体外分化胚胎干细胞为造血干细胞重建造血功能的研究

目的：探讨体外定向分化胚胎干细胞（ESCs）为造血干细胞（HSCs）对体内造血功能的重建作用。方法：将小鼠E14.1胚胎干细胞采用“三步诱导法”在体外分化发育为HSCs，造血克隆形成(CFU)实验观察其体外造血集落形成情况，免疫磁珠分选纯化HSCs移植给经亚致死剂量γ射线照射的雌性SCID小鼠，观察其植入及小鼠造血功能恢复情况。结果: 经过分阶段诱导，多种造血刺激因子联合应用能有效促进ESCs定向分化发育为HSCs,流式细胞仪检测HSCs特异性表面标志物CD34+/Sca-1+表达最高为（58.64±4.20）%，CFU培养能形成较多的红系、粒系/巨噬细胞系及混合细胞集落, Wright-Giemsa 染色显示为原始的造血细胞。此阶段的HSCs经分选纯化后移植给经γ射线照射后的小鼠，移植组小鼠+10 d造血功能开始恢复，观察40 d后除血小板恢复较慢外，白细胞、红细胞、血红蛋白等指标已接近正常，植入率为71.4%，存活率为43.0%，染色体检测证实已由受体鼠的XX转为供体鼠的XY。结论: 采用分阶段诱导的方法，可在体外定向诱导小鼠ESCs分化发育为HSCs，此来源的HSCs可以有效重建体内造血功能。     

电离辐射诱导造血干细胞损伤的分子机制研究进展

电离辐射(IR)诱导的造血干细胞(HSCs)损伤是IR后引发机体死亡的主要原因,其损伤机制主要涉及P53-Puma通路诱导HSCs凋亡;激活G-CSF/Stat3/BATF依赖性的分化检验点促进HSCs分化;ROS-P38通路诱导HSCs衰老以及损伤HSCs龛。近期研究为预防和治疗电离辐射引起的骨髓抑制提供了理论基础。     

造血细胞的发生和发育

胚胎期造血细胞发生分为原始造血和永久造血两部分 ,原始造血起源于卵黄囊已被普遍认可 ,但永久造血起源于何处仍存在争议 ,目前认为至少有两个部位与永久造血有关 ,即卵黄囊和胚内的PAS/AGM区。胚胎干细胞是来源于胚胎发育早期的全能干细胞 ,在诱导分化为造血细胞的过程中 ,一系列过程类似于在体胚胎发育 ,可作为研究造血发育调控的模型     

大鼠骨髓间充质干细胞体外诱导肝星状细胞系凋亡

目的 研究大鼠骨髓间充质干细胞（MSCs）体外诱导大鼠肝星状细胞（HSCs）凋亡及其机制。方法 分离培养MSCs, HSC-T6系及纤维原细胞系冻融后传代使用。用6孔塑料培养板,建立上下双层细胞共培养体系。分3组：⑴空白对照组⑵阴性对照组⑶实验组。以上体系培养观察24、48和72h,于倒置相差显微镜下动态观察HSCs细胞形态;免疫组化法检测HSCs a-SMA表达;WST-8法检测HSCs增殖抑制率;流式细胞仪Annexin-V-FITC/PI双染法和DNA凝胶电泳（DNA Ladder ）检测HSCs细胞凋亡;RT-PCR 、Western blot检测HSCs Caspase-3、Bax基因mRNA和蛋白表达。结果 实验组出现明显的DNA Ladder,24h后HSCs 增殖抑制率、凋亡率和Caspase-3, Bax mRNA和蛋白表达呈时间依赖性,显著高于对照组。（P<0.01）结论 MSCs可在体外抑制HSCs增殖,可能通过旁分泌途径诱导HSCs凋亡,其凋亡发生是通过上调Caspase-3、Bax表达发挥作用,本研究支持MSCs通过抑制HSCs产生抗肝纤维化机制。     

造血干细胞与β-地贫的基因治疗

造血干细胞 (ｈｅｍａｔｏｐｏｉｅｔｉｃｓｔｅｍｃｅｌｌｓ ,ＨＳＣｓ)是外周血中所有细胞成分的唯一来源。ＨＳＣｓ在体内的含量稀少 ,约占骨髓有核细胞总数的 0 .5 % ;主要存在于造血组织中 ,也有少量循环于外周血[1] 。实验证明 ,哺乳动物每天所需的大量成熟血细胞就是由其体内少量的ＨＳＣｓ产生的。ＨＳＣｓ是一种多能造血细胞 ,能分化出红系细胞、血小板、粒系细胞、单核血细胞和淋巴系细胞。ＨＳＣｓ可自我更新而不发生分化 ,并通过产生各系祖细胞来形成各种成熟的血细胞[2 -5] 。ＨＳＣｓ的特性可应用于移植方面 ,即把少量ＨＳＣ…     

胚胎期造血干细胞的发生及其调控研究进展

造血干细胞(HSCs)是一种能分化为成熟血细胞的组织特异性干细胞,并且在生命体内终生都存在。而脊椎动物胚胎的主动脉造血干细胞集落在成体血液系统形成的过程中起着重要的作用。近年来人们深入地研究了胚胎期造血干细胞的发生、发展及转位归巢机制,发现其发育过程中受到转录因子Runx1及Notch等信号通路的调控,而microRNA则对造血干祖细胞的自我更新及定向分化进行转录后水平的调控。在循环建立后,造血干细胞的形成及其向红细胞的分化还受到血液动力学的调控和影响。了解造血干细胞的发生机制及调控因素对我们在体外人工诱导生成造血干细胞并应用于临床血液疾病治疗具有重要的意义。本文将对胚胎造血干细胞发生及其调控的最新研究进展进行综述。     

细胞免疫表型对确定骨髓增生异常综合征髓系病态的作用

目的 探讨细胞免疫表型分析在确定骨髓增生异常综合征（MDS）患者是否存在髓系细胞异常中的价值.方法 以具有外周血一系以上血细胞减少、且骨髓原始细胞数＜5％的患者为研究对象,以免疫性血小板减少患者为对照,回顾性分析流式细胞术中患者骨髓髓系细胞的免疫表型,观察粒细胞群和单核细胞群SSC强度是否异常,是否存在抗原跨阶段表达、抗原跨系列表达和分化抗原表达异常,并与细胞形态学粒系病态造血的结果进行比较.结果 在符合研究条件的255例患者中,有127例确定诊断为低危MDS、95例为非MDS,另有33例为意义未明血细胞减少（ICUS）.在MDS患者中,髓系免疫表型异常的检出率（85.8％）显著高于形态学病态造血检出率（57.5％）（P=0.000）;在非MDS患者中,具有髓系免疫表型异常的比例（1.1％）显著低于形态学病态造血比例（4.2％） （P =0.042）;ICUS患者虽然形态学无病态造血,但48.5％的患者具有髓系免疫表型异常.结论 细胞免疫表型分析对髓系异常的确定,有助于MDS的早期诊断和鉴别诊断.     

胎肝条件培养液体外诱导人骨髓间充质干细胞定向分化为造血细胞的研究

目的：探讨人骨髓间充质干细胞（hMSCs）体外造血分化潜能。方法： 选用孕12.5-14.5 d（12.5-14.5 dpc）的昆明小鼠，分别制备小鼠胎肝基质细胞条件培养液（FLSC-CM）及胚胎成纤维细胞饲养层（FD），将体外扩增的CD34-CD45-hMSCs分别接种于含FLSC-CM、FD和IL-6及SCF组合的培养体系中，培养7 d后，通过形态学、表型、粒-单/巨噬细胞系集落培养（CFU-GM）对分化细胞进行鉴定。结果： hMSCs与FLSC-CM共培养组产生的非贴壁细胞明显增多，形态类似于单核或小淋巴细胞，部分细胞可表达人造血细胞特异性表面分子（CD34和CD45），在含人粒-单集落刺激因子（GM-CSF）的甲基纤维素培养体系中能够形成CFU-GM，而FD和IL-6+SCF诱导组无上述作用。结论： FLSC-CM可诱导CD34-CD45-hMSCs分化为造血细胞，提示hMSCs具有体外造血分化潜能。     

人AGM区及胎肝基质细胞促进小鼠胚胎干细胞向造血干细胞分化和体内造血重建

目的: 探讨小鼠胚胎干细胞(ESCs)经人主动脉-性腺-中肾(AGM)区及胎肝(FL)基质细胞程序诱导后,向造血干细胞(HSCs)分化的效率及其造血功能。方法: 将E14 ESCs诱导为拟胚体(EB),并在人AGM区及FL基质细胞饲养层上进一步诱导分化,培养6 d后收集细胞检测Sca-1⁺c-Kit⁺细胞含量、分析造血细胞集落形成能力及致瘤性。再将不同诱导阶段的EB来源细胞移植经致死量 γ射线辐照的BALB/c雌鼠,观察生存率、植入状况和造血重建。结果: (1)EB来源细胞经人AGM区及FL基质细胞程序诱导后Sca-1⁺c-Kit⁺细胞含量为(21.96±2.54)%,造血集落总数为(520±52)/10⁵cells,明显优于诱导前及人AGM区基质细胞初步诱导者(P＜0.05)。(2)NOD-SCID小鼠接种经人AGM区及FL基质细胞诱导的ESCs未见畸胎瘤。(3)BALB/c雌鼠移植经人AGM区及FL基质细胞诱导的EB来源细胞后生存率77.8%,14 d外周血细胞计数明显改善,存活受鼠均检测到供体来源sry基因,而移植人AGM区基质细胞诱导的EB细胞者15 d内全部死亡。结论: 人AGM区及FL基质细胞能促进小鼠ESCs定向分化为HSCs,有效重建体内造血功能。     

162白细胞介素11

白细胞介素１１（ＩＬ－１１）是在灵长类骨髓基质细胞中发现的一种新的细胞因子，分子量为２３ＫＤ，具有促进多谱系造血（包括缩短造血祖细胞的Ｇ０期，刺激巨核细胞、红细胞，粒细胞和巨噬细胞造血，抑制骨髓脂肪形成）；增强Ｔ细胞和单核细胞依赖的Ｂ细胞抗原特异应答；支持血液系统恶性肿瘤细胞的生长，刺激肝细胞表达急性相蛋白，诱导神经祖细胞的分化等生物学功能。对于骨髓移植后骨髓造血功能的重建以及放疗相关性血细胞缺乏     

Deletion of TLR2+ erythro-myeloid progenitors leads to embryonic lethality in mice

Early embryonic hematopoiesis in mammals is defined by three successive waves of hematopoietic progenitors which exhibit a distinct hematopoietic potential and provide continuous support for the development of the embryo and adult organism. Although the functional importance of each of these waves has been analyzed, their spatio-temporal overlap and the lack of wave-specific markers hinders the accurate separation and assessment of their functional roles during early embryogenesis. We have recently shown that TLR2, in combination with c-kit, represents the earliest signature of emerging precursors of the second hematopoietic wave, erythro-myeloid precursors (EMPs). Since the onset of Tlr2 expression distinguishes EMPs from primitive progenitors which coexist in the yolk sac from E7.5, we generated a novel transgenic “knock in” mouse model, Tlr2^Dtr , suitable for inducible targeted depletion of TLR2⁺ EMPs. In this model, the red fluorescent protein and diphtheria toxin receptor sequences are linked via a P2A sequence and inserted into the Tlr2 locus before its stop codon. We show that a timely controlled deletion of TLR2⁺ EMPs in Tlr2^Dtr embryos results in a marked decrease in both erythroid as well as myeloid lineages and, consequently, in embryonic lethality peaking before E13.5. These findings validate the importance of EMPs in embryonic development.     

Notch signaling and the emergence of hematopoietic stem cells

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.     

Hematopoietic stem cell fate is established by the Notch-Runx pathway

Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo.     

The HOXB4 homeoprotein differentially promotes ex vivo expansion of early human lymphoid progenitors

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.     

Commentary: the role of cell migration in the ontogeny of the lymphoid system

The foundations of experimental hematology were laid by histologists, and while their contributions were enormous, they were limited in their interpretation of very dynamic processes by the static nature of the methodology. The middle of the twentieth century saw the introduction of techniques for hematopoietic cell marking and development of in vitro and in vivo assays for primitive hematopoietic cells, allowing dynamic studies of hematopoiesis. Paralleling this was an understanding of cellular immunology with the discovery of the role of the thymus and the identification of T and B lymphocyte lineages. In the 1960s a series of ontogenetic studies in birds and subsequently in mice revealed that hematopoietic and lymphoid development involved migration streams of primitive cells that colonized developing primary lymphoid organs as well as spleen, marrow, and liver. The yolk sac was proposed as the ultimate origin of these lympho-hematopoietic precursors. Subsequent studies identified a region associated with the dorsal aorta as the primary site of "definitive" stem cells. These opposing views are currently achieving a compromise that recognizes that both sites contribute stem cells involved in seeding the developing tissues. The clear distinction between the local origin of the inducing microenvironment provided by the endoderm or by stroma derived from mesenchymal stem cells of mesodermal origin, and the immigrant origin of the hematopoietic stem cells and progenitors, raises intriguing questions in the current climate of stem cell plasticity, cell fusion, and discovery of stem cells in adult marrow with the capacity to generate hematopoiesis as well as other mesodermal, ectodermal, and endodermal lineages.     

B-1 B cell development in the fetus and adult

Models of hematopoiesis often depict lymphocyte production as a uniform process in which a homogenous population of hematopoietic stem cells (HSCs) generates progenitors from which all types of lymphocytes are derived. However, it is increasingly evident that these schemes are too simplistic and that the lymphoid potential of HSCs and precursors arising in the embryo, fetus, neonate, and adult is remarkably distinct. We review recent findings regarding the development of B lymphocytes, and the B-1 B cell lineage in particular, as a case in point. These studies show that B-1 and B-2 B cells involved in innate and adaptive immune responses, respectively, arise in staggered waves of development from distinct progenitors. We discuss the implications of this layered model of B cell development for understanding normal and dysregulated B lymphopoiesis.     

Isolation and characterization of hematopoietic stem cells in teleost fish

Despite 400 million years of evolutionary divergence, hematopoiesis is highly conserved between mammals and teleost fish. All types of mature blood cells including the erythroid, myeloid, and lymphoid lineages show a high degree of similarity to their mammalian counterparts at the morphological and molecular level. Hematopoietic stem cells (HSCs) are cells that are capable of self-renewal and differentiating into all hematopoietic lineages over the lifetime of an organism. The study of HSCs has been facilitated through bone marrow transplantation experiments developed in the mouse model. In the last decade, the zebrafish and clonal ginbuna carp (Carassius auratus langsdorfii) have emerged as new models for the study of HSCs. This review highlights the recent progress and future prospects of studying HSCs in teleost fish. Transplantation assays using these teleost models have demonstrated the presence of HSCs in the kidney, which is the major hematopoietic organ in teleost fish. Moreover, it is possible to purify HSCs from the kidney utilizing fluorescent dyes or transgenic animals. These teleost models will provide novel insights into the universal mechanisms of HSC maintenance, homeostasis, and differentiation among vertebrates.