Wharton’s jelly mesenchymal stem cells: Biological characteristics and therapeutic implications for cardiovascular diseases

doi:10.3969/j.issn.2095-4344.2013.23.020

Abstract

Abstract:

BACKGROUND: Wharton’s jelly mesenchymal stem cells are one type of perinatal stem cells which are capable of self-renewal and multi-differentiation. They are characterized by strong proliferative capability, low immunogenicity, abundant source, ease of production in industry, and free of ethical restriction.
OBJECTIVE: To review the biological characteristics of Wharton’s jelly mesenchymal stem cells and the application in cardiovascular disease treatment.
METHODS: A computer-based online retrieval of Weipu database and PubMed database was performed to search papers using the key words “Wharton’s jelly, umbilical cord, mesenchymal stem cell" both in Chinese and English. All included 69 papers can accurately reflect and describe the biological characteristics of Wharton’s jelly mesenchymal stem cells and the application in cardiovascular disease treatment.
RESULTS AND CONCLUSION: Wharton’s jelly mesenchymal stem cells bear a striking resemblance to embryonic stem cells while maintaining the properties of bone marrow mesenchymal stem cells. In addition, the morphology of Wharton’s jelly mesenchymal stem cells fits itself for the strong capacities for proliferation, migration and differentiation. Furthermore, Wharton’s jelly mesenchymal stem cells, which express more angiogenesis- and growth-related genes, are more suitable to treating ischemic lesions and reducing fibrosis and scarring. Wharton’s jelly mesenchymal stem cells, being hypoimmunogenic and non-tumorigenic, have the potential for safe cell-based therapies. However, there have been still some problems to be solved in the research of Wharton’s jelly mesenchymal stem cells, such as cell isolation, culture in vitro, grow/differentiation in vivo and clinical applications.

Key words: stem cells, stem cell academic discussion, umbilical cord, Wharton&, rsquo, sjelly, mesenchymal stem cells, biological characteristics, gene expression, protein secretion, immunoregulation, cardiovascular disease, regenerative medicine

CLC Number:

R318

Zhang Wei, Liu Xiao-cheng. Wharton’s jelly mesenchymal stem cells: Biological characteristics and therapeutic implications for cardiovascular diseases[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(23): 4320-4327.

Figures/Tables 1

2.1 来源目前已知人脐带中有4种间充质干细胞的来源。其一是沃顿胶体，其二是脐血管周，其三是脐带血，其四是脐静脉内皮下膜[30] ，见图1。其中，沃顿胶体是当前脐带源间充质干细胞的主要来源。原始的间充质干细胞在胚胎形成期间(在胚胎第10.5天前)，当通过发育中的脐带向主动脉-性腺-中肾区第一次迁移和从胎盘再次通过早期脐带向胎儿肝脏第二次迁移时陷于结缔组织的基质中，并在妊娠期间始终存留于此，并最终构成沃顿胶体间充质干细胞[32] 。沃顿胶体间充质干细胞表达胚胎干细胞的标记物Tra-1-60、Tra-1-81、SSEA-1、SSEA-4、碱性磷酸酶甚至能在体外形成类胚体[33] 。此外，沃顿胶体间充质干细胞还能表达多潜能干细胞的标记物Oct-4、Sox-2和Nanog[18,28,34-35] ，但与胚胎干细胞相比水平相对较低[33，36] 。如此，沃顿胶体间充质干细胞不仅保持成体间充质干细胞的特性，而且与胚胎干细胞还具有相似性。 2.2 分离与体外培养目前已有数种技术被用于沃顿胶体间充质干细胞的分离和原代培养。这些技术在一定程度上能影响所分离细胞的数量和质量。Salehinejad等[37]比较了4种分离细胞的方法：3种酶的方法；胶原酶-透明质酸酶-胰蛋白酶法，胶原酶-胰蛋白酶法和胰蛋白酶法，以及1种外植培养的方法。所有方法均能成功地分离出沃顿胶体间充质干细胞，胶原酶-胰蛋白酶法所分离的细胞密度高于其他组且多能干细胞标记(诸如C-kit和Oct-4)的表达比率更高，而外植细胞分离法所生成的细胞增殖速率和活性较其他方法更高。De Bruyn等[38]报道了一种不用酶解和解剖而仅基于间充质干细胞对塑料的黏附性而从沃顿胶体分离间充质干细胞的方法。此方法能从所有人脐带中分离出间充质干细胞，且第2代平均获得细胞1.4×108，而第3代则超过7×109。Nekanti等[39]发现DMEM-KO和DMEM-F12与其他支持沃顿胶体间充质干细胞在体外扩增的培养基相比更优。每条脐带 15 d内在DEMO-KO中增殖可超过1×108，且在培养基中加入碱性成纤维细胞生长因子不影响HLA-DR和CD44的表达水平[40] 。与常氧下培养的沃顿胶体间充质干细胞相比，在缺氧条件下沃顿胶体间充质干细胞的累积细胞群体倍增值与更短的细胞群体倍增时间表明其增殖能力更高[41] 。周建等[42]报道经50 Hz 3.6 mT的正弦交变电磁场处理亦能促进沃顿胶体间充质干细胞的增殖。 2.3 细胞形态与活性在组织学上，沃顿胶体间充质干细胞为成纤维细胞样细胞，呈漩涡状生长，细胞核大而不规则，核仁明显。朱少芳等[43]报道在透射电镜下沃顿胶体间充质干细胞的细胞器丰富，核浆比例大，甚至可见多个核仁，表明细胞代谢活跃，分化程度低，且细胞表面见微绒毛，可增加细胞的吸附能力和吸收能力。陈丽等[44]在原子力显微镜下观察到沃顿胶体间充质干细胞以长梭形为主，细胞骨架丝明显，并形成网状连接，这与其强大的增殖、迁移、分化功能相适应。Nekanti等[41]报道，在缺氧下培养沃顿胶体间充质干细胞的早期和晚期传代细胞中大的、扁平的细胞比例较常氧培养下高。在缺氧下细胞大小增大可能是由于对低氧的自然反应，增加的表面积使氧的弥散率得以增高。 Garzon等[45]发现沃顿胶体间充质干细胞从第1代到第3代细胞活性逐渐减低，接着直到第6代活性又逐渐增高，最后从第6代到第10代再次减低。最高的细胞活性水平相当于第5代和第6代。基因表达分析证实，细胞活性与调节凋亡细胞死亡的基因显著相关，尤其是促凋亡的FASTKD2、BNIP3L基因和抗凋亡的TNFAIP8和BCL2L2基因，提示在多代培养中的沃顿胶体间充质干细胞中可能有着复杂的生死平衡状态。 2.4 基因表达与分泌蛋白组学沃顿胶体间充质干细胞不仅表达作为多能干细胞标记的Oct-4、Nanog和Sox-2的mRNAs，而且还在mRNA和蛋白质水平表达起免疫调节作用的HLA-G[46] 。Nekanti等[41]通过比较缺氧和常氧下的沃顿胶体间充质干细胞的基因表达谱，证实了一种差异性基因表达的模式，数种干细胞标记和早期中胚层和内皮基因诸如DESMIN、CD34、ACTC在缺氧下被上调，提示在缺氧条件下体外培养沃顿胶体间充质干细胞导致向中胚层或内皮分化的结果。在早期和晚期的传代细胞中，沃顿胶体间充质干细胞较骨髓间充质干细胞高表达Nanog、DNMT3B和GABRB3等胚胎干细胞、多能干细胞和早期内胚层细胞的标记[47] 。沃顿胶体间充质干细胞与骨髓间充质干细胞间趋化因子和生长因子的表达亦存在差异。Balasubramanian等[48]发现沃顿胶体间充质干细胞上调具有谷氨酸-亮氨酸-精氨酸结构和ELR-阳性的CXC趋化因子基因的表达；同时，沃顿胶体间充质干细胞较骨髓间充质干细胞更强地表达VEGF-D、PDGF-AA、TGF-β、bFGF和HGF。这些结果提示沃顿胶体间充质干细胞可能较骨髓间充质干细胞更适于治疗缺血性病变及减轻纤维化和瘢痕形成。Hsieh等[49]也发现沃顿胶体间充质干细胞表达更多的与血管生成和生长相关的基因(如表皮生长因子和FLT1基因)，而骨髓间充质干细胞表达更多诸如RUNX2，DLX5和NPR3的成骨基因。骨髓间充质干细胞的基因表达类型较沃顿胶体间充质干细胞更类似于成骨细胞。相比之下，沃顿胶体间充质干细胞因其与胚胎干细胞共有某些基因而更原始。沃顿胶体间充质干细胞表达富含VEGF和PI3K-NFκ途径的基因，而骨髓间充质干细胞表达涉及抗原呈递和趋化因子/细胞因子途径的基因。因此。骨髓间充质干细胞在成骨和成脂分化上更有效，而沃顿胶体间充质干细胞则在血管生成和增殖方面更佳。Angelucci等[50]对人沃顿胶体间充质干细胞的蛋白组学谱进行了详尽的研究。他们发现包括Shootin1，腺苷酸激酶5同工酶和纤溶酶原激活物抑制剂2在内的数个重要的蛋白在早期细胞体外复制后不再表达，可能与其持久的多能性逐渐减低有关。此外，在细胞扩增的终末，新生的蛋白如ERO1样蛋白α、天冬氨酰-tRNA合成酶和脯氨酰-4-羟化酶α1和α2，可能参与损害复制期间的细胞生存和分化时间。 2.5 免疫原性与免疫调节功能沃顿胶体间充质干细胞表达免疫耐受基因HLA-E、HLA-G和HLA-F及免疫调节基因VEGF、TGFβ1、HGF、HMOX1、IL1β、IL-6、LIF、LGALS-1/3/8、COX1/2和PTGE，而不表达免疫应答相关的基因HLA-DR、HLA-DQ、HLA-DP、CD80、CD86、CD40和CD40L。在接受沃顿胶体间充质干细胞移植的心衰患者中观察到T淋巴细胞亚群和血浆IgG/IgM没有明显变化，提示沃顿胶体间充质干细胞的免疫特性是在缺乏免疫应答相关基因表达的同时表达免疫逃避与免疫调节基因[51] 。Zhou等[52]观察到沃顿胶体间充质干细胞与其培养上清均能抑制植物凝集素激发的人外周血淋巴细胞和小鼠脾细胞的增殖。此外，沃顿胶体间充质干细胞还能抑制人外周血淋巴细胞对转化生长因子β1和干扰素-γ的分泌。作者得出结论，沃顿胶体间充质干细胞的免疫调节作用可能与直接细胞接触和对人外周血淋巴细胞相关细胞因子的分泌有关。Valencic等[53]发现激活的淋巴细胞在沃顿胶体间充质干细胞面前虽然正常表达激活的标记物，但其增殖和细胞因子的生成均明显减低。当靶淋巴细胞被膜分开时抑制被减弱，但加入外源性L-色氨酸可部分恢复抑制，提示2、3-二氧生成酶具有重要的作用(可能的旁分泌效应)。免疫特许在接触与非接触背景下均能增强免疫抑制的效果。沃顿胶体间充质干细胞免疫特许的时机对免疫抑制作用来说是决定性的。预先单独激活24 h的淋巴细胞对未经免疫特许的沃顿胶体间充质干细胞的增殖反应是正常甚至是增强的，而另一方面，其增殖可被免疫特许的沃顿胶体间充质干细胞所强烈抑制。Najar等[54]研究了沃顿胶体间充质干细胞对白血病抑制因子的表达及其在间充质干细胞介导的抑制机制中的作用。证实在活化T细胞的刺激下，通过表达COX1/COX2酶和产生PGE2介导，沃顿胶体间充质干细胞对T细胞增殖产生强有力的剂量依赖性免疫抑制作用，沃顿胶体间充质干细胞较骨髓间充质干细胞在抑制淋巴细胞应答上更强有力，它们通过分泌高水平的白血病抑制因子介导这一效应。此外，沃顿胶体间充质干细胞维持和促进调节性T-细胞的扩增不依赖于间充质干细胞/T淋巴细胞比[55] 。 2.6 成瘤性沃顿胶体间充质干细胞低水平表达包括POUF1、NANOG、SOX2和LIN28在内的多潜能胚胎干细胞标记，从而解释了其为何不生成畸胎瘤。沃顿胶体间充质干细胞明显上调数种细胞因子的表达，其中包括IL12A(与诱导凋亡相关)，从而解释了其抗癌的特性[56] 。Gauthaman等[57]比较了人沃顿胶体间充质干细胞和胚胎干细胞在免疫缺陷小鼠中的肿瘤形成能力。经由3种途径：皮下、肌内和腹腔内注射人胚胎干细胞(2×106细胞/位点)和人沃顿胶体间充质干细胞(5×106细胞/位点)。所有接受人胚胎干细胞的动物在6周内均发生了肿瘤，而在接受人沃顿胶体间充质干细胞的动物没有肿瘤发生，甚至当观察20周时依然没有肿瘤发生。此外，接受人胚胎干细胞的动物呈现免疫应答，而接受人沃顿胶体间充质干细胞的动物却未出现。作者得出结论，人沃顿胶体间充质干细胞既不诱发肿瘤也不在移植时发生排斥反应，在细胞治疗上具有巨大的潜能。 2.7 向心肌细胞分化的能力人沃顿胶体间充质干细胞表现出分化为各种不同组织细胞的多潜能性，诸如心血管组织、骨、脂肪、软骨和神经细胞等。Wang等[17]用5-氮杂胞苷处理沃顿胶体间充质干细胞3周后，可表达出心肌细胞的标记物心脏肌钙蛋白I、缝隙连接蛋白43和结蛋白，并显示出心肌细胞的形态学。5-氮杂胞苷可能是通过调控GATA4、Nkx2.5基因的表达促进人脐带间充质干细胞分化为心肌样细胞，并促进其成熟[58] ，10 μmol/L是较佳的诱导浓度[59] 。Pereira等[60]报道，当培养3周时，某些分离的沃顿胶体间充质干细胞发育为带有心脏细胞形态学特征的表型，形成肌管结构和出现自发搏动。 2.8 在心血管系统疾病中的研究为了评估人沃顿胶体间充质干细胞与心脏组织的整合特性，将其与正常的或缺血的胚胎鼠心室切片共培养。对共培养制备的冰冻切片做免疫组织化学分析，在共培养中进行人类细胞的追踪。沃顿胶体间充质干细胞明显趋向心室切片，强烈整合到正常的与缺血的心脏组织的深部，在体外证实沃顿胶体间充质干细胞能定植于心脏组织[61] 。何红燕等[62]将用BrdU标记的人脐带间充质干细胞移植至心肌梗死的大鼠心肌，2周后取材检测，结果显示细胞移植后2周仍能存活，存活细胞能表达心肌细胞标记物心脏肌钙蛋白I和T。Wu等[63]在体外试验和大鼠心肌梗死模型中再次证实了沃顿胶体间充质干细胞能向心肌细胞分化，同时超声提示移植后2周和4周的左室功能较对照改善。Latifpour等[64]观察到，结扎冠状动脉前降支后30 d，接受人沃顿胶体间充质干细胞治疗动物的左室射血分数和短轴缩短率明显改善，瘢痕含量显著减低。组织病理学分析证明，在少数兔中，邻近和远离梗死区域的植入细胞表达肌钙蛋白I、F-actin和connexin-43，在梗死区内亦可见少数移植的细胞。"

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