Hematopoietic stem cells.

The adequate production of blood cells is maintained by a set of immature hematopoietic stem cells (HSC) located in the bone marrow after birth. HSC are able to reconstitute the hematopoietic system in disease-related bone marrow failure and bone marrow aplasia. Nowadays, HSC cells can be mobilized from the bone marrow into the peripheral blood using hematopoietic cytokines, allowing a convenient harvest of these cells for clinical transplantation. This review outlines the development of the hematopoietic system in the embryo and in adults and the characterization, enumeration, purification and ex vivo expansion of HSC for clinical use. Future directions include the genetic manipulation of HSC and the identification/expansion of bone marrow-derived stem cells capable of generating non-hematopoietic tissues.     

Hematopoietic stem cell processing and cryopreservation

Either bone marrow or peripheral blood may be harvested to provide hematopoietic stem cells (HSC) for autologous transplantation. Both, however, comprise heterogeneous cell populations. The HSC necessary for successful engraftment constitute a very small fraction of the cells harvested. After collection, the harvested cells usually undergo several processing steps to reduce the product volume, remove cells (such as mature blood cells or tumor cells), or to cryopreserve the cells for later reinfusion. Granulocytes and red blood cells, for example, survive cryopreservation poorly using freezing techniques designed for HSC. Therefore, bone marrows being cryopreserved must be depleted of mature blood cells to avoid toxicity from infusion of damaged mature blood cells. Mature blood cells may also impede the variety of tumor cell purging techniques currently being studied. These processings are designed to minimize the loss of HSC while achieving an appropriate HSC product for the individual patient. A number of apheresis devices and cell washers simplify the enrichment of HSC in the harvested cell products. In contrast, tumor cell purging techniques are not standardized between the various transplant centers. © 1992 Wiley-Liss, Inc.     

小鼠造血干细胞表型及其分离纯化的研究进展

无论在纯化标记方面还是生物功能方面,造血干细胞都是目前研究最深入的成体干细胞,具有很大的临床应用价值。但是由于造血干细胞以极低比例存在于小鼠骨髓中,这给其分离纯化及功能研究带来了一定的难度。利用细胞表型和功能染料,通过免疫磁珠和流式细胞术可分离纯化骨髓和外周血中的造血干细胞。40年来,已有很多表面免疫表型确定可以富集小鼠造血干细胞,并且不断有新的免疫表型被发现,CD34-c-Kit+Sca-1+lineage marker-(CD34-LSK)是最常用的小鼠造血干细胞表型。本文就小鼠造血干细胞表型及分离纯化的研究做一综述。     

The role of apoptosis in the regulation of hematopoietic stem cells: Overexpression of Bcl-2 increases both their number and repopulation potential

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2-transgenic mice have increased numbers of HSC in bone marrow (2.4x wild type), but fewer of these cells are in the S/G(2)/M phases of the cell cycle (0.6x wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.     

Bone marrow and peripheral blood stem cell harvesting.

A number of malignant diseases are responsive to supralethal myelotoxic high-dose chemoradiotherapy, and can be treated with hematopoietic stem cell rescue. The number of genetic diseases correctable by replacement of defective pluripotent stem cells with normal stem cells or through gene transfer is ever-increasing. In all these cases, pluripotent hematopoietic stem cells need to be infused, which can be obtained directly from the bone marrow or from the peripheral blood with or without the aid of mobilizing strategies. This article is a detailed review of the technical and medical aspects of stem cell collection from the bone marrow and the peripheral blood.     

Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro

In some rodent models, there is evidence that hematopoietic stem cells (HSC) can differentiate into neural cells. However, it is not known whether humans share this potential, and, if so, what conditions are sufficient for this transdifferentiation to occur. We addressed this question by assessing the ability of fetal human liver CD34(+)/CD133(+)/CD3(-) hematopoietic stem cells to generate neural cells and astrocytes in culture. We cultured fetal liver-derived hematopoietic stem cells in human astrocyte culture-conditioned medium or using a method wherein growing human astrocytes were separated from cultured, nonadherent hematopoietic stem cells by a semipermeable membrane in a double-chamber co-culture system. Hematopoietic stem cell cultures were probed for neural progenitor cell marker expression (nestin and bone morphogenic protein-2 [BMP-2]) during growth in both culture conditions. RT-PCR, western blotting, and immunocytochemistry assays showed that cells cultured in either condition expressed nestin mRNA and protein and BMP-2 mRNA. HSC similarly cultured in nonconditioned medium or in the absence of astrocytes did not express either marker. Cells expressing these neural markers were transferred and cultured on poly-D-lysine-coated dishes with nonconditioned growth medium for further study. Immunocytochemistry demonstrated that these cells differentiated into astrocytes after 8 days in culture as indicated by their morphology and expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and S100, as well as by their rate of proliferation, which was identical to that of freshly isolated fetal brain astrocytes. These findings demonstrate that neural precursor gene expression can be induced when human hematopoietic stem cells are exposed to a suitable microenvironment. Furthermore, the neural stem cells generated in this environment can then differentiate into astrocytes. Therefore, human hematopoietic stem cells may be an alternative resource for generation of neural stem cells for therapy of central nervous system defects resulting from disease or trauma.     

Hematopoietic stem cells: An overview

Considerable efforts have been made in recent years in understanding the mechanisms that govern hematopoietic stem cell (HSC) origin, development, differentiation, self-renewal, aging, trafficking, plasticity and transdifferentiation. Hematopoiesis occurs in sequential waves in distinct anatomical locations during development and these shifts in location are accompanied by changes in the functional status of the stem cells and reflect the changing needs of the developing organism. HSCs make a choice of either self-renewal or committing to differentiation. The balance between self-renewal and differentiation is considered to be critical to the maintenance of stem cell numbers. It is still under debate if HSC can rejuvenate infinitely or if they do not possess ‘‘true” self-renewal and undergo replicative senescence such as any other somatic cell. Gene therapy applications that target HSCs offer a great potential for the treatment of hematologic and immunologic diseases. However, the clinical success has been limited by many factors. This review is intended to summarize the recent advances made in the human HSC field, and will review the hematopoietic stem cell from definition through development to clinical applications.     

New agents for mobilizing peripheral blood stem cells

Transplantation with bone marrow (BM) hematopoietic stem cells (HSC) has been used for curative therapy of hematologic diseases and inborn errors of metabolism for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow and traffic to peripheral blood in response to external stimuli, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. Although a variety of agents can mobilize stem cells with different kinetics and efficiencies and these agents can be additive or synergistic when used in combination, currently G-CSF is the predominant stem cell mobilizer used clinically based upon potency, predictability and safety. Recent studies have demonstrated that the interaction between the chemokine stromal-derived factor 1 (SDF-1/CXCL12) and its receptor CXCR4 serves as a key regulator of HSC trafficking. AMD3100, a novel bicyclam CXCR4 antagonist, induces the rapid mobilization of HSC with both short- and long-term repopulation capacity. Mobilization with G-CSF and AMD3100 in clinical trials resulted in more patients achieving sufficient PBSC for transplantation than with G-CSF alone. Thus, chemokine axis-mobilization could allow rapid PBSC harvests with increased cell yields in difficult-to mobilize patients. Studies of autologous and allogeneic transplantation of AMD3100 mobilized grafts demonstrated prompt and stable engraftment.Enhanced homing properties of chemokine axis-mobilized PBSC suggest that these cells may have greater therapeutic utility in other areas including tissue repair and regeneration.     

按造血发育程序诱导小鼠胚胎干细胞为造血干细胞重建体内造血的实验研究

为研究小鼠胚胎干细胞(ESC)定向诱导分化为造血干细胞(HSC)在体内重建造血的功能,将小鼠E14ESC诱导为拟胚体(EB),EB细胞利用Transwell非接触共培养体系在人主动脉-性腺-中肾(AGM)区、胎肝(FL)及骨髓(BM)基质细胞饲养层上依次诱导,收获各阶段EB细胞,以流式细胞仪检测Sca-1+c-Kit+细胞含量,并接种于NOD-SCID小鼠以检测体内致瘤性。再将不同诱导阶段的EB来源细胞移植到经致死量60Coγ射线照射的BALB/c雌鼠,将受鼠随机分为5组:①AGM组,②AGM+FL组,③AGM+FL+BM组,④照射对照组,⑤正常对照组。观察各组生存率、造血重建和植入状况。结果显示:EB细胞经人AGM区和FL基质细胞共培养后Sca-1+c-Kit+细胞达到峰值(21.96±2.54)%;NOD-SCID小鼠在接种经人AGM区基质细胞诱导的EB细胞后可出现畸胎瘤,而接种经人AGM区+FL基质细胞诱导EB细胞后未见肿瘤形成;AGM组及照射对照组动物全部死亡,而AGM+FL组及AGM+FL+BM组生存率分别为77.8%、66.7%,移植后21天外周血象基本恢复,在存活受鼠检测到供体来源Sry基因。结论:按胚胎造血发育程序,体外经人AGM区、FL及BM基质细胞连续诱导小鼠ESC分化的HSC可安全、有效地重建体内造血。     

Gestational age-dependent changes in circulating hematopoietic stem cells in newborn infants

In the fetus, hematopoietic stem cells originate in the yolk sac and are believed to be transferred to all other hematopoietic organs via the circulation. In humans, the time course of this transfer has not been systematically evaluated in viable premature infants. We examined the cord blood of 13 preterm (25 to 36 weeks of gestation) and 10 term (38 to 42 weeks of gestation) infants for pluripotent (mixed colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte), erythroid (burst-forming unit-erythroid, colony-forming unit-erythroid) and myeloid (colony-forming unit-granulocyte, macrophage) stem cells. A gestational age-dependent decrease in all lineages of circulating hematopoietic stem cells was noted (p less than 0.001). By 34 weeks of gestation, preterm infant cord blood had a similar concentration of circulating stem cells compared with that of term infants. This gestational age-dependent decrease in hematopoietic stem cells of all lineages supports the hypothesis of a blood-borne transfer of hematopoiesis that appears largely complete by 34 weeks of gestation. Infants born after less than 32 weeks of gestation have high levels of circulating hematopoietic stem cells that may reflect the active transfer of hematopoiesis from liver to bone marrow.     

Hematopoietic stem cell

Hematopoietic stem cells (HSCs) maintain themselves over cell divisions (self-renewal) and produce all kinds of blood cells (multi-potency). Depletion of these cells eventually causes hematopoietic failure, while deregulated HSC division causes development of myeloproliferative disorders and leukemias. HSCs can be prospectively purified to nearly homogeneity in mice, but such a high-level purification has not been achieved in humans. HSCs are localized to an anatomical place called 'niche'. Specialized osteoblasts arrayed on the endosteum of cavernous bone and sinusoidal endothelial cells located at the distant position from the endosteum are the two representative candidates of such an HSC niche. A number of adhesion molecules and signaling molecules are thought to comprise the niche-HSC synapse. HSCs divide only once in 1-2 months. Both environmental signaling from the niche and HSC-autonomous molecular programs contribute to the quiescent state of HSCs, which is essential for the maintenance of HSC self-renewal capacity and homeostasis of blood production.     

RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation

Hematopoietic stem cells (HSCs) sustain lifelong production of all blood cell types through finely balanced divisions leading to self-renewal and differentiation. Although several genes influencing HSC self-renewal have been identified, to date no gene has been described that, when activated, enhances HSC self-renewal and, when inactivated [corrected] promotes HSC differentiation. We observe that the retinoic acid receptor (RAR)gamma is selectively expressed in primitive hematopoietic precursors and that the bone marrow of RARgamma knockout mice exhibit markedly reduced numbers of HSCs associated with increased numbers of more mature progenitor cells compared with wild-type mice. In contrast, RARalpha is widely expressed in hematopoietic cells, but RARalpha knockout mice do not exhibit any HSC or progenitor abnormalities. Primitive hematopoietic precursors overexpressing RARalpha differentiate predominantly to granulocytes in short-term culture, whereas those overexpressing RARgamma exhibit a much more undifferentiated phenotype. Furthermore, loss of RARgamma abrogated the potentiating effects of all-trans retinoic acid on the maintenance of HSCs in ex vivo culture. Finally, pharmacological activation of RARgamma ex vivo promotes HSC self-renewal, as demonstrated by serial transplant studies. We conclude that the RARs have distinct roles in hematopoiesis and that RARgamma is a critical physiological and pharmacological regulator of the balance between HSC self-renewal and differentiation.     

造血干细胞与骨髓微环境相互作用的研究进展

造血干细胞移植(HSCT)后,供者造血干细胞(HSC)的归巢和植入直接影响移植的效果.HSC位于骨髓微环境即HSC龛中,并与骨髓微环境相互作用调节其自我更新和多向分化,维持骨髓造血功能动态平衡.近期,研究者们采用不同的创新方法观察移植后骨髓中HSC与骨髓微环境的相互作用关系,发现骨髓微环境对HSC稳态起重要作用,同时HSC也对“龛”的完整性形成必不可少.现就HSC与骨髓微环境的相互作用作一简要综述.     

Proliferation and differentiation of highly enriched mouse hematopoietic stem cells and progenitor cells in response to defined growth factors

Three distinct hematopoietic populations derived from normal bone marrow were analyzed for their response to defined growth factors. The Thy-1loT- B- G- M-population, composing 0.2% of bone marrow, is 370-fold enriched for pluripotent hematopoietic stem cells. The two other populations, the Thy-1- T- B- G- M- and the predominantly mature Thy-1+ T+ B+ G+ M+ cells, lack stem cells. Thy-1loT- B- G- M- cells respond with a frequency of one in seven cells to IL-3 in an in vitro CFU-C assay, and give rise to many mixed colonies as expected from an early multipotent or pluripotent progenitor. The Thy-1- T- B- G- M- population also contains progenitor cells which responded to IL-3. However, colonies derived from Thy-1- T- B- G- M- cells are almost exclusively restricted to the macrophage/granulocyte lineages. This indicates that IL-3 can stimulate at least two distinct clonogenic early progenitor cells in normal bone marrow: multipotent Thy-1loT- B- G- M- cells and restricted Thy-1- T- B- G- M- cells. Thy-1loT- B- G- M-cells could not be stimulated by macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF) or IL-5 (Eosinophil-CSF). The hematopoietic precursors that react to these factors are enriched in the Thy-1- T- G- B- M- population. Thus, multipotent and restricted progenitors can be separated on the basis of the expression of the cell surface antigen Thy-1.     

Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region

Hematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Little is known about the embryonic development of human HSCs. We demonstrate that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the dorsal aorta, and only later appear in the yolk sac, liver, and placenta. AGM region cells transplanted into immunodeficient mice provide long-term high level multilineage hematopoietic repopulation. Human AGM region HSCs, although present in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates at least 300 daughter HSCs in primary recipients, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine.     

Perspectives on the morphology and biology of CD34-negative stem cells

The CD34 antigen is the classical indicator molecule of pluripotent hematopoietic stem cells. But there is more and more evidence that progenitors of a yet uncommitted stem cell population do not express this surrogate marker. The bone marrow and other sites of hematopoiesis consist also of fibroblast-like stromal cells, quiescent hematopoietic stem cells, and mesenchymal stem cells. Depending on their stage of differentiation, CD34- stem cells cannot only generate hematopoietic progenitors, but also more specified mesenchymal precursors, such as osteoblasts, chondrocytes, myocytes, adipocyts, and others. The stromal cell compartment produces not only matrix proteins, such as collagens, fibronectin and others, but also the essential growth factors, which initiate and support the differentiation of primary quiescent, but eventually activated CD34- stem cells into CD34+ hematopoietic progenitors. In vivo studies have shown that long-term hematopoietic and mesenchymal reconstitution can be achieved with CD34- stem cell lines, isolated from various sources, although the frequency of CD34- stem cells seams to be quite low among the progenitor population. Some authors deny the reconstitution ability of CD34- cells. The majority of CD34- stem cells are quiescent fibroblast-like cells, which can be identified in the bone marrow biopsy as "bone lining cells". Some of those bone lining cells show protein synthesis and contain secretory vesicles. Recent studies have demonstrated that there is a surprising plasticity of the earliest stem cell population, consisting of cells with stromal cell function as well as hematopoietic and mesenchymal progenitors. The new insights into the biology of totipotent stem cells give us novel perspectives for cell- and gene therapy of various malignant and nonmalignant diseases and the possibility to replace defective organ functions with autologous CD34- stem cells.     

以4种基因诱导产前诊断绒毛细胞建立诱导性多能干细胞

背景：诱导性多能干细胞因具有多能性特征,可以诱导分化为特定的细胞,包括神经细胞、造血细胞等。目的：建立产前诊断绒毛细胞来源的诱导性多能干细胞。方法：运用反转录病毒介导4种基因hOct4、hSox2、hc-Myc、hKlf4诱导产前诊断绒毛细胞,对建立的诱导性多能干细胞进行多能性、体内外分化能力、核型等鉴定。结果与结论：建立的诱导性多能干细胞能维持自我更新状态,在蛋白和mRNA水平上高表达全能性的标志基因,具有体内、外分化潜能;在体外长期培养能维持正常核型。说明4种全能性基因转入绒毛细胞可获得具有多能性的诱导性多能干细胞,这为胎儿的细胞自体移植治疗提供理想来源,为产前诊断疾病机制研究提供很好的细胞模型。     

岩藻糖基化修复脐血造血干细胞归巢缺陷研究的新进展

造血干细胞(HSC)植入延迟是脐血移植(UCBT)面临的主要问题,由于植入延迟导致患者移植后感染发生率高,移植相关死亡率增高,限制了UCBT的进行.脐血HSC存在归巢缺陷是造成植入延迟的原因之一.多项研究发现,岩藻糖基化处理能修复脐血HSC的归巢缺陷,并且其操作简便、快捷、安全、有效,对脐血HSC的自我更新与分化能力无损伤,是解决上述难题的可行方法.笔者拟就岩藻糖基化修复脐血造血干细胞归巢缺陷技术的相关研究与临床应用进展进行回顾与展望.     

Separation of pluripotent stem cells and early B lymphocyte precursors with antibody Fall-3

A major goal in the study of hematopoiesis is to obtain populations of primitive stem cells, free of restricted and mature cells. We previously showed that a small population of normal bone marrow, the Thy-1loLin- cells, was highly enriched for pluripotent stem cells that repopulate lethally irradiated mice. These cells also differentiated along the B lymphocyte lineage in response to the stromal elements in Whitlock-Witte cultures. These two hematopoietic activities were entirely contained in and were enriched to similar extents in the Thy-1loLin- population. Here we show for the first time that these two activities can be resolved functionally and phenotypically. The cells that respond to the stroma in lymphoid culture are more sensitive to the cytotoxic drug 5-Fluorouracil than are stem cells. Furthermore, we have derived a new monoclonal antibody, Fall-3, that detects primitive stem cells but does not label the B cell precursor. This indicates that the small Thy-1loLin- population is heterogeneous, containing precursors restricted to the B cell lineage as well as pluripotent stem cells. Antibody Fall-3 defines a novel stem cell antigen, expressed on all primitive stem cells and thus, will be useful in the further characterization and isolation of both stem cells and B cell precursors.