Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation

We report on a subset of cells that co-purify with CD45-positive/Lineage minus (CD45(pos)/Lin(minus)) hematopoietic cells that are capable of in vitro differentiation into multi-potential cells including cells with neuroectoderm properties. Although these cells are CD45 positive and have properties similar to CD45-negative mesenchymal progenitor cells (MPC) derived from bone marrow (BM), they are neither hematopoietic cells nor mesenchymal cells. These CD45(pos)/Lin(minus) cells can be expanded in vitro, express the stem cell genes Oct-4 and Nanog and can be induced to differentiate into endothelial cells, osteoblasts, muscle cells and neural cells at frequencies similar to those reported for bone marrow mesenchymal cells. Long-term culture of these cells followed by transplantation into NOD/SCID mice resulted in positive bone marrow stromal cell engraftment but not hematopoietic engraftment, suggesting that despite their CD45-positive status these cells do not have the same properties as hematopoietic stem cells. Clonal cell analysis determined that the culture period caused a broadening in the differentiation potential of the starting population.     

Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.     

Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration

Emerging evidence suggests that bone marrow-derived endothelial, hematopoietic stem and progenitor cells contribute to tissue vascularization during both embryonic and postnatal physiological processes. Recent preclinical and pioneering clinical studies have shown that introduction of bone marrow-derived endothelial and hematopoietic progenitors can restore tissue vascularization after ischemic events in limbs, retina and myocardium. Corecruitment of angiocompetent hematopoietic cells delivering specific angiogenic factors facilitates incorporation of endothelial progenitor cells (EPCs) into newly sprouting blood vessels. Identification of cellular mediators and tissue-specific chemokines, which facilitate selective recruitment of bone marrow-derived stem and progenitor cells to specific organs, will open up new avenues of research to accelerate organ vascularization and regeneration. In addition, identification of factors that promote differentiation of the progenitor cells will permit functional incorporation into neo-vessels of specific tissues while diminishing potential toxicity to other organs. In this review, we discuss the clinical potential of vascular progenitor and stem cells to restore long-lasting organ vascularization and function.     

Shear stress regulates adhesion and rolling of CD44+ leukemic and hematopoietic progenitor cells on hyaluronan

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm² is exceeded. At shear stress ∼1 dyn/cm², the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow.     

Contribution of marrow-derived progenitors to vascular and cardiac regeneration

Adult bone marrow is a rich reservoir of tissue-specific pluripotent stem and progenitor cells. Accumulating evidence suggest that these cells have the potential of contributing to tissue revascularization and cardiac regeneration. Physiological stress results in the release of specific chemokines and cytokines that promote mobilization of stem cells to the peripheral circulation. Incorporation of these mobilized cells contributes to formation of functional vasculature and sets up stage for tissue regeneration. Vascular Endothelial Growth Factor (VEGF) through interaction with its receptors VEGFR2 and VEGFR1 expressed on endothelial and hematopoietic stem cells promote recruitment of these cells into the sites of tissue injury accelerating vascular healing. Similarly, subset of CD34 + marrow derived cells are mobilized and recruited to the ischemic myocardium, differentiating into cardiac and vascular cells, restoring cardiac function. Identification of cellular mediators and tissue specific chemocytokines that facilitate selective recruitment of marrow-derived stem and progenitor cells to specific organs, will open up new avenues to accelerate cardiovascular regeneration and tissue revascularization.     

Do microRNAs regulate bone marrow stem cell niche physiology?

The adult bone marrow, situated within the bone cavity, comprises three distinct stem cell populations: hematopoietic stem cells (HSCs), mesenchymal stromal/stem cells (MSCs) and endothelial progenitor/stem cells (EPCs). HSCs are a well-characterized population of self-renewing cells that give rise to all blood cells. The definition of MSCs is more complex due to the limited understanding of MSC properties. In general, MSCs are considered multipotent stromal cells that are able to differentiate into various cell types, including osteoblasts, chondrocytes and adipocytes. Compared to HSCs and MSCs, EPCs are a newly discovered population of stem/progenitor cells with the capacity to differentiate into endothelial cells, the cells forming the inner lining of a blood vessel.     

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling

Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM-RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor β (TGFβ) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-γ receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM-RAMP2 signaling for development of LSECs.     

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1)

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.     

Molecular mechanisms underlying adhesion and migration of hematopoietic stem cells

Hematopoietic stem cell transplantation is the most powerful treatment modality for a large number of hematopoietic malignancies, including leukemia. Successful hematopoietic recovery after transplantation depends on homing of hematopoietic stem cells to the bone marrow and subsequent lodging of those cells in specific niches in the bone marrow. Migration of hematopoietic stem cells to the bone marrow is a highly regulated process that requires correct regulation of the expression and activity of various molecules including chemoattractants, selectins and integrins. This review will discuss recent studies that have extended our understanding of the molecular mechanisms underlying adhesion, migration and bone marrow homing of hematopoietic stem cells.     

Molecular mechanisms underlying adhesion and migration of hematopoietic stem cells

Hematopoietic stem cell transplantation is the most powerful treatment modality for a large number of hematopoietic malignancies, including leukemia. Successful hematopoietic recovery after transplantation depends on homing of hematopoietic stem cells to the bone marrow and subsequent lodging of those cells in specific niches in the bone marrow. Migration of hematopoietic stem cells to the bone marrow is a highly regulated process that requires correct regulation of the expression and activity of various molecules including chemoattractants, selectins and integrins. This review will discuss recent studies that have extended our understanding of the molecular mechanisms underlying adhesion, migration and bone marrow homing of hematopoietic stem cells.     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke

The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day) and 144 hrs (6th day) since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM) and erythroid (BFU-E)-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.     

Expression of stabilin-2, a novel fasciclin-like hyaluronan receptor protein,in murine sinusoidal endothelia,avascular tissues,and at solid/liquid interfaces

Stabilin-2, the hepatic hyaluronan receptor, has recently been cloned by us. Together with stabilin-1, stabilin-2 constitutes a novel family of fasciclin-like hyaluronan receptor homologues. Here, we analyzed expression of stabilin-2 (mStab-2) in a broad array of C57BL/6 mouse organs and tissues. While northern blot analysis showed positive expression of mStab-2 mRNA confined to liver and spleen, immunohistochemistry demonstrated mStab-2 protein expression in the endothelial sinuses of liver, lymph nodes, spleen, and bone marrow, and in specialized structures of eye, heart, brain, and kidney. Expression of mStab-2 was detected in corneal and lens epithelium, in mesenchymal cells of the heart valves, in the ependymal cells lining the ventricles in the brain, and in the prismatic epithelial cells covering the renal papillae. In pathological conditions, such as tumor growth or wound healing processes, mStab-2 was not expressed in the newly formed vasculature or other tissue components. Based on these results, we suggest that mStab-2 might be involved in the clearance of hyaluronan from the lymph or the blood circulation via the network of endothelial sinuses. At the other mStab-2-positive tissues sites that are either avascular and/or demarcate a solid/liquid interface, mStab-2 may serve to maintain tissue integrity by supporting extracellular matrix turnover or it may contribute to maintaining fluidity of bodily liquids by resorption of hyaluronan.     

内皮祖细胞分离培养及应用技术新进展

内皮祖细胞(Endothelial progenitor cells,EPCs)是能够增殖、迁移、粘附并分化为血管内皮细胞潜能的一种原始细胞,在修复血管内皮和促进血管生成中具有重要的作用。EPC来源于骨髓,存在于骨髓、外周血、脐带血,新研究表明在脂肪组织、心肌中也能发现EPC的存在。EPC与干细胞的细胞表面标志物相似,功能上亦接近干细胞,但不具有自我更新的功能。近年来EPC已成为热点问题,对疾病诊断,预后判断和靶向治疗方面发挥重要作用,在冠状动脉粥样硬化性疾病、糖尿病血管病变、恶性肿瘤等治疗中全身或局部注射EPC具有更广泛的前景和应用价值。但关于分离培养EPC的方法及细胞表面标志物不完全相同,报道较少,至今尚没有形成统一的标准,本文就对于内皮祖细胞基本现状、分离培养技术、分选鉴定及临床应用方面做一综述。     

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.     

Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization

Adult bone marrow is a rich reservoir of hematopoietic and vascular stem and progenitor cells. Mobilization and recruitment of these cells are essential for tissue revascularization. Physiological stress, secondary to tissue injury or tumor growth, results in the release of angiogenic factors, including vascular endothelial growth factor (VEGF), which promotes mobilization of stem cells to the circulation, contributing to the formation of functional vasculature. VEGF interacts with its receptors, VEGFR2 and VEGFR1, expressed on endothelial and hematopoietic stem cells, and thereby promotes recruitment of these cells to neo-angiogenic sites, accelerating the revascularization process. The mobilization of stem cells from marrow is a dynamic process, regulated by shear stress imparted by blood flow, and the activation of metalloproteinases that induce the release of 'Kit ligand', facilitating egress from the marrow to the circulation. Identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells will open up new avenues for the design of clinical trials to accelerate tissue vascularization and organogenesis.     

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age-related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34(+) CD38(-) cells increases in the bone marrow of elderly (>70 years) individuals. CD34(+) CD38(+) CD90(-) CD45RA(+/-) CD10(-) and CD34(+) CD33(+) myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34(+) CD38(+) CD90(-) CD45RA(+) CD10(+) and committed CD34(+) CD19(+) B-lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL-2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age-related decrease in the capacity of CD34(+) cells to generate myeloid cells was also seen in colony-forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.     

Endothelial cells mediate the regeneration of hematopoietic stem cells

Recent studies suggest that endothelial cells are a critical component of the normal hematopoietic microenvironment. Therefore, we sought to determine whether primary endothelial cells have the capacity to repair damaged hematopoietic stem cells. Highly purified populations of primary CD31⁺ microvascular endothelial cells isolated from the brain or lung did not express the pan hematopoietic marker CD45, most hematopoietic lineage markers, or the progenitor marker c-kit and did not give rise to hematopoietic cells in vitro or in vivo. Remarkably, the transplantation of small numbers of these microvascular endothelial cells consistently restored hematopoiesis following bone marrow lethal doses of irradiation. Analysis of the peripheral blood of rescued recipients demonstrated that both short-term and long-term multilineage hematopoietic reconstitution was exclusively of host origin. Secondary transplantation studies revealed that microvascular endothelial cell-mediated hematopoietic regeneration also occurs at the level of the hematopoietic stem cell. These findings suggest a potential therapeutic role for microvascular endothelial cells in the self-renewal and repair of adult hematopoietic stem cells.     

Stabilin-1, a homeostatic scavenger receptor with multiple functions

The multifunctional scavenger receptor stabilin-1 (STAB1, FEEL-1, CLEVER-1, KIAA0246) was originally identified as the MS-1 antigen, expressed by sinusoidal endothelial cells in human spleen. Extensive histological studies revealed that stabilin-1 is also expressed by tissue macrophages and sinusoidal endothelial cells in the healthy organism; its expression on both macrophages and different subtypes of endothelial cells is induced during chronic inflammation and tumorigenesis. In vitro induction of stabilin-1 in macrophages requires the presence of glucocorticoids. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and recycling; and shuttling between the endosomal compartment and trans-Golgi network (TGN). The latter intracellular pathway of stabilin-1 trafficking is mediated by GGAs, clathrin adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. When expressed by alternatively activated macrophages, stabilin-1 mediates the uptake and targeting for degradation of acLDL and SPARC, a regulator of tissue remodeling. Likewise, stabilin-1 in macrophages is involved in intracellular sorting and lysosomal delivery of the novel stabilin- 1-interacting chitinase-like protein (SI-CLP). Indirect evidence suggests that stabilin-1 is involved in adhesion and transmigration in various cell types (including tumor cells, leukocytes, and lymphocytes); however, its rapid recycling and scant level of surface expression argue against its universal role in cell adhesion. In summary, stabilin-1 is a homeostatic receptor which links signals from the extracellular environment to intracellular vesicular processes, creating a potential impact on the macrophage secretion profile.     

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.