A critical role for Apc in hematopoietic stem and progenitor cell survival

The adenomatous polyposis coli (Apc) tumor suppressor is involved in the initiation and progression of colorectal cancer via regulation of the Wnt signaling cascade. In addition, Apc plays an important role in multiple cellular functions, including cell migration and adhesion, spindle assembly, and chromosome segregation. However, its role during adult hematopoiesis is unknown. We show that conditional inactivation of Apc in vivo dramatically increases apoptosis and enhances cell cycle entry of hematopoietic stem cells (HSCs)/ hematopoietic progenitor cells (HPCs), leading to their rapid disappearance and bone marrow failure. The defect in HSCs/HPCs caused by Apc ablation is cell autonomous. In addition, we found that loss of Apc leads to exhaustion of the myeloid progenitor pool (common myeloid progenitor, granulocyte-monocyte progenitor, and megakaryocyte-erythroid progenitor), as well as the lymphoid-primed multipotent progenitor pool. Down-regulation of the genes encoding Cdkn1a, Cdkn1b, and Mcl1 occurs after acute Apc excision in candidate HSC populations. Together, our data demonstrate that Apc is essential for HSC and HPC maintenance and survival.     

The stem cell niches in bone

The stem cell niche is composed of a specialized population of cells that plays an essential role in regulating adult stem cell self-renewal and differentiation. In adults, osteoblasts, responsible for osteogenesis, and hematopoietic cells, responsible for hematopoiesis, are closely associated in the bone marrow, suggesting a reciprocal relationship between the two. It was recently discovered that a subset of osteoblasts functions as a key component of the HSC niche (namely, the osteoblastic niche), controlling HSC numbers. HSCs interact not only with osteoblasts but also with other stromal cells, including endothelial cells. Sinusoidal endothelial cells in bone marrow have been revealed as an alternative HSC niche called the vascular niche. In this Review we compare the architecture of these 2 HSC niches in bone marrow. We also highlight the function of osteoblasts in maintaining a quiescent HSC microenvironment and the likely role of the vascular niche in regulating stem cell proliferation, differentiation, and mobilization. In addition, we focus on studies of animal models and in vitro assays that have provided direct insights into the actions of these osteoblastic and vascular niches, revealing central roles for numerous signaling and adhesion molecules. Many of the discoveries described herein may contribute to future clinical treatments for hematopoietic and bone-related disorders, including cancer.     

HOXB4基因在造血干细胞自我更新中的调控作用

造血干细胞（hematopoietic stem cell,HSC）具有高度自我更新能力和多向分化潜能,HSC的自我更新是由促进生长的正调控信号和导致凋亡的负调控信号之间的平衡来调控的。在正调控信号中,HOXB4通过激活不同的信号通路增强HSC的自我更新,同时又不影响维持正常稳态造血的调控机制。提高HOXB4的表达水平,能够极大地增强HSC的自我更新功能,但基本不影响细胞的分化、系特异性及终末细胞的形态和功能。不仅如此,HOXB4还可增强胚胎干细胞（embryonic stem cell,ESC）的造血潜能,促进ESC向造血细胞分化。因此,HOXB4和（或）HOXB4的靶基因的表达上调可能在干细胞移植和基因治疗等方面具有广阔的应用前景。本文就HOXB4基因参与调控造血干细胞的自我更新,HOXB4对HSC的分化特异性及终末分化的“零”效应及HOXB4调控HSC自我更新的分子机制等进行综述。     

The Stromal Activity of Mesenchymal Stromal Cells

SUMMARY: The mechanism that regulates self-renewal and differentiation of hematopoietic stem cells (HSC) is a central question in stem cell biology that might ultimately lead to reliable protocols for in vitro expansion of HSC. Cellular fate is governed by cell-cell interaction with the microenvironment in the bone marrow, the stem cell niche. Mesenchymal stromal cells (MSC) are precursors of the cellular components, and they secrete extracellular matrix proteins of the bone marrow stroma. Therefore, MSC feeder layer might provide a suitable in vitro model system for the stem cell niche. In vitro assays demonstrate that MSC maintain the stem cell function of HSC and that MSC from bone marrow have a higher hematopoiesis supportive activity than MSC from adipose tissue. Co-cultivation with MSC might pave the way for expansion of long-term repopulating HSC, and various clinical trials indicate that co-transplantation of HSC and MSC might enhance engraftment. Thus, MSC are promising tools to elucidate the underlying mechanism of the cellular microenvironment. The large variety of preparative protocols for isolation and cultivation of MSC affects their stromal activity. Standardized isolation methods and molecular characterization of MSC are of utmost importance for reproducible isolation of hematopoiesis supportive stromal cells and for their potential clinical application.     

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.     

脐带血中造血干/祖细胞的体外扩增研究与临床应用进展

脐带血因具有来源广泛、采集方便、免疫配型要求低、移植后移植物抗宿主病(GVHD)发生率低等优点,为造血干细胞(HSC)的重要来源之一,为需要接受异基因造血干细胞移植(allo-HSCT)的患者提供1种可靠的HSC来源.然而,脐带血干细胞移植(UCBT)亦存在一定的缺陷,由于脐带血的体积小、细胞数量极有限,其所含的造血干/祖细胞(HS/PC)数量有限,往往不能满足高体重儿童及成年患者allo-HSCT的需要,从而限制了UCBT在临床上的广泛应用.HS/PC体外扩增方法为解决上述难题的关键,笔者拟就HS/PC体外扩增方法的相关研究与临床应用进展进行综述.     

采用多功能流式细胞术分析分选造血干细胞和髓系定向分化祖细胞

目的 探讨富集纯化造血干细胞(HSC)和髓系定向分化祖细胞的新实验方案.方法 根据造血干细胞和定向分化祖细胞在发育过程中表达某些特异性分化抗原的特性,通过免疫磁珠分选技术结合四色和六色流式细胞术分析14只健康小鼠的骨髓造血干细胞、造血祖细胞及定向分化祖细胞系列的表达,并对其进行分选,以进一步通过集落细胞培养和传代试验对分选后细胞的活性进行检测.结果 经上述实验方案分析,14只健康小鼠骨髓造血祖细胞(HPC)的表达率约为HSC的10倍;但其牛成活性远不如造血干细胞,共同髓系祖细胞(CMP)的传代能力仅为HSC的1/2,且次级分化的粒系单核系祖细胞(GMP)和红系巨核系祖细胞(MEP)的生成活性更弱,其传代次数为零.结论 通过多色流式细胞术实验方案可以分析纯化HSC和髓系定向分化祖细胞的表达,并精确计数HSC和祖细胞.     

The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in

Stem cell niches are defined as the cellular and molecular microenvironments that regulate stem cell function together with stem cell autonomous mechanisms. This includes control of the balance between quiescence, self-renewal, and differentiation, as well as the engagement of specific programs in response to stress. In mammals, the best understood niche is that harboring bone marrow hematopoietic stem cells (HSCs). Recent studies have expanded the number of cell types contributing to the HSC niche. Perivascular mesenchymal stem cells and macrophages now join the previously identified sinusoidal endothelial cells, sympathetic nerve fibers, and cells of the osteoblastic lineage to form similar, but distinct, niches that harbor dormant and self-renewing HSCs during homeostasis and mediate stem cell mobilization in response to granulocyte colony-stimulating factor.     

SDF-1/CXCR4信号通路在造血干细胞归巢中作用的研究进展

造血干细胞移植(HSCT)是治疗白血病、淋巴瘤、再生障碍性贫血等恶性血液病的一种有效治疗手段.但是,HSCT后造血功能的恢复与归巢至骨髓造血微环境中的造血干细胞(HSC)数目有关.HSCT输注入受者体内的HSC不会立刻发挥作用,而是先经历一系列复杂的过程归巢至骨髓造血微环境后,才能继续增殖并分化为相应的效应细胞发挥作用.其中,供者HSC与众多细胞及细胞因子间的相互作用是决定HSC归巢、增殖及分化的关键因素.基质细胞衍生因子(SDF)-1及其唯一的受体CXC趋化因子受体(CXCR)4所构成的SDF-1/CXCR4信号通路,可能在HSC归巢中发挥重要作用.为了更深入地研究HSC归巢的具体过程和SDF-1/CXCR4信号通路在该过程中发挥的作用,现对SDF-1、CXCR4的结构、作用机制,以及SDF1/CXCR4信号通路在HSC归巢中作用的研究进展进行综述.     

间充质干细胞在造血调控中的作用

间充质干细胞（MSC）作为造血微环境主要细胞成分的来源，具有自我更新和多向分化的潜能，通过与造血细胞直接接触、分泌细胞外基质及多种细胞因子维持造血微环境结构和功能的完整性，进而实现对造血的精细调控。本文结合近几年来国内外MSC的研究进展，就MSC在造血调控中的作用，诸如MSC分泌多种支持造血的细胞因子，MSC表达与造血细胞相互作用的黏附分子，联合移植MSC对造血重建的支持作用及其临床应用前景作一综述。     

CXCR4 is required for the quiescence of primitive hematopoietic cells

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4(-/-) HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.     

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.     

Recombinant IL-7/HGFβ efficiently induces transplantable murine hematopoietic stem cells

Difficulty obtaining sufficient hematopoietic stem cells (HSCs) directly from the donor has limited the clinical use of HSC transplantation. Numerous attempts to stimulate the ex vivo growth of purified HSCs with cytokines and growth factors generally have induced only modest increases in HSC numbers while decreasing their in vivo reconstituting ability. We previously developed a recombinant single-chain form of a naturally occurring murine hybrid cytokine of IL-7 and the β chain of hepatocyte growth factor (rIL-7/HGFβ) that stimulates the in vitro proliferation and/or differentiation of common lymphoid progenitors, pre-pro-B cells, and hematopoietic progenitor cells (day 12 spleen colony-forming units) in cultures of mouse BM. Here we used the rIL-7/HGFβ in culture to induce large numbers of HSCs from multiple cell sources, including unseparated BM cells, purified HSCs, CD45^– BM cells, and embryonic stem cells. In each instance, most of the HSCs were in the G₀ phase of the cell cycle and exhibited reduced oxidative stress, decreased apoptosis, and increased CXCR4 expression. Furthermore, when injected i.v., these HSCs migrated to BM, self-replicated, provided radioprotection, and established long-term hematopoietic reconstitution. These properties were amplified by injection of rIL-7/HGFβ directly into the BM cavity but not by treatment with rIL-7, rHGF, and/or rHGFβ.     

The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.     

Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.     

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.