组织工程中应力对骨髓间充质干细胞分化为成骨细胞的影响

骨髓间充质干细胞具有自我复制、未分化的特点,并可在不同条件下分化为中胚层起源的多种细胞,是一种成体多能干细胞。就组织工程而言,良好的种子细胞是组织工程技术的关键,骨髓间充质干细胞的性质决定了其在骨组织工程领域中的重要地位。此外,骨骼系统属于机体的运动系统,承担体重是骨骼的重要功能之一;而且,人体内几乎所有的细胞都会受到力学因素的影响,故有必要研究力学因素对骨髓间充质干细胞诱导分化为成骨细胞的作用,为骨髓间充质干细胞的体外扩增、诱导分化及培养提供一种新途径。     

马的骨髓间充质干细胞诱导为软骨细胞和成骨细胞及其鉴定

本研究旨在将建立的马(Equuscaballus)骨髓间充质干细胞诱导分化为成骨细胞和软骨细胞。通过原代细胞培养获取马的骨髓间充质干细胞,并对第3代(P3)纯化细胞进行干细胞特性鉴定,之后诱导其向不同细胞分化并对诱导分化的细胞进行染色和特异性基因表达的鉴定。实验结果显示,获得的马骨髓细胞表达了干细胞转录因子和间充质干细胞表面标记物,确定获得的细胞为马骨髓间充质干细胞。P3代细胞经诱导培养后由长梭形转变为"骨结节"形态的成骨细胞和"铺路石"形态的软骨细胞。茜素红将诱导的成骨细胞团染成红色,并随着时间的递增红色"骨结节"逐步增大;阿尔新蓝则将蛋白聚糖和透明质酸等含量丰富的诱导细胞染为蓝色,并且随着诱导天数的增加被染成蓝色的软骨细胞逐渐增多,而对照组细胞未见着色。实时荧光定量PCR检测发现,成骨细胞中Col和ALPL基因的表达量随诱导时间的延长发生明显变化;普通PCR结果显示,在诱导的软骨细胞中扩增获得了collagenⅡ、aggrecan和Sox9软骨特异基因,而对照组细胞不表达特异基因。综上所述,本实验建立了马骨髓间充质干细胞并成功将其诱导分化为成骨细胞和软骨细胞,为骨组织缺损修复和软骨...     

影响骨髓间充质干细胞分离效率的若干因素的研究

骨髓间充质干细胞来源有限,数量稀少,极大地限制了其在组织修复、细胞治疗等方面的应用。为了解决细胞数量限制的问题,从提高分离效率的角度对骨髓间充质干细胞的分离过程进行了研究,考察了培养基种类、初次换液时间、起始接种密度对兔骨髓间充质干细胞分离效率的影响。结果表明,α-MEM培养基较DMEM、DMEM-LG培养基更适于兔MSCs的分离;从骨髓中分离得到的单个核细胞以1×106cells／ml的密度接种,接种24h后换液在短时间内得到的MSCs数目最多。采用以上分离条件,能够获得形态均一、可稳定传代10代以上仍保持旺盛增殖能力的MSCs,并具有向骨、脂肪分化能力。     

骨髓间充质干细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的具有高度自我更新能力和多向分化潜能的干细胞群体 ,具有支持造血、多向分化潜能以及在细胞和基因工程中具有潜在应用前景等特点 ,将在医学上具有重要的临床应用价值。     

机械拉伸对骨髓间充质干细胞迁移行为的影响

目的:研究机械拉伸刺激对大鼠骨髓间充质干细胞迁移行为的影响并探讨其相关分子机制。方法:应用单轴机械拉伸加载装置考察不同条件的周期拉伸刺激对大鼠骨髓间充质干细胞迁移行为的影响,采用Transwell和划痕法评价细胞迁移能力,采用明胶酶谱法检测基质金属蛋白酶-2,-9(MMP-2,-9)表达的变化。结果:适宜的拉伸刺激可以明显促进大鼠骨髓间充质干细胞的迁移能力,1 Hz、10%应变拉伸8 h后可以使细胞迁移数量增加到对照组的1.58倍。拉伸刺激诱导骨髓间充质干细胞基质金属蛋白酶-2,-9(MMP-2,-9)表达。抑制剂GM6001抑制了拉伸诱导的MMP-2,-9分泌增加,同时抑制了拉伸刺激对细胞迁移的促进作用。结论:机械拉伸刺激影响大鼠骨髓间充质干细胞的迁移行为,MMP-2,-9在此过程中可能起着重要介导作用。     

骨髓间充质干细胞可塑性研究

骨髓间充质干细胞（bone marrow mesenchymal stem cells,MSCs）具有跨系统、甚至跨胚层分化的特性,称为MSCs的可塑性（plasticity）,成为细胞工程、再生医学中的主要选择细胞。但随着对成体干细胞可塑性质疑的出现,使MSCs是否具有转分化能力,存在着极大的分歧。对MSCs可塑性是否真正存在的进一步明确,越加显得急切和重要,也对干细胞基础理论及其临床应用具有指导性意义。     

丙二醛对小鼠骨髓间充质干细胞成骨分化的影响

为了探索氧化应激活性中间产物丙二醛对小鼠骨髓间充质干细胞成骨分化的影响及机制,体外培养的间充质干细胞经丙二醛处理后,在第4、7、14、21 d分别提取等份细胞进行碱性磷酸酶活性检测,第7 d时提取RNA通过实时定量RT-PCR测定ALP和Runx2/Cbfal mRNA表达,并在第21 d进行von Kossa染色,茜素红染色.研究发现:丙二醛通过降低碱性磷酸酶活性及ALP和Runx2/Cbfal mRNA的表达,抑制矿化骨节形成.这些结果表明:丙二醛可通过抑制ALP和Runx2/Cbfal通路,抑制小鼠骨髓间充质干细胞的成骨分化.     

Periostin表达上调对去势大鼠骨髓间充质干细胞生物学行为的作用

目的:探究Periostin(骨膜蛋白)表达上调对雌性去势大鼠骨髓间充质干细胞(BMSCs)成骨分化、细胞增殖与凋亡特性的作用。方法:通过去势手术建立雌性大鼠骨质疏松模型,待建模成功后分离培养并鉴定BMSCs,利用含有增强型绿色荧光蛋白(EGFP)和大鼠Periostin基因的重组慢病毒转染P3代BMSCs,成骨诱导后鉴定其成骨分化能力改变,流式细胞仪检测其细胞周期以及细胞凋亡率的变化。结果:成功建立骨质疏松模型;荧光显微镜下观察到绿色荧光提示慢病毒载体实现转染并表达目的蛋白;慢病毒转染组BMSCs成骨诱导后ALP及茜素红染色较去势组BMSCs染色加深;慢病毒转染组BMSCs的S期细胞比例为(17.07±0.56)%,显著高于去势组BMSCs的S期细胞比例(8.42±0.02)%,差异具有统计学意义(P0.05);慢病毒转染组BMSCs的细胞凋亡率为(7.3±0.1)%,显著低于去势组BMSCs的凋亡率(12.05±0.55)%,其差异具有统计学意义(P0.05)。结论:Periostin表达上调可提高去势骨髓间充质干细胞的成骨分化及细胞增殖能力,并对其凋亡有抑制作用。     

骨髓间充质干细胞治疗脑梗死

目的:研究骨髓间充质干细胞(MSC)对大鼠脑缺血再灌注损伤的治疗机制。方法:20只Wistar大鼠随机分为对照组和MSC治疗组。应用GFP阳性MSC,再灌注1d后经尾静脉注射MSC(1×106),对照组则注射PBS。采用线栓法建立脑缺血再灌注模型。术后每天由双盲于试验组的研究人员应用爬杆计分法评定大鼠神经功能。缺血2h再灌注8d取脑组织,用免疫组织化学方法检测脑组织中bFGF的表达。结果:MSC治疗组大鼠的神经功能缺损评分明显低于手术组和对照组(P<0.05)。MSC治疗组缺血侧缺血周边区脑组织中观察到GFP阳性与bFGF免疫组化染色阳性细胞。结论:经尾静脉给予的MSC可促进脑缺血再灌注大鼠的运动功能恢复;bFGF表达升高,可能是MSC脑保护作用机制之一。     

Effects of mechanical stimulation on the proliferation of bone marrow-derived human mesenchymal stem cells

To support and enhance thein vitro growth and activity of mesenchymal stem cells (MSCs), the cell culture medium may be supplemented with various proteins and factors to mimic the physiological environment in which the cells optimally proliferate and differentiate. In this study, the effects of mechanical factors on cellular metabolic responses were investigated experimentally using a bioreactor. The effects of various chemical factors, such as growth factors, cytokines, and hormones, were also investigated. Based on previous reports demonstrating the important roles of mechanical factors in the growth and activity of MSCs, we sought to evaluate the effects of mechanical stimuli on the proliferation of bone marrow-derived MSCs using a cell training bioreactor that imposed cyclic mechanical stretch, with parameters of 240 min/day, 0.03 Hz, and 5–15% strain. The application of cyclic stretch (5–15% strain) to the MSCs enhanced their proliferation during the early stage (3 days), but not the late stage (14 days), of batch culture. Mechanical stretch did not increase the release of lactate dehydrogenase (LDH) from the MSCs during culture. Appropriate levels of mechanical stretch (5–10% strain) increased collagen synthesis, but did not alter MSC surface antigen expression. It is thought that the appropriate level of mechanical stretch was able to serve as a potent positive modulator of MSC proliferation during the initial stages of culture.     

The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.     

Mitophagy promotes the stemness of bone marrow-derived mesenchymal stem cells

Previous studies demonstrated that mitochondrial fission arguments the stemness of bone marrow-derived mesenchymal stem cells (BMSCs). Because mitophagy is critical in removing damaged or surplus mitochondrial fragments and maintaining mitochondrial integrity, the present study was undertaken to test the hypothesis that mitophagy is involved in mitochondrial fission-enhanced stemness of BMSCs. Primary cultures of rat BMSCs were treated with tyrphostin A9 (TA9, a potent inducer of mitochondrial fission) to increase mitochondrial fission, which was accompanied by enhanced mitophagy as defined by increased co-staining of MitoTracker Green for mitochondria and LysoTracker Deep Red for lysosomes, as well as the increased co-localization of autophagy markers (LC3B, P62) and mitochondrial marker (Tom20). A mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to promote mitophagy, which was confirmed by an increased co-localization of mitochondrial and lysosome biomarkers. The argumentation of mitophagy was associated with enhanced stemness of BMSCs as defined by increased expression of stemness markers Oct4 and Sox2, and enhanced induction of BMSCs to adipocytes or osteocytes. Conversely, transfection of BMSCs with siRNA targeting mitophagy-essential genes Pink1/Prkn led to diminished stemness of the stem cells, as defined by depressed stemness markers. Importantly, concomitant promotion of mitochondrial fission and inhibition of mitophagy suppressed the stemness of BMSCs. These results thus demonstrate that mitophagy is critically involved in mitochondrial fission promotion of the stemness of BMSCs.     

Differentiation into neurons of rat bone marrow-derived mesenchymal stem cells

Purpose

It has been reported that mesenchymal stem cells (MSCs) can differentiate into neurons as an effect of adding extraneous factors, such as β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanisole. However, many of these compounds could harm MSCs and the human body, which restricts their application. We examined whether MSCs could differentiate into neuron-like cells under the influence of natural growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1, and neurotrophin 3 (NT-3).

Methods

MSCs were collected from rat bone marrow using the plastic adherent selection method, and induced in culture media to which was added different combinations of EGF, bFGF, IGF-1 and NT-3. The shape of the induced cells was observed daily and the differentiated cells were characterized by immunocytochemistry with neural-specific markers.

Result

With bFGF and NT-3 in the medium, the induced cells became slim, gradually developing protruding processes, with parts of them forming net- or ring-like structures. Cells with processes showed expression of microtubule-associated protein 2 (MAP2) and nestin (NES), which was enhanced when bFGF and NT-3 were added in combination. However, with IGF-1 added to the medium, there was no evidence of neurite-like processes or any net- or ring-like structures; the MSCs retained their round or slim shape.

Conclusion

Using natural cytokines in vitro, MSCs successfully differentiated into neuron-like cells. Our study confirms that bFGF and NT-3 exerts a neural-induction effect on the differentiation of MSCs, but that IGF has a rather negative effect on this process.

     

Immunomodulatory effects of bone marrow-derived mesenchymal stem cells in a swine hemi-facial allotransplantation model

Background

In this study, we investigated whether the infusion of bone marrow-derived mesenchymal stem cells (MSCs), combined with transient immunosuppressant treatment, could suppress allograft rejection and modulate T-cell regulation in a swine orthotopic hemi-facial composite tissue allotransplantation (CTA) model.

Methodology/Principal Findings

Outbred miniature swine underwent hemi-facial allotransplantation (day 0). Group-I (n = 5) consisted of untreated control animals. Group-II (n = 3) animals received MSCs alone (given on days −1, +1, +3, +7, +14, and +21). Group-III (n = 3) animals received CsA (days 0 to +28). Group-IV (n = 5) animals received CsA (days 0 to +28) and MSCs (days −1, +1, +3, +7, +14, and +21). The transplanted face tissue was observed daily for signs of rejection. Biopsies of donor tissues and recipient blood sample were obtained at specified predetermined times (per 2 weeks post-transplant) or at the time of clinically evident rejection. Our results indicated that the MSC-CsA group had significantly prolonged allograft survival compared to the other groups (P<0.001). Histological examination of the MSC-CsA group displayed the lowest degree of rejection in alloskin and lymphoid gland tissues. TNF-α expression in circulating blood revealed significant suppression in the MSC and MSC-CsA treatment groups, as compared to that in controls. IHC staining showed CD45 and IL-6 expression were significantly decreased in MSC-CsA treatment groups compared to controls. The number of CD4+/CD25+ regulatory T-cells and IL-10 expressions in the circulating blood significantly increased in the MSC-CsA group compared to the other groups. IHC staining of alloskin tissue biopsies revealed a significant increase in the numbers of foxp3⁺T-cells and TGF-β1 positive cells in the MSC-CsA group compared to the other groups.

Conclusions

These results demonstrate that MSCs significantly prolong hemifacial CTA survival. Our data indicate the MSCs did not only suppress inflammation and acute rejection of CTA, but also modulate T-cell regulation and related cytokines expression.     

Integration potential of mouse and human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.