Cell cycle dynamics and complement expression distinguishes mature haematopoietic subsets arising from hemogenic endothelium

The emergence of haematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium results in the formation of sizeable HSPC clusters attached to the vascular wall. We evaluate the cell cycle and proliferation of HSPCs involved in cluster formation, as well as the molecular signatures from their initial appearance to the point when cluster cells are capable of adult engraftment (definitive HSCs). We uncover a non-clonal origin of HSPC clusters with differing cell cycle, migration, and cell signaling attributes. In addition, we find that the complement cascade is highly enriched in mature HSPC clusters, possibly delineating a new role for this pathway in engraftment.     

SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis

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Reduced repression of cytokine signaling ameliorates age‐induced decline in hematopoietic stem cell function

Aging causes profound effects on the hematopoietic stem cell (HSC) pool, including an altered output of mature progeny and enhanced self‐propagation of repopulating‐defective HSCs. An important outstanding question is whether HSCs can be protected from aging. The signal adaptor protein LNK negatively regulates hematopoiesis at several cellular stages. It has remained unclear how the enhanced sensitivity to cytokine signaling caused by LNK deficiency affects hematopoiesis upon aging. Our findings demonstrate that aged LNK^?/? HSCs displayed a robust overall reconstitution potential and gave rise to a hematopoietic system with a balanced lineage distribution. Although aged LNK^?/? HSCs displayed a distinct molecular profile in which reduced proliferation was central, little or no difference in the proliferation of aged LNK^?/? HSCs was observed after transplantation when compared to aged WT HSCs. This coincided with equal telomere maintenance in WT and LNK^?/? HSCs. Collectively, our studies suggest that enhanced cytokine signaling can counteract functional age‐related HSC decline.     

Developmental regulation of replication-coupled protein destruction

Comment on: Lee HO, et al. Mol Biol Cell 2010; 21:3639-53.     

Establishment and regulation of the HSC niche: Roles of osteoblastic and vascular compartments

Hematopoietic stem cells (HSC) are multi-potent cells that function to generate a lifelong supply of all blood cell types. During mammalian embryogenesis, sites of hematopoiesis change over the course of gestation: from extraembryonic yolk sac and placenta, to embryonic aorta-gonad-mesonephros region, fetal liver, and finally fetal bond marrow where HSC reside postnatally. These tissues provide microenviroments for de novo HSC formation, as well as HSC maturation and expansion. Within adult bone marrow, HSC self-renewal and differentiation are thought to be regulated by two major cellular components within their so-called niche: osteoblasts and vascular endothelial cells. This review focuses on HSC generation within, and migration to, different tissues during development, and also provides a summary of major regulatory factors provided by osteoblasts and vascular endothelial cells within the adult bone marrow niche.     

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.     

Maladaptive innate immune training of myelopoiesis links inflammatory comorbidities

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Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Shear stress during early embryonic stem cell differentiation promotes hematopoietic and endothelial phenotypes

Pluripotent embryonic stem cells (ESCs) are a potential source for cell‐based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm² and for cells seeded on collagen‐, fibronectin‐ or laminin‐coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force‐mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies. Biotechnol. Bioeng. 2013; 110: 1231–1242. © 2012 Wiley Periodicals, Inc.     

A complex linkage in the developmental pathway of endothelial and hematopoietic cells

During normal vertebrate development, hematopoietic and endothelial cells form closely situated and interacting populations. Although the close proximity of cells to each other does not necessarily mean that they are relatives, accumulating evidence indicates that hematopoietic and endothelial cells are indeed close kin; they share common progenitors and each is able to become the other under certain circumstances. This article summarizes recent advances in the developmental relationship between hematopoietic and endothelial cells.     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

小鼠骨髓内皮细胞条件培养液复合FL及TPO对HPP—CFC及CFU—GM增殖的影响

通过传代培养小鼠骨髓内皮细胞,收集无血清条件培养液（ECM）,并经超滤得到大于10kD的浓缩液,分别观察ECM和大于10kD的浓缩液复合flt3ligand(FL)及thrombopoietin(TPO)对体外培养HPP-CFC、CFU-GM的影响,结果表明：ECM或大于10kD的浓缩液对HPP-CFC、CFU-GM的生长均有支持作用;FL或/和TPO与ECM或大于10kD的浓缩液合用能加强对HPP-CFC、CFU-GM生长的刺激作用;FL加TPO与ECM或大于10kD的浓缩液合用对HPP-CFC、CFU-GM生长的刺激作用更加明显;选择FL和TPO特异性的引物,用RT-PCR技术未能检测到小鼠骨髓内皮细胞有FL和TPOmRNA的表达。     

lncRNA GAS5/miR-223/NAMPT axis modulates the cell proliferation and senescence of endothelial progenitor cells through PI3K/AKT signaling

Endothelial progenitor cells (EPCs) have been reported to replace the damaged endothelial cells to repair the injured or dead endothelium. However, EPC senescence might lead to the failure in EPC function. Thus, developing an in-depth understanding of the mechanism of EPC senescence might provide novel strategies for related vascular disorders’ treatments. Herein, nicotinamide phosphoribosyltransferase (NAMPT) overexpression could increase cell proliferation and suppress cell senescence in EPCs. miR-223 directly bound to the 3′-untranslated region of NAMPT to inhibit its expression, therefore modulating EPC proliferation and senescence through NAMPT and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling. Long noncoding RNA (lncRNA) GAS5 sponges miR-223, consequently downregulating miR-223 expression. GAS5 knockdown inhibited EPC proliferation and promoted senescence. GAS5 might serve as a competing endogenous RNA for miR-223 to counteract miR-223-mediated suppression on NAMPT, thus regulating EPC proliferation and senescence via the PI3K/AKT signaling pathway. In summary, our findings provide a solid experimental basis for understanding the role and mechanism of lncRNA GAS5/miR-223/NAMPT axis in EPC proliferation and senescence.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.     

The role of Wnt signaling in hematopoietic stem cell development

Hematopoietic stem cells (HSCs) can self-renew and differentiate into all cell types of the blood. This is therapeutically important as HSC transplants can provide a curative effect for blood cancers and disorders. The process by which HSCs develop has been the subject of extensive research in a variety of model organisms; however, efforts to produce bonafide HSCs from pluripotent precursors capable of long-term multilineage reconstitution have fallen short. Studies in zebrafish, chicken, and mice have been instrumental in guiding efforts to derive HSCs from human pluripotent stem cells and have identified a complex set of molecular signals and cellular interactions mediated by such developmental regulators as fibroblast growth factor, Notch, transforming growth factor beta (TGFβ), and Wnt, which collectively promote the stepwise developmental progression toward mature HSCs. Tight temporal and spatial control of these signals is critical to generate the appropriate numbers of HSCs needed for the life of the organism. The role of the Wnt family of signaling proteins in hematopoietic development has been the subject of many studies owing in part to the complex nature of its signaling mechanisms. By integrating cell fate specification with cell polarity establishment, Wnt is uniquely capable of controlling complex biological processes, including at multiple stages of embryonic HSC development, from HSC specification to emergence from the hemogenic epithelium to subsequent expansion. This review highlights key signaling events where specific Wnt signals instruct and guide hematopoietic development in both zebrafish and mice and extend these findings to current efforts of generating HSCs in vitro.     

视黄酸核受体γ重组腺病毒构建及其鉴定

目的构建携带大鼠视黄酸核受体γ（retinoic acid receptor γ,RARγ）的重组腺病毒,为研究RAR3,在骨髓间充质干细胞（mesenchymal stem cells,MSCs）成神经分化中的作用奠定基础。方法体外扩增大鼠础研基因,将其定向克隆至腺病毒穿梭质粒pAd Trace—TOX构建重组质粒pAdTrace—RARγ,并在BJ5183菌中与骨架质粒pAd Easy-1重组获得腺病毒载体pad—RARγ,Pac I酶切后转染HEK293细胞包装腺病毒。腺病毒Ad—RARγ感染大鼠MSCs,Real—time PCR和Western印迹检测RARγ的表达。结果PCR,酶切及测序均证实础研正确克隆至腺病毒质粒载体中,Ad—RARγ对MSCs感染率达60％～70％,并明显增强础研基因和蛋白的表达。结论成功构建携带RARγ的重组腺病毒,并具有上调大鼠MSCsRA脚基因和蛋白表达的功能。