|Alternative Title||Mechano-biological coupling of embryonic stem cells in stemness maintenance and directed differentiation|
|Place of Conferral||北京|
|Keyword||胚胎干细胞 硅酸盐浸提液 力学组学 基质硬度 干性维持 定形内胚层分化|
胚胎干细胞（embryonic stem cells, ESCs）是一类具有分化全能性和无限增殖能力的特殊干细胞，可以在体外特定培养条件下诱导分化成为目标细胞，是细胞治疗的潜在细胞来源。体外ESCs的增殖、分化均与局部的物理与化学微环境相关，认识其特定的干性维持和定向分化规律，对于体外扩增和诱导分化具有重要的意义。本文采用力学-生物学、细胞生物学及力学-蛋白质组学等相结合的方法，对人胚胎干细胞（hESCs）H1/H9细胞系的干性维持和定向分化的力学-生物学耦合规律及机制进行研究。主要开展如下工作：
1、硅酸钙（calcium silicate, CS）浸提液对H9细胞的细胞毒性、干性维持及早期分化潜能的影响。探索有别于传统生化方法调控H9细胞干性和分化的简易方法是干细胞工程的重要途径。采用高（1/64）、低（1/256）两种浓度的CS浸提液（主要功能成分为硅离子）作用于H9细胞，对克隆铺展速度和铺展面积，凋亡细胞的比例及分布等指标进行检测。结果表明，不同浓度CS浸提液对H9细胞不但均未表现出明显细胞毒性，反而使克隆内细胞-细胞间产生更强的胞间相互作用。短时程培养条件下（3天），CS浸提液有利于H9细胞干性维持，而长时程培养条件下（6天）则驱动H9细胞分化。实验证实CS浸提液对H9细胞的作用具有双向调节：短时程时有利于H9细胞的干性维持；长时程又有利于H9细胞分化的启动。上述研究为采用无机硅离子调控H9细胞的干性或分化提供新的思路和方法。
2、多种力学刺激的多参数组合对H1细胞干性维持及早期分化潜能的影响。体内力学因素具有不同的类型、模式和参数，对细胞功能的影响远较体外单一力学刺激复杂。利用定量蛋白质组学方法，在拉伸、流动剪切和压缩三种力学刺激类型及不同加载模态和参数下，检测到共表达蛋白856种。聚类分析说明力学刺激类型对H1细胞影响最大。对于同类型力学刺激，不同参数及模态影响不同：拉伸组，时间>类型>频率；流体剪切组，时间>幅值>类型；压缩组，时间>幅值>频率。实验结果表明H1细胞在响应力学刺激时蛋白质表达变化具有力学种类及参数依赖性。采用Three Way ANOVA、GO、GSEA、WGCNA等方法对力学组学获取数据进行生物信息学分析，结果表明：拉伸条件下共得到37个模态敏感蛋白质、41个频率敏感蛋白质和23个时间敏感蛋白质；流体剪切条件下共得到13个模态敏感蛋白质、18个幅值敏感蛋白质和41个时间敏感蛋白质；压缩条件下得到4个幅值敏感蛋白质、183个时间敏感蛋白质。大部分力学差异表达蛋白质可跨细胞不同区域行使功能，细胞外到细胞质99种，细胞质到细胞核53种，从细胞外到细胞质再到细胞核110种。在所有的力学差异表达蛋白质中，拉伸组和流体剪切组共有的蛋白质是EXOSC5、MDC1、ACY1、STON2、BASP1，流体剪切组和压缩组共有的蛋白质是MTHFD1、GART，拉伸组和压缩组共有的蛋白质是UHRF1、RPL35A、HIST1H1B。拉伸组和流体剪切组及流体剪切组和压缩组均共有的关键枢纽蛋白分别是GART和EXCSC5。上述研究证实了力学刺激的不同类型、模态和参数协同影响H1细胞力学敏感蛋白的表达，为力学组学实验体系及分析方法的建立、干细胞力学-生物学耦合机制的研究提供方法参考、基础数据和下游研究思路。
3、胞间粘附对H1细胞干性维持及向定形内胚层（definitive endoderm, DE）分化的影响及调控规律。克隆内细胞-细胞间粘附是H1细胞维持干性和启动分化的关键物理作用、并与基质硬度协同调控其功能。实验结果表明，诱导H1细胞分化1天时硬基质（46.7 kPa）上分化的DE细胞比例显著高于其它二种较软基质（6.1 kPa、0.14 kPa）。分化1天或3天后，DE细胞标志物CXCR4的表达量随着基质硬度的增加而显著增加，说明硬基质更易于启动H1细胞的DE向分化并助其成熟。软胶（0.14 kPa）基质上H1克隆中细胞-细胞间E-cadherin和β-catenin的表达量显著高于其它两种基质，而阻断胞间粘附能够增加分化1天后DE细胞的比例，说明细胞间粘附不利于H1细胞的DE向分化。分化标志和力学敏感蛋白YAP的总量和核质比以及核区表达量，在DE分化1天和3天两个时间点均随着硬度的增加而增加。相关性分析表明YAP激活后的入核可正向调控CXCR4的表达。因此，硬基质通过引起YAP核转位进入细胞核来促进H1细胞定向分化为DE细胞，E-cadherin形成的胞间粘附能够结合YAP、拮抗其对DE向分化的正向调控作用。
Embryonic stem cells (ESCs) are specialized stem cells with the ability of totipotential differentiation and infinite proliferation. They can be induced into any target cells in vitro under specific inducing conditions and serve as potential sources for cell therapy. Their proliferation and differentiation in vivo or in vitro are affected by physical and chemical features of localized microenvironment. It is thus significant to understand the specific mechanisms of ESC’s stemness maintenance and directed differentiation in vitro. In this dissertation, the mechano-biological coupling of human embryonic stem cells (hESCs) H1/H9’s stemness maintenance and directed differentiation were studied by coordinating the approaches of mechanobiology, cell biology and mechanomics. The major works have beed conducted as follows:
1. Effects of calcium silicate (CS) extract on cytotoxicity, stemness maintenance and early differentiation potentials of H9 cells. It is critical in stem cell engineering to explore a simple assay, that is different from those conventional biochemical methods, to regulate the stem cell differentiation and stemness maintenance. Here H9 cells were cultured under two concentrations of CS (1/64 and 1/256) and the spreading rate, spreading area, fraction and distribution of apoptotic cells were measured. Data showed that different concentrations of CS did not exert significant cytotoxicity to H9 cells, but resulted in stronger cell-cell interaction within the clone. Under the condition of short-term culture (3 days), CS extract was beneficial to the stemness maintenance of H9 cells, while CS extract was favorable for H9 cells differentiation under long-term culture (6 days). These results suggested that CS extract has biphasic regulation on the function of H9 cells: short-term is conducive to the stemness maintenance of H9 cells and long-term is favorable in initiating H9 cells the differentiation. This work provides a new insight in regulating the stemness and differentiation of H9 cells via inorganic silicon ions.
2. Impacts of multiple mechanical stimuli on the stemness maintenance and early differentiation potentials of H1 cells. Mechanical cues in vivo present different types, patterns, and parameters, and their effects on cell responses is far more complex than that under a single mechanical stimulation in vitro. Combining typical mechanical stimuli with quantitative proteomics analysis, total 856 proteins were screened for H1 cells under three types of tensile stretch, shear flow and mechanical compression with different loading patterns and parameters. Cluster analysis showed that the type of mechanical stimuli yielded the greatest impact on mapping mechanosensitive proteins. Under a specific loading type, different loading parameters presented distinct roles in an order of duration > pattern > frequency for tensile stretch, duration > amplitude > pattern for shear flow, or duration > amplitude > frequency for mechanical compression. These measurements indicated that protein expressions of H1 cells in response to mechanical stimuli were strongly dependent on loading types and parameters. Three Way ANOVA, GO, GSEA, WGCNA and other bioinformatics analyses were conducted for the data obtained from the above mechanomics tests. Briefly, tensile stretch presented 37 pattern-sensitive, 41 frequency-sensitive and 23 duration-sensitive proteins; shear flow had 13 pattern-sensitive proteins, 18 amplitude-sensitive proteins and 41 duration-sensitive proteins; mechanical compression yielded 4 amplitude-sensitive and 183 duration-sensitive proteins. Most of the differentially-expressed mechanosensitive proteins were distributed over various regions inside the cell, of which 99 were from extracellular to cytoplasmic, 53 from cytoplasmic to nuclear, and 110 from extracellular thru cytoplasmic to nuclear regions. Among these differentially-expressed proteins, EXOSC5, MDC1, ACY1, STON2, BASP1 were shared between tensile stretch and shear flow, MTHFD1, GART were shared between shear flow and mechanical compression, and UHRF1, RPL35A, HIST1H1B were shared between tensile stretch and mechanical compression. GART and EXOSC5 were the key hub proteins shared by tensile stretch, shear flow and mechanical compression. These results confirmed that different types and parameters of mechanical stimuli synergistically affect the mechanosensitive protein’s expression in H1 cells. This work also provides the bases for developing a mechanomics platform to elucidate the mechano-biological coupling mechanisms of hESCs and other cell types.
3. Regulation of intercellular adhesion of H1 cells on the stemness maintenance and the definitive endoderm (DE) differentiation on varied-stiffness substrates. Intercellular adhesion plays the key roles in regulating stemness maintenance and differentiation initiation of H1 cells, which is also cooperated with substrate stiffness. Here the fraction of DE cells on stiff PA gel substrate (46.7 kPa) was significantly higher than that of the other two softer substrates (6.1 kPa and 0.14 kPa) after 1-day DE induction. After 1- or 3-day DE induction, the expression of CXCR4 in DE cells increased remarkably with the increase of substrate stiffness, indicating that stiff substrate more likely initiated DE differentiation of H1 cells and favored their maturization. Expressions of E-cadherin and β-catenin in H1 colonies on soft substrate (0.14 kPa) were significantly higher than that of the other two substrates while blocking intercellular adhesion after 1-day DE induction enhanced the fraction of DE cells, suggesting that intercellular adhesion was not conducive to DE differentiation of H1 cells. Expressions of CXCR4, total YAP, nuclear-to-cytosol (N/C) ratio of YAP and absolute nuclear YAP all increased with the increase of substrate stiffness. Correlation analysis showed that YAP could upregulate CXCR4 expression. Collectively, the stiff substrate promotes H1 cell differentiate into DE cells by translocating cytoplasmic YAP into the nucleus. These intercellular adhesions formed upon E-cadherins can interact with YAP and antagonize its positive effect on DE differentiation.
In conclusion, this work cooperatively elucidated the roles of CS extract and different mechanical stimuli on fate-decision of H1/H9 cells. The results uncovered the impacts of these physicochemical cues on stemness maintenance and early differentiation of H1 cells and revealed the regulating mechanisms of intercellular adhesion on directed DE differentiation of H1 cells via YAP signaling. This work sheds light on understanding the mechano-biological coupling mechanisms in stemness maintenance and directed differentiation of H1 cells.
|张帆. 胚胎干细胞干性维持与定向分化的力学-生物学耦合机制[D]. 北京. 中国科学院大学,2020.|
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