Targeting the Spleen as an Alternative Site for Hematopoiesis

Bone marrow is the main site for hematopoiesis in adults. It acts as a niche for hematopoietic stem cells (HSCs) and contains non‐hematopoietic cells that contribute to stem cell dormancy, quiescence, self‐renewal, and differentiation. HSC also exist in resting spleen of several species, although their contribution to hematopoiesis under steady‐state conditions is unknown. The spleen can however undergo extramedullary hematopoiesis (EMH) triggered by physiological stress or disease. With the loss of bone marrow niches in aging and disease, the spleen as an alternative tissue site for hematopoiesis is an important consideration for future therapy, particularly during HSC transplantation. In terms of harnessing the spleen as a site for hematopoiesis, here the remarkable regenerative capacity of the spleen is considered with a view to forming additional or ectopic spleen tissue through cell engraftment. Studies in mice indicate the potential for such grafts to support the influx of hematopoietic cells leading to the development of normal spleen architecture. An important goal will be the formation of functional ectopic spleen tissue as an aid to hematopoietic recovery following clinical treatments that impact bone marrow. For example, expansion or replacement of niches could be considered where myeloablation ahead of HSC transplantation compromises treatment outcomes.     

Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches

Specific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.     

The location and cellular composition of the hemopoietic stem cell niche

While it is accepted that hemopoietic stem cells (HSC) are located in a three-dimensional microenvironment, termed a niche, the cellular and extracellular composition, as well as the multifaceted effects the components of the niche have on HSC regulation, remains undefined. Over the past four decades numerous advances in the field have led to the identification of roles for some cell types and propositions of potentially a number of HSC niches. We present evidence supporting the roles of multiple cell types and extracellular matrix molecules in the HSC niche, as well as discuss the potential significant overlap and intertwining of previously proposed distinct HSC niches.     

Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells

Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non‐SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non‐SP cells. From a cell regulation point of view, we showed that SP activity depended on O₂ concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia‐induced factors HIF‐1α and HIF‐2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non‐SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Hematopoietic stem cells: generation and self-renewal

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.     

Uncertainty in the niches that maintain haematopoietic stem cells

Haematopoietic stem cell (HSC) niches are specialized microenvironments that contain stem cells and regulate their maintenance. Cells at the interface of bone and the bone marrow (the endosteum) contribute to the creation of HSC niches. It remains uncertain whether this interface itself is a niche, or whether endosteal cells secrete factors that diffuse to nearby niches. Vascular and/or perivascular cells may also create niches as many HSCs are observed around sinusoidal blood vessels, and perivascular cells secrete factors that regulate HSC maintenance. Do endosteal and perivascular cells create distinct niches, or do they contribute to a common niche? We discuss a range of niche models consistent with recent evidence.     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Bone-marrow haematopoietic-stem-cell niches

Adult stem cells hold many promises for future clinical applications and regenerative medicine. The haematopoietic stem cell (HSC) is the best-characterized somatic stem cell so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells. Here we review recent progress in characterizing the composition of the HSC bone-marrow microenvironment, known as the HSC niche. During homeostasis, HSCs, and therefore putative bone-marrow HSC niches, are located near bone surfaces or are associated with the sinusoidal endothelium. The molecular crosstalk between HSCs and the cellular constituents of these niches is thought to control the balance between HSC self-renewal and differentiation, indicating that future successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem-cell-niche unit.     

The effects of zoledronic acid in the bone and vasculature support of hematopoietic stem cell niches

Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin‐ cells demonstrated increased expression of self‐renewal‐related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long‐term self‐renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA‐induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche. J. Cell. Biochem. 114: 67–78, 2012. © 2012 Wiley Periodicals, Inc.     

Transcriptional regulation of Rex1 (zfp42) in normal prostate epithelial cells and prostate cancer cells

The enormous regenerative capacity of the blood system to sustain functionally mature cells are generated from highly proliferative, short‐lived progenitors, which in turn arise from a rare population of pluripotent and self‐renewing hematopoietic stem cells (HSC). In the bone marrow, these stem cells are kept in a low proliferative, relatively quiescent state in close proximity to stromal cells and osteoblasts, forming specialized niches. The interaction in particular to bone is crucial to prevent exhaustion of HSCs from uncontrolled cell‐cycle entry and to excessive proliferation. In addition, the niche and its components protect stem cells from stress, such as accumulation of reactive oxygen species and DNA damage. One of the key issues is to identify conditions to increase the number of HSCs, either in vivo or during ex vivo growth cultures. This task has been very difficult to resolve and most attempts have been unsuccessful. However, the mechanistic insights to HSC self‐renewal and preservation are gradually increasing and there is now hope that future research will enable scientists and clinicians to modulate the process. In this review, we will focus on the molecular mechanisms of self‐renewal and HSC maintenance in the light of novel findings that HSCs reside at the lowest end of an oxygen gradient. Hypoxia appears to regulate hematopoiesis in the bone marrow by maintaining important HSC functions, such as cell cycle control, survival, metabolism, and protection against oxidative stress. To improve the therapeutic expansion of HSCs we need to learn more about the molecular mechanisms of hypoxia‐mediated regulation. J. Cell. Physiol. 222:17–22, 2010. © 2009 Wiley‐Liss, Inc.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009. © 2009 Wiley‐Liss, Inc.     

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。     

Haematopoietic stem cell niche in Drosophila

Development and homeostasis of the haematopoietic system is dependent upon stem cells that have the unique ability to both self-renew and to differentiate in all cell lineages of the blood. The crucial decision between haematopoietic stem cell (HSC) self-renewal and differentiation must be tightly controlled. Ultimately, this choice is regulated by the integration of intrinsic signals together with extrinsic cues provided by an exclusive microenvironment, the so-called haematopoietic niche. Although the haematopoietic system of vertebrates has been studied extensively for many decades, the specification of the HSC niche and its signals involved are poorly understood. Much of our current knowledge of how niches regulate long-term maintenance of stem cells is derived from studies on Drosophila germ cells. Now, two recently published studies by Mandal et al.1 and Krezmien et al.2 describe the Drosophila haematopoietic niche and signal transduction pathways that are involved in the maintenance of haematopoietic precursors. Both reports emphasize several features that are important for controlling stem cell behavior and show parallels to both the vertebrate haematopoietic niche as well as the Drosophila germline stem cell niches in ovary and testis. The findings of both papers shed new light on the specific interactions between haematopoietic progenitors and their microenvironment.     

Autophagy fuels tissue fibrogenesis

Activation of hepatic stellate cells (HSC), a resident pericytic cell in liver, into a proliferative and fibrogenic cell type, is the principal event underlying hepatic fibrosis following injury. Release of lipid droplets (LD) containing retinyl esters and triglyceride is a defining feature of HSC activation, yet the basis for this release has remained mysterious. Here we offer a surprising discovery that autophagy is the missing link underlying LD release, by stimulating metabolism of their contents to provide the energy vital to fuel HSC activation. By specifically inhibiting the autophagic pathway in activated HSC, LD release is impaired and cellular ATP levels are decreased. Moreover, animals with HSC-specific deletion of Atg7 display attenuated activation following liver injury, leading to reduced fibrosis in vivo. We further demonstrate that fibrogenic cells from other organs, including kidney and lung, also rely on autophagy as a core pathway driving the scarring response. Our results provide a novel framework for understanding pathways underlying fibrogenic cell responses to tissue injury.     

Autophagy fuels tissue fibrogenesis

Activation of hepatic stellate cells (HSC), a resident pericytic cell in liver, into a proliferative and fibrogenic cell type, is the principal event underlying hepatic fibrosis following injury. Release of lipid droplets (LD) containing retinyl esters and triglyceride is a defining feature of HSC activation, yet the basis for this release has remained mysterious. Here we offer a surprising discovery that autophagy is the missing link underlying LD release, by stimulating metabolism of their contents to provide the energy vital to fuel HSC activation. By specifically inhibiting the autophagic pathway in activated HSC, LD release is impaired and cellular ATP levels are decreased. Moreover, animals with HSC-specific deletion of Atg7 display attenuated activation following liver injury, leading to reduced fibrosis in vivo. We further demonstrate that fibrogenic cells from other organs, including kidney and lung, also rely on autophagy as a core pathway driving the scarring response. Our results provide a novel framework for understanding pathways underlying fibrogenic cell responses to tissue injury.     

Differential requirements for Wnt and Notch signaling in hematopoietic versus thymic niches

All blood cells are derived from multipotent stem cells, the so-called hematopoietic stem cells (HSCs), that in adults reside in the bone marrow. Most types of blood cells also develop there, with the notable exception of T lymphocytes that develop in the thymus. For both HSCs and developing T cells, interactions with the surrounding microenvironment are critical in regulating maintenance, differentiation, apoptosis, and proliferation. Such specialized regulatory microenvironments are referred to as niches and provide both soluble factors as well as cell-cell interactions between niche component cells and blood cells. Two pathways that are critical for early T cell development in the thymic niche are Wnt and Notch signaling. These signals also play important but controversial roles in the HSC niche. Here, we review the differences and similarities between the thymic and hematopoietic niches, with particular focus on Wnt and Notch signals, as well as the latest insights into regulation of these developmentally important pathways.     

Asymmetric Cell Divisions of Human Hematopoietic Stem and Progenitor Cells Meet Endosomes

Hematopoietic stem cells (HSC) are undifferentiated cells, which self-renew over a long period of time and give rise to committed hematopoietic progenitor cells (HPC) containing the capability to replenish the whole blood system. Since both uncontrolled expansion as well as loss of HSC would be fatal, the decision of self-renewal versus differentiation needs to be tightly controlled. There is good evidence that both HSC niches as well as asymmetric cell divisions are involved in controlling whether HSC self-renew or become committed to differentiate. In this context, we recently identified four proteins which frequently segregate asymmetrically in dividing HSC/HPC. Remarkably, three of these proteins, the tetraspanins CD53 and CD63, and the transferrin receptor are endosome-associated proteins. Here, we highlight these observations in conjunction with recent findings in model organisms which show that components of the endosomal machinery are involved in cell-fate specification processes.