造血干细胞在心脏中的归巢与分化

利用造血干细胞治疗心脏损伤尤其是由退化、缺血和炎症引起的损伤已被认为是新近发展的重要治疗心脏病变的手段之一。造血干细胞在生理及病理条件下均能归巢于心肌,并能被动员至外周血且参与心脏的自稳态和组织维持。现就造血干细胞在心脏中归巢与分化的机制以及造血干细胞在治疗心脏病变中的应用进行简要综述。     

Hematopoietic differentiation of rhesus monkey embryonic stem cells

Several lines of embryonic stem cells (ESC) have been established from rhesus monkey blastocysts. We have examined two of these cell lines for their potential for generating hematopoietic progenitors in cell culture, and we identified culture conditions, including supplementation with bone morphogenetic proteins (BMP), that result in hematopoietic differentiation of rhesus ESC with high efficiency. We have also characterized the resulting hematopoietic progenitor cells for their patterns of gene expression, as compared to those of hematopoietic progenitor cells harvested from rhesus monkey bone marrow. Of more than 60 genes examined in this manner, CD34+/CD38- cells derived from embryonic stem cells and those obtained from bone marrow demonstrated very similar patterns of gene expression. However, with integrin alphaL, IL-6 receptor, and flt-3 gene expression was greatly diminished or absent in CD34+/CD38- cells derived from the ESC, whereas the bone marrow-derived progenitors showed substantial expression of all of these genes. When the same type of comparison was done with mouse (D3 and CCE) as well as human (H1) embryonic stem cells, in each case comparing ESC-derived hematopoietic progenitors with those harvested from bone marrow, the only consistent deficiency of gene expression was that of flt-3. In hematopoietic precursors derived from mouse ESC, globin-gene expression has previously been shown to be a useful index of the embryological maturity of the cells, and we also examined globin-gene expression in rhesus monkey ESC-derived hematopoietic precursor cells, using a semiquantitative technique. CD34+/CD38- cells demonstrated expression of the epsilon- and gamma-globin genes, but negligible levels of beta globin, suggesting that these cells were at the developmental stage in which the yolk sac and fetal liver are the primary sites of hematopoiesis.     

The mammary microenvironment alters the differentiation repertoire of neural stem cells

A fundamental issue in stem cell biology is whether adult somatic stem cells are capable of accessing alternate tissue sites and continue functioning as stem cells in the new microenvironment. To address this issue relative to neurogenic stem cells in the mouse mammary gland microenvironment, we mixed wild-type mammary epithelial cells (MECs) with bona fide neural stem cells (NSCs) isolated from WAP-Cre/Rosa26R mice and inoculated them into cleared fat pads of immunocompromised females. Hosts were bred 6–8 weeks later and examined postinvolution. This allowed for mammary tissue growth, transient activation of the WAP-Cre gene, recombination, and constitutive expression of LacZ. The NSCs and their progeny contributed to mammary epithelial growth during ductal morphogenesis, and the Rosa26-LacZ reporter gene was activated by WAP-Cre expression during pregnancy. Some NSC-derived LacZ⁺ cells expressed mammary-specific functions, including milk protein synthesis, whereas others adopted myoepithelial cell fates. Thus, NSCs and their progeny enter mammary epithelium–specific niches and adopt the function of similarly endowed mammary cells. This result supports the conclusion that tissue-specific signals emanating from the stroma and from the differentiated somatic cells of the mouse mammary gland can redirect the NSCs to produce cellular progeny committed to MEC fates.     

Myeloma plasma cells alter the bone marrow microenvironment by stimulating the proliferation of mesenchymal stromal cells

Multiple myeloma is an incurable hematologic cancer characterized by the clonal proliferation of malignant plasma cells within the bone marrow. Numerous studies suggest that the myeloma plasma cells occupy and alter the stromal tissue of the bone marrow as a means of enhancing their survival and growth. However, the nature and magnitude of the changes to the stromal cell tissue remain to be determined. In this study, we used mesenchymal stromal cell and osteoblast-related cell surface marker expression (STRO-1 and alkaline phosphatase, respectively) and flow cytometry to enumerate mesenchymal stromal cell and osteoblast numbers in bone marrow recovered from myeloma patients at the time of diagnosis. Using this approach, we identified an increase in the number of STRO-1 positive colony forming mesenchymal stromal cells and a concomitant decrease in alkaline phophatase osteoblasts. Notably, this increase in mesenchymal stromal cell numbers correlated closely with plasma cell burden at the time of diagnosis. In addition, in comparison with the osteoblast population, the STRO-1+ mesenchymal stromal cell population was found to express higher levels of plasma cell- and osteoclast-activating factors, including RANKL and IL-6, providing a mechanism by which an increase in mesenchymal stromal cells may promote and aid the progression of myeloma. Importantly, these findings were faithfully replicated in the C57BL/KaLwRij murine model of myeloma, suggesting that this model may present a unique and clinically relevant system in which to identify and therapeutically modulate the bone microenvironment and, in turn, alter the progression of myeloma disease.     

Hematopoietic stromal cells and megakaryocyte development

Abstract

The hematopoietic microenvironment, and in particular the hematopoietic stromal cell element, are intimately involved in megakaryocyte development. The process of megakaryocytopoiesis occurs within a complex bone marrow microenvironment where adhesive interactions, chemokines, as well as cytokines play a pivotal role. Here we review the effect of stromal cells and cytokines on megakaryocytopoiesis with the aim of exploring new therapeutic strategies for platelet recovery after hematopoietic stem cell transplantation (HSCT).     

Hematopoietic stromal cells and megakaryocyte development

The hematopoietic microenvironment, and in particular the hematopoietic stromal cell element, are intimately involved in megakaryocyte development. The process of megakaryocytopoiesis occurs within a complex bone marrow microenvironment where adhesive interactions, chemokines, as well as cytokines play a pivotal role. Here we review the effect of stromal cells and cytokines on megakaryocytopoiesis with the aim of exploring new therapeutic strategies for platelet recovery after hematopoietic stem cell transplantation (HSCT).     

Total body irradiation tremendously impair the proliferation,differentiation and chromosomal integrity of bone marrow-derived mesenchymal stromal stem cells

Total body irradiation (TBI) is frequently used in hematopoietic stem cell transplantation (HSCT) and is associated with many complications due to radiation injury to the normal cells, including normal stem cells. Nevertheless, the effects of TBI on the mesenchymal stromal stem cell (MSC) are not fully understood. Bone marrow-derived MSCs (BM-MSCs) isolated from normal adults were irradiated with 200 cGy twice daily for consecutive 3 days, a regimen identical to that used in TBI-conditioning HSCT. The characteristics, differentiation potential, cytogenetics, hematopoiesis-supporting function, and carcinogenicity of the irradiated BM-MSCs were then compared to the non-irradiated control. The irradiated and non-irradiated MSCs shared similar morphology, phenotype, and hematopoiesis-supporting function. However, irradiated MSCs showed much lower proliferative and differentiative potential. Irradiation also induced clonal cytogenetic abnormalities of MSCs. Nevertheless, the carcinogenicity of irradiated MSCs is low in vitro and in vivo. In parallel with the ex vivo irradiation experiments, decreased proliferative and differentiative abilities and clonal cytogenetic abnormalities can also be found in MSCs isolated from transplant recipients who had received TBI-based conditioning previously. Thus, TBI used in HSCT drastically injury MSCs and may contribute to the development of some long-term complications associated with clonal cytogenetic abnormality and poor adipogenesis and osteogenesis after TBI.     

心肌微环境对骨髓间充质干细胞的诱导分化作用

目的通过与心肌细胞(CMs)共同培养和采用含有CMs裂解液的培养基两种方法体外模拟心肌微环境,探讨心肌微环境对骨髓间充质干细胞(MSCs)分化的诱导作用。方法自新生乳鼠的心脏分离CMs,自成年大鼠的骨髓分离MSCs,将MSCs与CMs按1∶4的比例共同培养1周,观察细胞形态的改变,并通过免疫荧光方法检测共培养后MSCs表达心脏特异性肌钙蛋白T(cTnT)及CD31的情况;将分离的CMs反复冻融制成CMs裂解液,将MSCs在含有4倍CMs裂解液的培养基中培养1周,观察细胞形态的改变,并通过免疫化学方法检测MSCs表达心脏特异性肌钙蛋白T(cTnT)及CD31的情况;以仅用普通培养基所培养的MSCs作为对照。结果与CMs共培养的MSCs和CMs裂解液培养的MSCs逐渐伸展变长,形成肌细胞形态,培养1周后经抗cTnT和抗CD31免疫染色均呈阳性;对照组MSCs没有明显形态变化,抗cTnT和抗CD31免疫染色成阴性。结论与CMs共培养和采用含有CMs裂解液的培养基两种方法均可在体外模拟心肌微环境,诱导MSCs向心肌样细胞和内皮样细胞方向分化。     

Hematopoietic stem cells: effect of preirradiation, bleeding, and erythropoietin on thrombopoietic differentiation.

A method of measuring differentiation of stem cells towards platelets is described using syngeneic bone marrow injected into lethally irradiated mice. Fourteen days after such injection, the platelet counts are found to be proportional to the number of bone marrow cells injected and can be used as a measure of platelet progenitors. Perturbation of the milieu in which the transplanted marrow is placed by host preirradiation, bleeding, or erythropoietin administration leads to enhanced thrombopoiesis. It has been shown previously that similar perturbation favors erythropoiesis at the expense of granulopoiesis. The data from these and other experiments appear to be consistent, with a model of the stem cell compartment as a continuum with proliferative activity increasing as commitment is restricted. These functions vary inversely with the capacity for self-renewal. The various stem cell assays measure different ranges of stem cells, but overlap within this continuum.     

Hematopoietic stem cells and aging.

The question of whether hematopoietic stem cells are altered in aging has been the subject of considerable controversy for over two decades. The substantial advancement of knowledge on hematopoietic stem cells and developmental hematology in the last few years has reopened this issue for critical analysis. Dynamic changes have been noted regarding the anatomic site and the function of hematopoietic cells, from the early embryo to old age. Whereas basal hematopoietic potential is maintained in aging. the capacity for recovery from hematological stress and for stem cell self-renewal appears to decline gradually. A distinction is thus made between the steady-state hematopoiesis in aging and the developmental potential of stem cells. The establishment of proper tools to identify and to study purified stem cells and committed cell populations offers a direct approach to further elucidate aging across the axis from primitive stem cells to the mature blood cells. The present article represents a brief review of this area.     

Intestinal stem cells and the colorectal cancer microenvironment

Colorectal cancer(CRC)remains a highly fatal condition in part due to its resilience to treatment and its propensity to spread beyond the site of primary occurrence.One possible avenue for cancer to escape eradication is via stem-like cancer cells that,through phenotypic heterogeneity,are more resilient than other tumor constituents and are key contributors to cancer growth and metastasis.These proliferative tumor cells are theorized to possess many properties akin to normal intestinal stem cells.Not only do these CRC"stem"cells demonstrate similar restorative ability,they also share many cell pathways and surface markers in common,as well as respond to the same key niche stimuli.With the improvement of techniques for epithelial stem cell identification,our understanding of CRC behavior is also evolving.Emerging evidence about cellular plasticity and epithelial mesenchymal transition are shedding light onto metastatic CRC processes and are also challenging fundamental concepts about unidirectional epithelial proliferation.This review aims to reappraise evidence supporting the existence and behavior of CRC stem cells,their relationship to normal stem cells,and their possible dependence on the stem cell niche.     

Hematopoietic stem cells and the aging hematopoietic system

The etiology of the age-associated pathophysiological changes of the hematopoietic system including the onset of anemia, diminished adaptive immune competence, and myelogenous disease development are underwritten by the loss of normal homeostatic control. As tissue and organ homeostasis in adults is primarily mediated by the activity of stem and progenitor cells, it has been suggested that the imbalances accompanying aging of the hematopoietic system may stem from alterations in the prevalence and/or functional capacity of hematopoietic stem cells (HSCs) and progenitors. In this review, we examine evidence implicating a role for stem cells in the aging of the hematopoietic system, and focus on the mechanisms suggested to contribute to stem cell aging.     

急性肾损伤微环境对培养骨髓间充质干细胞分化及分裂增殖的影响

目的:观察体外模拟急性肾损伤(acute kidney injury,AKI)的微环境下,小鼠骨髓间充质干细胞(mouse mesenchymal stem cells,mMSCs)分化及分裂增殖情况。方法:采用夹闭雄性C57BL/6小鼠双侧肾蒂30min再开放30min的方法制作缺血再灌注(I/R)性AKI鼠模型,即刻取双侧肾脏皮质制作I/R肾脏匀浆上清。抽取C57BL/6小鼠的骨髓,经Percoll密度梯度离心联合贴壁培养法分离纯化出mMSCs,以流式细胞仪鉴定。取扩增3代的mMSCs分组培养:(1)对照组:含10%胎牛血清的低糖DMEM培养基;(2)干预组:含10%胎牛血清的低糖DMEM培养基+I/R肾脏匀浆上清。诱导1d、3d、5d、7d后倒置显微镜下观察细胞形态学变化;透射电镜观察细胞超微结构;流式细胞仪检测角蛋白18(cytokeratin18,CK18);CCK-8法检测培养mMSCs的增生;TUNEL法检测mMSCs凋亡。结果:分离获得的P3-mMSCs高表达CD29和CD44,低表达CD34和CD45。与对照组长梭形细胞相比,干预组第3天可见部分细胞为椭圆形、短梭形,至第7天大部分细胞呈圆形、椭圆形、短胖梭形;透射电镜也观察到胞质内开始出现较多的粗面内质网、溶酶体、线粒体。流式细胞仪检测发现,对照组mMSCs内仅有极微量CK18表达,而干预组CK18阳性表达率显著增加。经I/R肾脏匀浆上清干预后,不同时间点mMSCs的增殖效应均显著减弱,而TUNEL检测显示胞核染色阳性的细胞百分比有显著升高(P0.01)。结论:体外模拟的AKI微环境可诱导mMSCs部分分化为肾小管上皮样细胞,但同时也会导致培养的mMSCs凋亡,增殖能力减弱,进而减少了可肾向分化的mMSCs数量,推测这可能是MSCs体内移植促肾修复能力有限的原因之一。     

小鼠骨髓基质干细胞的鉴定及体外诱导分化为肝细胞的可行性

目的:分离、鉴定小鼠骨髓基质干细胞(MSCs)并探讨在体外多种细胞因子的诱导下分化为肝细胞的可行性.方法:获取小鼠骨髓干细胞,进行体外贴壁培养、纯化,观察不同传代次数细胞形态特点.流式细胞法检测不同传代细胞的表面标志物CD45和CD90.分离后的MSCs再经含有HGF,FGF-4,EGF三种细胞因子的诱导体系继续培养21 d,分别以半定量逆转录聚合酶链反应(RT-PCR)和Western blot方法检测诱导后细胞的白蛋白(ALB)、细胞角化蛋白18(Cg18)、以及甲胎蛋白(AFP)在基因和蛋白水平的表达.结果:培养的骨髓干细胞随传代次数增多细胞形态趋向为长梭形.传代到第5代,基质干细胞的表面标志CD90阳性细胞从原代的25.42%增加到93.47%,造血干细胞的表面标志CD45表达阳性细胞从原代的86.49%降低到2.77%.通过RT-PCR可检测出诱导第7天细胞表达AFP mRNA,ALB mRNA及CK18 mRNA;通过Western blot可检测出诱导第21天的细胞表达ALB和CK18.结论:小鼠MSCs可以在体外被有效地分离纯化,可以被诱导为表达肝细胞表面标志的肝细胞样细胞.     

The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.