Hepatic stellate cells modulate the differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells

Differentiation of stem cells is tightly regulated by the microenvironment which is mainly composed of nonparenchymal cells. Herein, we investigated effect of hepatic stellate cells (HSCs) in different states on mesenchymal stem cells (MSCs) differentiation. Rat HSCs were isolated and stayed quiescent within 5 days. Primary HSCs were activated by being in vitro cultured for 7 days or cocultured with Kupffer cells for 5 days. MSCs were cocultured with HSCs of different states. Expression of hepatic lineage markers was analyzed by RT-PCR and immunofluorescence. Glycogen deposition was detected by periodic acid-schiff staining. MSCs cocultured with HSC-T6 or Kupffer cell activated HSCs were morphologically transformed into hepatocyte-like cells. Hepatic-specific marker albumin was expressed in 78.3% of the differentiated MSCs 2 weeks after initiation of coculture. In addition, the differentiated MSCs also expressed alpha-fetoprotein, cytokeratin-18, glutamine synthetase and phosphoenolpyruvate carboxykinase. Glycogen deposition was detectable in 55.4% of the differentiated MSCs 6 weeks after initiation of coculture. However, the quiescent HSCs or culture activated HSCs did not exert the ability to modulate the differentiation of MSCs. Moreover, Kupffer cell activated HSCs rather than culture activated HSCs expressed hepatocyte growth factor mRNA. We draw the conclusion that fully activated HSCs could modulate MSCs differentiation into hepatocyte-like cells.     

Function of oxidative stress in the regulation of hematopoietic stem cell-niche interaction

During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized niches, such as osteoblastic niche and vascular niche. A cell adhesion molecule, N-cadherin expressed in the HSCs and osteoblasts, suggesting that homophylic binding of N-cadherin induce the adhesion of HSCs to the niche cells. Here we demonstrate that an anti-cancer drug, 5-fuluorouracil induces reactive oxygen species (ROS) in HSCs, which suppressed N-cadherin expression. These events result in the shift of side population (SP) cells to non-SP cells, indicating that quiescent HSCs are detached from the niche. Administration of a potent anti-oxidant, N-acetyl cystein (NAC) suppressed the shift from SP cells. These data suggest that ROS suppressed the N-cadherin-mediated cell adhesion, and induce the exit of HSCs from the niche.     

造血干细胞相关microRNAs的筛选及其促分化研究

为了获得人脐血造血干细胞(HSCs)的microRNAs(miRNAs)表达谱,并对相关miRNAs功能进行初步鉴定.利用免疫磁珠(MACS)和流式细胞仪(FACS)细胞分选技术分离人脐血造血干细胞(HSCs),分别提取细胞总RNA并分离小分子RNA,经荧光标记后与miRNAs基因芯片杂交,获得HSCs的miRNAs表达谱,集落形成实验(CFC)研究在HSC中高表达miR-520h对HSC的促分化作用.成功分离人脐血CD34 细胞和HSC,经基因芯片杂交获得31个造血干细胞相关miRNAs,其中22个为低表达,9个为高表达;经实时定量RT-PCR验证miR-520h显著升高,CFC实验表明其可增加多种集落形成,具有促进HSC向祖细胞分化的作用.上述结果表明,人脐血HSC具有自身特征性miRNAs,参与并调控HSC生物学功能,为深入探讨miRNAs在造血系统发育中的作用打下基础.     

A sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 μg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34(+) hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34(+)CD38(-) cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34(+)CD38(+) cells). In contrast, mouse quiescent HSCs (Pyronin-Y(low) LKS(+) cells) and cycling HSCs (Pyronin-Y(hi) LKS(+) cells) repaired the damage more efficiently than HPCs (LKS(-) cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs.     

SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells

To improve our ability to identify hematopoietic stem cells (HSCs) and their localization in vivo, we compared the gene expression profiles of highly purified HSCs and non-self-renewing multipotent hematopoietic progenitors (MPPs). Cell surface receptors of the SLAM family, including CD150, CD244, and CD48, were differentially expressed among functionally distinct progenitors. HSCs were highly purified as CD150(+)CD244(-)CD48(-) cells while MPPs were CD244(+)CD150(-)CD48(-) and most restricted progenitors were CD48(+)CD244(+)CD150(-). The primitiveness of hematopoietic progenitors could thus be predicted based on the combination of SLAM family members they expressed. This is the first family of receptors whose combinatorial expression precisely distinguishes stem and progenitor cells. The ability to purify HSCs based on a simple combination of SLAM receptors allowed us to identify HSCs in tissue sections. Many HSCs were associated with sinusoidal endothelium in spleen and bone marrow, though some HSCs were associated with endosteum. HSCs thus occupy multiple niches, including sinusoidal endothelium in diverse tissues.     

A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells

Hematopoietic stem cells (HSCs) isolated from mouse fetal liver, like adult HSCs, are Thy-1lo Lin- Sca-1+. Donor-derived V gamma 3+ T cells were detected in fetal thymic lobes repopulated in vitro with fetal liver HSCs, but not in those with adult bone marrow HSCs. Single clonogenic fetal HSCs gave rise to thymic progeny that include V gamma 3+, other gamma delta+, and alpha beta+ T cells. No V gamma 3+ T cells were detected in adult thymus injected intrathymically with either fetal or adult HSCs. These results support the hypothesis that only fetal HSCs have the capacity to differentiate into V gamma 3+ T cells in the fetal thymic microenvironment and that the developmental potential of HSCs may change during ontogeny.     

Hematopoietic reconstitution of CD34<Superscript>+</Superscript> cells grown in static and stirred culture systems in NOD/SCID mice

The hematopoietic reconstitution of cord blood (CB) CD34⁺ cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.     

On signaling pathways: hematopoietic stem cell specification from hemogenic endothelium

Hematopoietic stem cells(HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells(PSCs-embryonic stem cells(ESCs) and induced pluripotent stem cells(i PSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.     

应用SYBR荧光实时定量RT-PCR法检测BMSCs诱导大鼠肝星状细胞中DR5mRNA表达

目的应用SYBR荧光实时定量RT-PCR法检测骨髓间充质干细胞（BMSCs）对大鼠肝星状细胞（HSCs）的死亡受体5（DR5）mRNA表达的影响,探讨BMSCs诱导HSCs凋亡及其机制。方法采用贴壁筛选法培养、纯化SD大鼠BMSCs,传至第4代使用;大鼠原代HSCs细胞及肝纤维原细胞系冻融后传代使用。应用6孔塑料培养板,建立上下双层细胞共培养体系,常规培养。实验分为3组：（1）实验组：BMSCs与HSCs共培养;（2）空白对照组：HSCs单独培养;（3）阴性对照组：大鼠肝纤维原细胞与HSCs共培养。以上培养体系动态观察24、48、72h,应用流式细胞仪检测HSCs细胞凋亡率,采用SYBRGreenI荧光实时定量RT-PCR法检测,以β-actin基因作为内参,计算各组DR5mRNA的相对表达量。结果在共培养组中,BMSCs促进了HSCs凋亡,与其他两组比较差异有显著统计学意义（P〈O．01）,空白对照组与阴性对照组比较无统计学意义（P〉0．05）。实验组BMSCs能明显上调HSCs中DR5mRNA的表达,与空白对照组和阴性对照组比较差异有显著统计学意义（P〈O．01）;空白对照组与阴性对照组DR5mRNA的表达比较无统计学意义（P〉O．05）。结论利用SYBR荧光实时定量RT-PCR法检测BMSCs诱导大鼠肝星状细胞中DR5mRNA表达,为进一步研究BMSCs通过死亡受体途径调控HSCs凋亡以及为BMSCs用于治疗肝纤维化的机制研究提供了理论基础。     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.     

The journey of developing hematopoietic stem cells

Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites. Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs. During this process, developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. These processes have to be recapitulated to generate HSCs from embryonic stem cells. Elucidating the interactions between developing HSCs and their niches should facilitate the generation and expansion of HSCs in vitro to exploit their clinical potential.     

mTOR activation induces tumor suppressors that inhibit leukemogenesis and deplete hematopoietic stem cells after Pten deletion

Pten deficiency depletes hematopoietic stem cells (HSCs) but expands leukemia-initiating cells, and the mTOR inhibitor, rapamycin, blocks these effects. Understanding the opposite effects of mTOR activation on HSCs versus leukemia-initiating cells could improve antileukemia therapies. We found that the depletion of Pten-deficient HSCs was not caused by oxidative stress and could not be blocked by N-acetyl-cysteine. Instead, Pten deletion induced, and rapamycin attenuated, the expression of p16(Ink4a) and p53 in HSCs, and p19(Arf) and p53 in other hematopoietic cells. p53 suppressed leukemogenesis and promoted HSC depletion after Pten deletion. p16(Ink4a) also promoted HSC depletion but had a limited role suppressing leukemogenesis. p19(Arf) strongly suppressed leukemogenesis but did not deplete HSCs. Secondary mutations attenuated this tumor suppressor response in some leukemias that arose after Pten deletion. mTOR activation therefore depletes HSCs by a tumor suppressor response that is attenuated by secondary mutations in leukemogenic clones.     

Fibronectin dependent upregulation of matrix metalloproteinases in hepatic stellate cells

Activation and transition of hepatic stellate cells (HSCs) to myofibroblast (MFB)-like cells is influenced by growth factors, cytokines and matrix proteins like fibronectin (FN). To examine whether the FN-dependent transition of HSCs is mediated through FN receptor, a marker function, such as matrix metallo-proteinase (MMP) production by HSCs in primary culture was studied. An upregulation of MMP production by HSCs maintained on FN was observed. FN-dependent upregulation of MMPs was significantly reduced when cells were pre-treated with antibodies to alpha5beta1 integrin. Treatment of cells with genistein, a protein kinase C inhibitor completely blocked the gelatinase production by HSCs, indicating that the FN-dependent upregulation of MMPs is mediated through integrins and it involves tyrosine phosphorylation dependent signaling pathways.     

Adult mouse hematopoietic stem cells: purification and single-cell assays

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.     

In Vivo Tracking and Comparison of the Therapeutic Effects of MSCs and HSCs for Liver Injury

Background

Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have been studied for damaged liver repair; however, the conclusions drawn regarding their homing capacity to the injured liver are conflicting. Besides, the relative utility and synergistic effects of these two cell types on the injured liver remain unclear.

Methodology/Principal Findings

MSCs, HSCs and the combination of both cells were obtained from the bone marrow of male mice expressing enhanced green fluorescent protein(EGFP)and injected into the female mice with or without liver fibrosis. The distribution of the stem cells, survival rates, liver function, hepatocyte regeneration, growth factors and cytokines of the recipient mice were analyzed. We found that the liver content of the EGFP-donor cells was significantly higher in the MSCs group than in the HSCs or MSCs+HSCs group. The survival rate for the MSCs group was significantly higher than that of the HSCs or MSCs+HSCs group; all surpassed the control group. After MSC-transplantation, the injured livers were maximally restored, with less collagen than the controls. The fibrotic areas had decreased to a lesser extent in the mice transplanted with HSCs or MSCs+HSCs. Compared with mice in the HSCs group, the mice that received MSCs had better improved liver function. MSCs exhibited more remarkable paracrine effects and immunomodulatory properties on hepatic stellate cells and native hepatocytes in the treatment of the liver pathology. Synergistic actions of MSCs and HSCs were most likely not observed because the stem cells in liver were detected mostly as single cells, and single MSCs are insufficient to provide a beneficial niche for HSCs.

Conclusions/Significance

MSCs exhibited a greater homing capability for the injured liver and modulated fibrosis and inflammation more effectively than did HSCs. Synergistic effects of MSCs and HSCs were not observed in liver injury.