鉴定标准的造血干/祖细胞(hematopoietic stem/progenitor cell,HSC/HPC)和间充质干细胞(mesenchymal stem cell,MSC),本研究采用Ficoll淋巴细胞分离液方法从脐带血中分离出脐带血单个核细胞,MACS免疫磁珠法分离出CD34~+的造血干细胞。分离出的细胞与MSC同时接种在培养瓶中,利用15%AB型脐血浆的IMDM培养基添加白细胞介素3(interleukin-3,IL-3)、IL-6、血小板生成素(thrombopoietin,TPO)、干细胞因子(stem cell factor,SCF)和Flt-3配体(FMSlike tyrosine kinase 3 ligand,Flt-3L)因子培养体系来同时培养扩增HSC/HPC和MSC。为了评估扩增出的HSC/HPC和MSC是否符合细胞鉴定标准,本研究通过倒置显微镜观察HSC/HPC和MSC生长状态,并计数细胞。流式细胞仪检测HSC/HPC表面抗原CD34阳性百分率,检测第4代(P4)MSC表面抗原CD105、CD90、CD73、CD45、CD34和HLA-DR阳性表达率。半固体集落培养检测HSC/HPC的粒细胞-巨噬细胞集落形成能力。将共培养至P4的MSC行成骨、成软骨、成脂肪诱导鉴定。通过秋水仙素法进行MSC核型分析。结果显示,体外共培养的HSC/HPC依然有高增殖能力、集落形成能力和CD34阳性百分率。MSC具有向成骨细胞、脂肪细胞和软骨细胞诱导分化的能力。MSC染色体核型维持稳定。以上结果提示本研究成功建立了一种合适的MSC和HSC/HPC混合培养体系,通过该体系可同时获得两种干细胞,共培养后的MSC依然有典型的MSC的生物学功能;共培养后的HSC/HPC的粒细胞-巨噬细胞集落培养、细胞数量和流式表型符合鉴定标准,HSC/HPC生物学功能没有受到影响。     

造血干细胞生物学——研究与展望

造血干细胞（hematopoietic stem cell,HSC）是目前研究方法最为多样、研究技术手段最为成熟的一类组织干细胞,并且已经被成功运用于临床上对白血病以及先天性免疫缺陷等疾病的治疗。近年来,通过对一系列“转基因”与“基因敲除”小鼠模型的分析,人们对造血干细胞在胚胎早期发育过程中的发生与起源、造血干细胞“自我更新”与“定向分化”的调节机制、骨髓中造血干细胞的微环境（niche）对造血干细胞功能维持的调控,以及造血干细胞与白血病干细胞之间的相互关系等诸多方面都取得了很大的进展。如何实现造血干细胞的体外长期培养与扩增,实现胚胎干细胞（embryonic stem cell,ESC）或诱导多能干细胞（induced pluripotent stem cell,iPS细胞）向造血干细胞进行有效的定向分化,以及探索造血干细胞在病理状态（如癌症、贫血、衰老等）或应激状态下（如炎症与感染、组织损伤、代谢异常等）的功能变化,都将会是今后造血干细胞研究的重要方向。     

造血干细胞相关microRNAs的筛选及其促分化研究

为了获得人脐血造血干细胞(HSCs)的microRNAs(miRNAs)表达谱,并对相关miRNAs功能进行初步鉴定.利用免疫磁珠(MACS)和流式细胞仪(FACS)细胞分选技术分离人脐血造血干细胞(HSCs),分别提取细胞总RNA并分离小分子RNA,经荧光标记后与miRNAs基因芯片杂交,获得HSCs的miRNAs表达谱,集落形成实验(CFC)研究在HSC中高表达miR-520h对HSC的促分化作用.成功分离人脐血CD34 细胞和HSC,经基因芯片杂交获得31个造血干细胞相关miRNAs,其中22个为低表达,9个为高表达;经实时定量RT-PCR验证miR-520h显著升高,CFC实验表明其可增加多种集落形成,具有促进HSC向祖细胞分化的作用.上述结果表明,人脐血HSC具有自身特征性miRNAs,参与并调控HSC生物学功能,为深入探讨miRNAs在造血系统发育中的作用打下基础.     

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。     

Proliferation of human hematopoietic bone marrow cells in simulated microgravity

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.     

胚胎干细胞体外分化为造血干细胞

造血干细胞移植已成为治疗白血病、再生障碍性贫血、重症免疫缺陷征、地中海贫血、急性放射病、某些恶性实体瘤和淋巴瘤等造血及免疫系统功能障碍性疾病的成熟技术和重要手段,另外这一技术还被尝试用于治疗艾滋病,已取得积极的效果。但是由于移植需要配型相同的供体,并且过程复杂,使得造血干细胞移植因缺少配型相同的供体来源以及费用昂贵而不能被广泛应用。胚胎干细胞是一种能够在体外保持未分化状态并且能进行无限增殖的细胞,在适合条件下能够分化为体内各种类型的细胞,研究胚胎干细胞分化为造血干细胞,不仅可作为研究动物的早期造血发生的模型,而且可以增加造血干细胞的来源,还可以通过基因剔除、治疗性克隆等方法来解决移植排斥的问题,从而为造血干细胞移植的发展扫除了障碍,因此有着重要的研究价值和应用前景。现对胚胎干细胞体外分化为造血干细胞的诱导方法,诱导过程中的调控机制,并对胚胎干细胞分化为造血干细胞的存在问题和发展前景进行讨论。     

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.     

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.     

具有维持造血干/祖细胞能力的新型豆类凝集素的分离、纯化及功能鉴定

一种分离自豆类的新型凝集素不仅具有凝集活性,还具有体外长期维持造血干/祖细胞的能力.由眉豆（Dolichos lablab）中分离得到了这种多亚基的凝集素——FRIL（Flt3 receptor-interacting lectin）,并对它进行了核酸和蛋白质序列分析.免疫细胞分析显示,它的受体是CD34⁺造血干/祖细胞所特有的.在培养基中添加这种凝集素可长期维持CD34⁺细胞存活和增殖能力.以Flt3配基（FL）作为对照,在28天的培养时间内,相对于FL,FRIL可维持细胞较高的G0/G1期比例（80%以上G0/G1期）和长期培养中（14天以上）1.5倍以上的集落形成量.可见FRIL通过滞留造血干/祖细胞于G0/G1期而维持它们的自我更新潜能.     

Delivering cellular therapies: lessons learned from ex vivo culture and clinical applications of hematopoietic cells

Advances in stem cell biology and cellular therapy have led to promising treatments in a range of incurable diseases. However, it is unclear whether primitive stem cells can be delivered to damage tissue for regeneration of functional mature cells or stem cells must be stimulated to differentiate into mature cells in vitro and these cells delivered to patients. A range of other questions remains to be determined including how to formulate cellular products for in vivo delivery and how to undertake pharmacological testing of cellular products. Insights into these questions can be obtained from hematopoietic stem cells (HSC) which have been used for the past 50 years in bone marrow transplantation for regeneration of blood cells in patients undergoing high dose chemotherapy to treat cancer. The differentiation of HSC into mature blood cells is controlled by proteins called hematopoietic growth factors and these factors have been used to generate cellular products in vitro for clinical applications. This chapter will review some of the results of cellular therapies performed with HSC and the lessons that can be learned from these studies.