Mesenchymal stem cells isolated from different sources differentiate into vascular endothelial cells under induction

doi:10.3969/j.issn.2095-4344.2016.36.021

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) can be differentiated into vascular endothelial cells to construct ideal blood vessel grafts in vitro that have wide prospective utility in the clinical treatment of ischemic diseases, vascular engineering and regeneration medicine.
OBJECTIVE: To review the recent research progress and related issue in biological characteristics of MSCs as well as differentiation of MSCs isolated from different sources into vascular endothelial cells.
METHODS: The first authors retrieved PubMed, Sciencedirect and Medline databases for relevant articles published from January 2000 to June 2015. The key words were “mesenchymal stem cells; vascular endothelial cell; cell differentiation” in English. Initially, 156 articles were retrieved, and finally 51 articles were included in result analysis.
RESULTS AND CONCLUSION: The MSCs sources are abundant. The research about bone marrow-derived MSCs is the earliest and maximum, but with the increasing donor age, their proliferation and differentiation ability decreases. MSCs from umbilical cord, placenta and amniotic membrane are easy to obtain, and meanwhile both neonatal and maternal does not feel any pain and psychological burden. Given these, these cells are more acceptable for recipients, and there are no ethical, moral and legal disputes. MSCs from the umbilical cord blood are abundant with weak cell immunogenicity and low viral and bacterial contamination probability. However, the clinical application of amniotic fluid marrow-derived MSCs is limited because of its sampling methods.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Endothelial Cells, Cell Differentiation, Tissue Engineering

CLC Number:

R318

Wang Jun-li, Xu Hui-fang, Feng Li, Wei Dan, Chen Guo-hua . Mesenchymal stem cells isolated from different sources differentiate into vascular endothelial cells under induction[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(36): 5466-5472.

Figures/Tables 1

2.1 间充质干细胞的生物学特征 2.1.1 间充质干细胞的定义 Friedenstein[3]最早在骨髓中发现了间充质干细胞，1976年其研究报道：骨髓提取物中的小部分黏附细胞可在一定的培养条件下分化成类似骨或软骨的细胞集群，推测这些类似骨或软骨的细胞集群可能为间质细胞的前体部分，1991年Caplan[4]将这些细胞集群命名为间充质干细胞。近几年来因其具有高度再生功能、多向分化潜能、造血支持功能及免疫调控等特点而受到众多研究者的广泛关注[5-6]。间充质干细胞没有特定的细胞表面标志物，不同实验室的分离扩增方法、生物学特征和功能应用无法统一，实验结果也不具有可比性[7]。为了更好的鉴定间充质干细胞，2006年国际细胞疗法协会认定间充质干细胞需满足以下3个标准：①间充质干细胞在标准组织培养条件下能贴壁生长；②细胞表达一些特定的表面抗原，高表达CD90、CD73、CD105，不表达CD14/CD11b、CD79a/CD19、CD45、CD34、HLA class II；③多向分化潜能，指在体外特定的诱导条件下可以向成骨细胞、脂肪细胞、软骨细胞等不同种类的组织细胞分化[8]。 2.1.2 间充质干细胞的分离目前常用的间充质干细胞分离方法有4种：全骨髓贴壁法[9]、组织消化法[10]、密度梯度分选法[11]、免疫磁珠分选法[12]。全骨髓贴壁法具有操作简单，获得的间充质干细胞数量较多且细胞贴壁时间较早，细胞分化潜能保存好等优点，但为了纯化需多次换液传代，换液传代后细胞间难免残留消化间充质干细胞的胰消化酶、EDTA等物质，其可以影响间充质干细胞的增殖和分化潜能，甚至会导致间充质干细胞生物学特征消失。组织消化法短时间内能高效、稳定地获得大量存活的间充质干细胞，且间充质干细胞生物学特征与其体内相似并能反映其生长特性，但是此种方法步骤繁多、容易污染，难以把握合适的消化时间，所以会直接影响获得的间充质干细胞的纯度，甚至会破坏其细胞结构、增殖及分化能力，获得异种细胞集群。Rosca等[13]运用密度梯度分选法在1.067-1.070 g/mL的密度区间发现间充质干细胞，说明合适密度的介质可分离获得纯度较高的间充质干细胞。MACS、FACS组织培养瓶铺展贴壁(Cellector培养瓶)和亲和吸附柱(CEPRATE)为目前常用的免疫磁珠分选方法[12]，这些方法都具有特异性分离出高纯度目的细胞的优点，但也有技术要求高、实验设备昂贵、易污染等不足。目前研究表明CD133、CD271、CD105和CD11b作为间充质干细胞分离的表面标记物[14-16]，仍需一些阴性标记物作为辅助条件，比如CD3、CD19、CD14、CD38、CD34、CD66b等，如果只用单一特异性标记物理论上会造成其他间充质干细胞亚群的丢失；如果用多种特异性标记物则显著提高研究成本。Phinney等[17]设计了一种新的分离方法——免疫耗竭法(immunodepletion)，此方法从大鼠骨髓中分离获得去掉血液系及内皮系细胞后的成纤维基质细胞，因为不用长时间的体外贴壁增殖，所以不会产生永生细胞系。Pierini等[18]运用BD真空采血细胞制备管收集到了间充质干细胞。Xing等[19]通过组织块改良法和直接铺种法相结合，可从人骨髓中简单高效的提取出间充质干细胞。 2.1.3 间充质干细胞的鉴定及表面标志物细胞表面标记物可以体现细胞的一些基本特征，与其特定的功能相关[20-21]，不同组织来源的间充质干细胞虽然保持了间充质干细胞的表面标记，但相互之间仍有差异性[22-23]。如脐带来源的间充质干细胞表达CD29、CD44、CD51、CD105、SH2、SH3，不表达CD34、CD45[24]；胎盘来源的间充质干细胞表达的标记物有CD105、SH-2、SH-3及SH-4，同时表达部分胚胎干细胞表面标记物，比如SSEA-4、TRA-1-61及TRA-1-80等，不表达造血细胞、内皮细胞及滋养层细胞的特殊细胞标记物[25]；脂肪来源的间充质干细胞表达CD49，不表达CD106，而骨髓来源的间充质干细胞表达CD106，不表达CD49[26]。Lee等[27]应用密度梯度离心法从脐血中分离获得间充质干细胞，证明脐血来源的间充质干细胞不表达CD90，低表达CD166，有别于骨髓来源的间充质干细胞。Tantrawatpan等[28]和Teotia等[29]研究也表明，不同来源组织间充质干细胞的表面标记物、免疫表型等生物功能均有差异性，这直接影响干细胞在再生、组织工程、细胞修复治疗等方面的临床应用。 2.2 不同组织来源间充质干细胞诱导分化为血管内皮细胞 2.2.1 间充质干细胞诱导分化为血管内皮细胞的理论基础研究表明间充质干细胞具有部分血管内皮细胞的表型。Haynesworth等[30]1992年首次用免疫小鼠制备单克隆抗体SH2，SH2仅识别骨髓来源的间充质干细胞，而对造血细胞无反应，因此SH2被认为是间充质干细胞的特征性抗原。Barry等[31]1999年研究发现endoglin蛋白(CD105)，endoglin蛋白主要存在于血管内皮细胞中，其功能作用与血管发育相关，而endoglin蛋白是Barry等[31]运用SH2单克隆抗体从人间充质干细胞中免疫沉淀出来的，因此间充质干细胞表达此特异性抗原说明人间充质干细胞与血管内皮细胞存在着一定的内在联系。目前一些研究检测发现细胞间黏附分子(ICAM-1)、血管内皮细胞黏附分子(VCAM-1)和整合素(Integrinβ3)参与了血管的生成，与血管内皮细胞有关。其他一些研究结果也提示间充质干细胞与血管的形成有关。例如Tremain等[32]分析人间充质干细胞RNA发现干细胞中存在着血管内皮细胞的某些特征性基因，如果人间充质干细胞培养在可以向血管内皮细胞分化的微环境下，干细胞的这些特征性血管内皮细胞基因将被激活，并可能表达出相关性蛋白，从而使干细胞直接稳定地分化为血管内皮细胞。 2.2.2 骨髓来源的间充质干细胞及其分化条件骨髓来源的间充质干细胞是目前研究最多、最深入的间充质干细胞，关于其诱导分化为血管内皮细胞的文献报道有很多。比如Jianguo等[33]运用经典的血管内皮细胞生长因子方案对骨髓间充质干细胞进行内皮细胞分化并获得成功；Yan等[34]在高密度低扩增条件下，加入血管内皮细胞生长因子和牛垂体提取物对间充质干细胞进行诱导培养，可诱导分化出血管内皮细胞。Raida等[35]还认为血管内皮细胞生长因子诱导骨髓间充质干细胞可促进重组人骨形成蛋白2增加，重组人骨形成蛋白2再反作用于骨髓间充质干细胞，从而加速骨髓间充质干细胞向血管内皮细胞转化。 2.2.3 脐带来源的间充质干细胞及其分化条件脐带来源的间充质干细胞是一种大部分存在于脐带华通胶和血管周围组织中的多能成体干细胞。2003年Mitchell等[36]首次成功地从人脐带基质中分离培养出脐带来源间充质干细胞，同年Romanov等[37]也从脐带华通胶、血管周围组织、人脐静脉内皮和人脐静脉内皮下分别培养获得脐带来源的间充质干细胞，同时建立了脐带来源间充质干细胞植块法和酶解法的分离培养体系。人血管生成素1是脐带来源间充质干细胞诱导分化为血管内皮细胞过程中不可或缺的调节因子。2006年Baffert等[38]提出血管生成素1(Ang-1/Tie系统)能与血管内皮细胞特异性表达的酪氨酸激酶受体结合，通过信号转导来使Ang-1/Tie系统磷酸化发挥其调节人脐带间充质干细胞诱导分化为血管内皮细胞的作用。因为Ang-1/Tie系统可缓解血管渗漏，阻碍新生血管的形成，所以其在调节人脐带间充质干细胞诱导分化为血管内皮细胞过程中的地位是不可或缺的，这一发现也从理论上验证了脐带来源间充质干细胞可以直接诱导分化为血管内皮细胞。 2.2.4 脐血来源的间充质干细胞及其分化条件 Erices等[39]2000年第1次报道了脐血中可以分离培养出间充质干细胞。Gang等[40]发现在血管内皮细胞生长因子、表皮生长因子和氢化可的松的培养条件下，脐血来源的间充质干细胞可诱导分化具有血管内皮细胞形态的细胞集群，通过流式细胞仪、RT-PCR和免疫荧光法分析检测后发现脐血来源间充质干细胞诱导分化的细胞集群可表达FIT-1、FIK-1、血管内皮细胞黏附分子1、血管内皮钙黏蛋白、酪氨酸激酶1，2(Tie-1、Tie-2)、vWF等血管内皮细胞特异性的表面标志物，且诱导分化的细胞集群可以形成血管管状结构；2007年Wu等[41]同样证实了脐血来源间充质干细胞能在含有血管内皮细胞生长因子和碱性成纤维生长因子的培养基微环境中，诱导分化出血管内皮细胞，经检测证实诱导分化出的细胞群落可以表达血小板内皮细胞黏附分子(PECAM)和CD34等血管内皮细胞的特异性蛋白。目前关于脐血来源间充质干细胞存在较大的分歧，主要集中于脐血来源间充质干细胞容易丧失SH-2、SH-3和SH-4等间充质干细胞的特异性表面标记物、多向分化潜能低和脐血中含有间充质干细胞个数少(脐血中每105-108个单核细胞才含有1个间充质干细胞)等方面，但脐血来源较充足，免疫原性较弱，且由于病毒、细菌等难以透过胎盘屏障使其成分受污染的概率较低，这些都是其临床实际应用中的先天优势，同时脐血有望作为间充质干细胞的自体来源，成为临床上间充质干细胞的最佳组织来源。 2.2.5 脂肪来源间充质干细胞及其分化条件脂肪来源间充质干细胞研究起步较晚，直到2001年Zuk等[42]才首次从人类脂肪组织中分离出一种干细胞，这种干细胞在形态结构上与骨髓间充质干细胞相似，且具有多向分化潜能，故将其命名为脂肪间充质干细胞。Amos等[43]首先建立缺血肠系膜大鼠模型，然后体内分离获得脂肪来源间充质干细胞并将其在体外进一步培养、扩增，同时用免疫荧光法对其进行标记，最后注射至缺血的肠系膜大鼠模型体内。经过观察检测发现大量脂肪来源间充质干细胞表达血管管状系统的亚型和特异性的表面标志物。Konno等[44]将脂肪来源间充质干细胞注入至下肢缺血的裸鼠模型体内也得到了与Amos等[43]研究相同的结论，同时论证了脂肪来源间充质干细胞可诱导分化为血管内皮细胞。 2.2.6 胎盘来源间充质干细胞及其分化条件 2004年Zhang等[45]研究报道胎盘灌流液可以分离培养出一种成体干细胞，研究发现其生物学特征和功能等方面与间充质干细胞相似。近年来有动物实验表明胎盘来源间充质干细胞体外可以分化为血管内皮细胞，并促进血管生成，且与丝素蛋白/羟基磷灰石复合材料联合移植能够较好的促进血管生成，还可提高周围性动脉病模型小鼠的运动能力[46]。同样有最新研究显示，成纤维细胞生长因子2及血小板源性生长因子BB转基因的人胎盘来源间充质干细胞可以促进血管再生[47]。 2.2.7 羊水来源间充质干细胞及其分化条件近年来关于羊水来源间充质干细胞文献报道较少，Kaviani等[48]首次报道了羊水细胞的一个亚群具有间充质干细胞的特性，在体外比成体间充质干细胞增殖更快，称这个亚群细胞为羊水来源间充质干细胞。In’t Anker等[49]研究发现孕中期(17-22周)和孕晚期(平均38+周)羊水中都能够分离培养出间充质干细胞，但孕中期(17-22周)羊水中分离培养出间充质干细胞的成功率远高于孕晚期(平均38+周)。Mauro等[50]和Antonucci等[51]研究证实羊水来源间充质干细胞在特定的情况下可以分化为血管内皮细胞，但羊水来源间充质干细胞诱导分化为血管内皮细胞的条件有待进一步研究证实。"

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