Microenvironmental cues influence the reprogramming of somatic cells to induced pluripotent stem cells

doi:10.3969/j.issn.2095-4344.0455

Abstract

Abstract:

BACKGROUND: Induced pluripotent stem cells hold enormous potential as a tool to generate cells for disease therapy and modeling. However, the lower efficiency of cell reprogramming using exogenous transcription factors limits the clinical use of induced pluripotent stem cells. Recent studies have indicated that microenvironmental cues can promote the production of pluripotent cells. Further summarization on these reports will provide new strategies to enhance the safety and efficiency of the reprogramming technology.
OBJECTIVE: To summarize the effect of microenvironmental cues on the reprogramming of somatic cells to induced pluripotent stem cells and the maintenance of pluripotency.
METHODS: Scientific papers from 2006 to present included in Web of Science were searched by the first author. Eighty papers focusing on the effect of microenvironmental cues on the reprogramming of somatic cells to induced pluripotent stem cells and the maintenance of pluripotency were included.
RESULTS AND CONCLUSION: Cell-matrix interaction and cell-cell adhesion regulate gene expression and have effects on the epigenetic states of the chromatin through a direct connection between actin cytoskeletons and the nuclear membrane; thus, manipulation of the cell-matrix interaction and cell-cell adhesion can improve the induction of pluripotency. In addition, other environmental factors including hypoxia, dynamic culture and electrical stimulation can also promote cell reprogramming.

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: Induced Pluripotent Stem Cells, Electric Stimulation, Cell Hypoxia, Cell Adhesion, Tissue Engineering

CLC Number:

R394.2

Chen Zhong-yao, Cao Ze-yu, Huang Yan, Ji Jing, Chen Xiao-fang. Microenvironmental cues influence the reprogramming of somatic cells to induced pluripotent stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 807-814.

Figures/Tables 2

2.1 纳入文献基本情况细胞处在复杂的环境中，受到可溶性因子和不可溶环境因素的共同影响。许多不同来源和不同种类的信号都作用在共同的信号通路上，比如培养基质的软硬程度和表面形貌都会影响细胞黏着斑的形成和细胞骨架的排列，以及下游的一系列信号传导过程[6]。文章从材料和实验方法的角度对检索到的文献进行简单分类，探讨以下3个方面因素对多能干细胞干性维持和体细胞重编程过程的影响。 2.2 培养基质的理化特征培养基质的理化特征直接影响细胞黏着斑的形成和细胞肌动蛋白骨架的排列，进而影响下游基因表达和细胞形态、贴壁、增殖、迁移和分化等一系列行为[6]。细胞与基质间的相互作用能够对几乎所有细胞类型产生影响，包括多能干细胞。另一方面，由于细胞种类的不同、形貌特征的差别和基质材料本身理化性质的差异，导致培养基质对细胞的影响有所不同[7]。 2.2.1 培养基质的表面微纳结构目前应用一些成熟的加工技术，比如光刻、热压、腐蚀及合成等，能够在不同的材料表面形成几十纳米到几百微米范围的微纳结构，这些方法也用于对细胞培养基质材料进行处理[8]。研究表明，表面粗糙度、不规则微纳颗粒、微纳纤维、不同尺寸和形状的微纳结构等都能够影响多能干细胞的自我更新和干性维持。 Jeon等在polydimethylsiloxane(PDMS)材料上加工出了粗糙度为8的表面并在其上培养小鼠胚胎干细胞。结果表明，相比于粗糙度为1的光滑PDMS材料，粗糙度为8的PDMS材料促进了小鼠胚胎干细胞的自我更新，小鼠胚胎干细胞能够在PDMS材料表面长期维持多能性，而在普通培养皿表面经过长期传代后出现随机分化。粗糙的PDMS材料表面有利于小鼠胚胎干细胞的干性维持，不利于细胞贴壁和分化[9]。相反的，Chen等[10]利用腐蚀的方法增加玻璃表面粗糙度，他们发现光滑的玻璃表面(粗糙度为1)更有利于人胚胎干细胞贴壁、增殖和多能性的长期维持，而粗糙的玻璃表面(粗糙度为100)会引起人胚胎干细胞分化。2篇报道中结果的差异可能与所用材料本身的差异有关，另外，小鼠胚胎干细胞与人胚胎干细胞不同，其维持干性所需条件也有很大差别。Kong等[11]在聚苯乙烯材料上加工了不同形状的纳米柱，结果表明在没有碱性成纤维细胞生长因子的情况下，培养在具有纳米结构的基底上的人胚胎干细胞能够更好的维持干性。Bae等[12]在聚苯乙烯材料上加工了不同直径的纳米柱(100-350 nm)，他们发现相比于光滑表面，具有纳米柱的表面不利于细胞形成稳定的黏着斑，限制了细胞迁移，增强了钙黏附蛋白E(E-cadherin)介导的细胞间连接，促进了多能性的维持。此外，微米和纳米级的纤维结构及合成的不规则颗粒结构都会影响胚胎干细胞的多能性[13-16]。最新的研究发现，培养基底表面形貌对重编程过程也有显著影响。Song Li研究小组在PDMS基底上加工出了宽度10 μm的沟槽，他们发现，沟槽表面能够显著提高细胞重编程的效率，如图1所示。机制研究发现沟槽结构抑制了细胞组蛋白脱乙酰激酶HDAC2活性，增强了组蛋白H3甲基转移酶亚单位WDR5的表达，进而使核小体组蛋白H3的乙酰化(AcH3)修饰以及H3上赖氨酸4的二甲基化(H3K4me2)和三甲基化(H3K4me3)修饰都显著增强，这些表观遗传的变化促进了细胞重编程。微沟槽与小分子表观遗传修饰剂valproic acid(VPA)和Tranyl-cypromine hydrochloride(TCP)作用效果类似，并且能够取代这两个小分子促进细胞的命运转变。此外，沟槽结构促进上皮相关基因的表达，增强了细胞的间质-上皮转化(mesenchynal to epithelial transition，MET)。作者还证明了微沟槽结构引起的一系列变化可能是由细胞内肌动蛋白骨架的重构以及肌动蛋白-肌球蛋白间的相互作用力引起[17]。另有报道指出敲低丝氨酸/苏氨酸激酶Tesk1和Limk2能够降低Cofilin的磷酸化程度，去磷酸化的Cofilin与肌动蛋白骨架结合，导致其解聚，进而促进细胞重编程[18]。对间充质干细胞的研究也表明表面形貌刺激能够直接改变细胞的表观遗传状态[19]，而表观遗传状态对细胞重编程至关重要[20]。表面微纳结构也能够促进成纤维细胞向其他细胞类型的转变。Leong小组发现表面微沟槽能够促进转录因子Ascl1、Brn2和Myt1L诱导的成纤维细胞向神经细胞重编程[21]。表面结构对神经轴突的分支数也有显著影响，在微沟槽表面轴突分支减少，而在微柱表面分支增加。轴突分支数目的变化与成纤维细胞对外界力学环境的感受有关，非肌肌球蛋白Ⅱ在其中发挥重要作用[21]。Kim等发现具有纳米沟槽的聚氨酯丙烯酸酯表面能够有效提高转录因子Ascl1、Pitx3、Nurr1和Lmx1a介导的成纤维细胞向多巴胺神经元重编程。其分子机制是纳米沟槽引起的细胞骨架重构促进了细胞的间质-上皮转化和组蛋白H3K4me3修饰[22]。此外，微沟槽表面促进成纤维细胞向心肌细胞方向重编程也有报道，原理是微沟槽促进组蛋白乙酰化(AcH3)修饰[23]。这些研究说明了表面微结构引起的表观遗传变化有助于细胞克服重编程过程中的阻碍因素[20]，进而提高重编程的效率。"

2.2.2 基底的软硬程度二维和三维培养的细胞都能够感受基底或周围基质的软硬度，并做出相应的反应以调节分化、形态、增殖和迁移等生命活动。研究表明基底硬度对胚胎干细胞的多能性有重要影响。Chowdhury 等[24]报道小鼠胚胎干细胞能够在硬度为0.6 Pa的水凝胶上更好的维持多能性，无需在培养液中添加白血病抑制因子。软质基底上，细胞与基质之间的结合减弱，干性能够更好的维持。同样，培养在三维聚乙二醇水凝胶中的小鼠胚胎干细胞在较软的基质中具有较高的增殖和自我更新能力[25]。与小鼠胚胎干细胞相反，人胚胎干细胞在软基底上会发生分化，而在较硬的基底上(10-25 kPa)能够较好的维持干性[26-28]。在较硬基底上，人胚胎干细胞形成更加致密的肌动蛋白网络，细胞骨架产生的收缩力增加，E-cadherin和Oct4表达上调。小鼠胚胎干细胞和人胚胎干细胞对基底力学性质的不同反应可能与两种细胞本身的差异有关[29-30]。另一方面，研究表明软基底有可能促进细胞重编程。培养在软的聚丙烯酰胺凝胶上的小鼠和人成纤维细胞，其胚胎干细胞标志基因Oct4和Nanog的表达水平比培养在硬凝胶上的同类细胞显著提高[31]。培养在硬度较低的PDMS上的人胚胎肾细胞(HEK)形成具有碱性磷酸酶活性的细胞团，并且Oct4和Nanog表达上调，而培养在硬度更大的玻璃基底上的人胚胎肾细胞不形成细胞团，也不具有碱性磷酸酶活性。细胞团内大部分细胞的肌动蛋白骨架产生了更大的收缩力，利用细胞松弛素使骨架收缩力降低则无法形成细胞团，因此骨架收缩力与多能性标志基因的表达上调有关[32]。此外，软基底能够提高小鼠胚胎成纤维细胞向诱导多能干细胞方向重编程的效率。小鼠胚胎成纤维细胞感染OSKM四因子后，在软基底上(100 Pa)培养1 d再转移到种有滋养层细胞的培养皿内继续培养，重编程效率提高1倍[33]。此外，当小鼠胚胎成纤维细胞种在三维水凝胶内进行重编程时，硬度较小的水凝胶具有较高的重编程效率[33]。 2.2.3 培养基质的化学特征培养基质的化学特征包括其成分和表面修饰都会影响细胞的贴壁、增殖和分化等行为，特定的基质材料能够帮助胚胎干细胞和诱导多能干细胞维持干性，甚至促进体细胞向诱导多能干细胞方向重编程。早期的人胚胎干细胞和诱导多能干细胞培养以及人体细胞向诱导多能干细胞方向重编程，需要利用小鼠胚胎成纤维细胞作为滋养细胞或者在培养皿上包被一层Matrigel(一种由小鼠肉瘤细胞产生的细胞外基质)，这些培养方法引入了动物来源的物质，存在安全性和稳定性等问题，无法满足临床应用和大规模工业生产的需求，因此许多研究探索利用合成材料或者细胞外基质蛋白替代滋养细胞和Matrigel。多个研究小组采用细胞微阵列的方法，将几百或上千种待测物质点在玻片上，观察人多能干细胞在这些物质上的生长状况[34-37]，筛选出了多种能够支持人多能干细胞贴壁和干性维持的高分子聚合物以及细胞外基质蛋白[38]。基于这些研究成果，利用成分确定的且无动物来源的物质进行人多能干细胞的培养技术已经广泛应用[2-3]。除了支持人多能干细胞生长，很多合成材料比如poly2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)[39]、聚多巴胺等[40]，以及细胞外基质蛋白比如玻连蛋白(Vitronectin)[41-42]、层连蛋白(Laminin)[43-44]、胶原蛋白(Collagen)等还能够支持人体细胞向诱导多能干细胞方向重编程[45]。然而在这些报道中，基质材料都是与特定的培养液成分配合使用，基质材料本身能否加快重编程过程，提高重编程效率还有待研究。特殊的培养基质材料改变重编程过程、影响重编程效率的报道目前还不多见。Yoo等[46]发现石墨烯能够促进小鼠成纤维细胞向诱导多能干细胞方向重编程，在石墨烯材料上，具有碱性磷酸酶活性和表达Oct4-GFP的克隆数量相比玻璃基底都有显著提高，并且内源的胚胎干细胞标志基因Oct4，Nanog，Sox2和Esrrb的表达都明显上调。石墨烯促进了小鼠成纤维细胞的间质-上皮转化，提高了组蛋白H3K4me3修饰，进而促进细胞重编程。Chang等[47]发现，从幼年小鼠尾部提取的胶原蛋白能够使年长小鼠的成纤维细胞表现出与幼年小鼠细胞类似的活力，增殖速度提高，凋亡降低，并且重编程为诱导多能干细胞的效率也显著提高。此外，Smith等[48]发现，接枝Laminin的PEG材料能够提高小鼠成纤维细胞向心肌细胞重编程的效率。 2.3 细胞间黏附细胞间黏附对于多能干细胞生存和干性维持有非常重要的作用，由于细胞间相互黏附，多能干细胞形成紧密的克隆。人胚胎干细胞具有上皮细胞的特点，具有细胞极性、E-cadherin介导的细胞间黏附和integrin介导的细胞-基质间黏附等特征。当上皮结构被破坏后，上皮细胞通常会启动凋亡程序[49]。人胚胎干细胞处于克隆生长时，细胞黏附和肌动蛋白-肌球蛋白引起的收缩达到平衡，维持着稳定的形态和功能[50]，当单个人胚胎干细胞无法与其他细胞接触时，肌动蛋白-肌球蛋白的过度收缩会引起细胞凋亡。在传代过程中，通常利用温和的蛋白质水解酶比如Accutase或者是机械力将人胚胎干细胞分散成小团以保护细胞间黏附，避免凋亡。Rho相关激酶(ROCK)抑制剂Y-27632能够减弱肌动蛋白-肌球蛋白之间的收缩，避免肌动蛋白-肌球蛋白的过度收缩引起的细胞凋亡[51-52]。E-cadherin是维持细胞间黏附的最主要分子，当人胚胎干细胞过表达E-cadherin或者在培养基底表面修饰E-cad能够显著增强单个人胚胎干细胞存活和形成克隆的能力[53-54]。胚胎干细胞存在两种多能性状态，即原始态多能性和始发态多能性[30]。小鼠胚胎干细胞和人胚胎干细胞的形态及维持多能性所需的信号分子有很大差别，通常认为小鼠胚胎干细胞处于原始态多能性，需要白细胞介素来维持其未分化状态；而人胚胎干细胞处于始发态多能性，需要碱性成纤维细胞生长因子来维持自我更新。细胞间黏附对维持两种细胞多能态都有重要作用，对于原始态小鼠胚胎干细胞，细胞间黏附能够稳定白细胞介素受体，维持细胞对白细胞介素的敏感性，破坏细胞间黏附会使原始态小鼠胚胎干细胞转变为始发态小鼠胚胎干细胞。而对于始发态小鼠胚胎干细胞，破坏细胞间黏附会引起细胞分化[55]。在成纤维细胞重编程为诱导多能干细胞的过程中，细胞间黏附发挥着关键作用。重编程初始阶段会发生间质-上皮转化，E-cadherin表达升高和细胞间黏附增强[56-57]。间质-上皮转化是重编程过程中的重要环节，许多促进间质-上皮转化的分子比如BMP、miR-200和Tets等能够提高重编程效率[56，58]，上皮细胞重编程成功率明显优于成纤维细胞[59]，而抑制间质-上皮转化的分子比如转化生长因子β则阻碍重编程[57]。实时动态成像证明细胞间黏附在重编程早期就能够观察到，并且相互黏附的细胞团大多数最终形成了Oct4-EGFP阳性的诱导多能干细胞克隆[60]。E-cadherin是介导细胞间黏附的主要分子，降低E-cadherin表达明显阻碍重编程，而增强E-cadherin表达则显著提高重编程效率[61]，甚至可以取代四因子中的Oct4。另有研究发现，ESKM诱导的诱导多能干细胞和OSKM诱导的诱导多能干细胞其功能和基因表达都非常类似[62]，用多肽或抗体破坏E-cadherin介导的细胞间黏附显著降低重编程效率[61]。值得一提的是，N-cad能够替代E-cadherin重新建立细胞间黏附并恢复细胞重编程能力[63]，这些研究说明细胞间黏附，而并非某种特定的钙黏素(cadherin)分子是成纤维细胞转变为诱导多能干细胞过程中必不可少的关键因素。另一方面，增强细胞间黏附有助于分化细胞获得干细胞特征，达到重编程的效果。在低吸附表面或者悬浮培养条件下，细胞团聚成三维小球，细胞间黏附增强。聚团培养的RT4膀胱癌细胞系表现出肿瘤干细胞的特点，具有更强的成瘤和转移能力。Hek293细胞团则表现出肾祖细胞和一些胚胎干细胞的特征，比如碱性磷酸酶活性，Oct4、Nanog等基因的高表达和体内成瘤能力[64]。对成纤维细胞进行成团培养，能够促进间质-上皮转化转化，并引起Oct4启动子区域的去甲基化和Oct4的表达。尽管当细胞重新被接种在培养皿上平面培养时，Oct4启动子区域再次被甲基化，其表达也被抑制，但是细胞仍然表达一些成体干细胞相关基因[65]。在小鼠胚胎干细胞培养基中培养的小鼠成纤维细胞团表现出神经祖细胞的特征，表达神经祖细胞相关因子Sox2、Pax6等，并且能够分化成神经元和胶质细胞[66]。三维成团培养的人成纤维细胞也能够在无外源基因物质的条件下重编程为神经祖细胞[67]。对小鼠睫状体上皮细胞进行短期的成团培养，能够使其表达视网膜前体细胞基因，并在只导入一个外源转录因子Oct4的情况下重编程为诱导多能干细胞[68]。此外，在PEG水凝胶内进行三维重编程，增强了细胞间黏附，促进了间质-上皮转化，并且提高了组蛋白H3K4me3修饰，促进干性相关基因的表达，重编程效率比二维条件下显著提高[33]。该研究表明，与表面微沟槽类似，增强细胞间黏附也能够提高细胞的可塑性，促进细胞命运转变。 2.4 其他理化因素 2.4.1 低氧环境胚胎发育处在低氧环境中(体积分数为1.5%-5.3% O2)，低氧环境下(体积分数为5% O2)培养的人胚胎干细胞相比常氧环境(体积分数为21% O2)干性维持更好，自发分化减少，并且更容易形成拟胚体[69]。低氧对小鼠胚胎干细胞克隆生长也有促进作用，尽管目前报道的研究结果仍有一些矛盾[70]。低氧环境还有利于造血干细胞、神经干细胞和间充质干细胞的自我更新[71]。Yamanaka实验室证明低氧环境(体积分数为5% O2)能够使人和小鼠体细胞重编程为诱导多能干细胞的效率提高3倍以上，而且可以取代转录因子Sox2和c-Myc，实现二因子重编程。转染了OSKM四因子的小鼠胚胎成纤维细胞在低氧环境下，基因表达向小鼠胚胎干细胞方向转变[72]。Mathieu等[73]把发生随机分化的人胚胎干细胞置于体积分数为2% O2的培养条件下，细胞能够重新恢复多能性。另有研究表明低氧对成纤维细胞重编程为神经祖细胞和类心肌细胞也有促进作用[74-75]。这些研究表明，氧气体积分数对多能干细胞自我更新以及体细胞重编程具有调控作用，尽管其分子机制还有待进一步研究。 2.4.2 动态培养 Junren等发现，旋转振荡能够显著提高重编程效率，其原因是液体的对流促进营养物质和细胞因子等的交换，而并非流体剪切力的作用。重编程中期，当细胞过于密集时p57表达提高，抑制细胞增殖和重编程，旋转振荡降低了p57的表达，克服了接触抑制引起的细胞老化，这说明处在重编程过程中的部分细胞能够分泌某些有助于克服细胞老化和凋亡的因子[76]。与这一现象一致的是频繁换液降低重编程效率，而在体积微小的微流管道内重编程效率显著提高[77]。 2.4.3 电磁场 Baek等[78]发现50 Hz、1 mT的低频磁场能够使四因子重编程效率提高30倍，而且只需要Oct4一个转录因子就可以成功重编程。磁场作用下，细胞增殖速度加快，组蛋白赖氨酸甲基转移酶MLL2表达增加，Oct4，Nanog，Esrrb等干性相关基因的组蛋白H3K4me3修饰水平提高。Jin等[79]发现280 nA、1 Hz电信号促进转录因子介导的小鼠胚胎成纤维细胞向神经元重编程。外环境因素影响细胞重编程重要文献如表1所示。"

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