干细胞是一种具有自我更新、高度增殖和多向分化潜能的早期未分化细胞，在特定条件下可分化成不同的功能细胞，形成组织和器官。胚胎干细胞（ESCs）是可以在体内外分化为内、中、外三个胚层的几乎所有类型细胞的多潜能干细胞，既可作为细胞分化和发育调控机制的体外研究模型，亦可用作细胞治疗及组织器官构建的种子细胞，使其成为研究的热点。模拟体内ESCs 生长微环境中化学、物理因素，可探究其对ESCs 定向分化的影响，提升分化效率和细胞成熟度。无机纳米粒子硅酸钙（CS），已被证实能够促进间充质干细胞的成骨向分化，但其对人ESCs（hESCs）的分化（尤其是肝向分化）是否具有调控作用还鲜有研究；基底硬度作为典型的生理力学调控因素，对hESCs 分化调控的作用亦不明朗。因此本文从细胞生长的生物化学因素（CS 浸出液）及基于生理病理的力学微环境（基底硬度）两方面分别探究hESCs 的定向分化，分述如下：

1. CS 浸出液对hESCs 肝向分化的影响

在优化的hESCs（H9细胞系）分化诱导方案，即干性维持阶段（STEM）→定型内胚层阶段（DE）→前体肝细胞阶段（Pre-H）→成熟肝细胞阶段（M-H），的不同阶段，分别加入高（1/64）、低（1/256）两种不同稀释浓度CS浸出液，研究hESCs不同分化阶段对CS添加顺序和浓度的依赖性。结果表明，分化的前两个阶段，高浓度浸出液可迅速启动分化，而低浓度浸出液则有利于持续性的DE 向分化。基于此，筛选出hESCs 肝向分化的四个阶段最适的CS 添加方式及浓度组合条件，所得到的肝细胞能显著改善CCl₄ 注射小鼠的早期肝纤维化症状。上述研究揭示了CS 浸出液浓度与作用阶段对hESCs 肝向分化的贡献，确定CS 促分化的最适添加条件，验证了所获得的肝细胞对疾病模型的治疗作用。

2. 基底-细胞间粘附影响hESCs 的DE 向分化机制

基底-细胞间粘附是hESCs（H1 细胞系）维持干性和启动分化的重要力学因素。使用硬度可调的聚丙烯酰胺水凝胶、制备了可模拟生理/病理条件下肝脏硬度的三种基底（0.14 kPa、6.1 kPa、46.7 kPa），将接种于其上的H1细胞系定向诱导分化为DE 细胞。结果表明，随着硬度的增加，DE 分化标志物增加，基底-细胞间β₁-integrin、粘着斑组成蛋白vinculin、磷酸化FAK 的表达量增加、细胞受到基底的牵引力变大，力学敏感蛋白YAP 的活化入核比例升高。阻断β₁-integrin 后DE 分化减弱、解聚F-actin 纤维、或使用YAP小分子抑制剂，均可促进YAP 活化、增强hESCs 的DE 向分化。硬基底可增强YAP 活化并入核、进而促进H1分化为DE 细胞；在ESCs 的DE 向分化过程中不同硬度基底上细胞所受牵引力与YAP 活化呈现正相关。上述研究深化了对基底硬度调控ESCs 向DE 细胞分化的生物力学机制的认识。

综上，本文通过CS 浸出液和基底硬度对胚胎干细胞H9/H1 细胞系肝向分化进行调控的研究，证实了CS 能够促进ESCs 体外肝向分化，验证了基底硬度通过β₁-整合素调控细胞牵引力、并影响干细胞分化这一作用机制，揭示了YAP 活化对ESCs 向DE 细胞分化的促进作用及YAP 活化与牵引力之间的正相关关系。以上研究丰富了对ESCs 肝向分化的力学-化学-生物学耦合规律的认识。

干细胞是一种具有自我更新、高度增殖和多向分化潜能的早期未分化细胞，在特定条件下可分化成不同的功能细胞，形成组织和器官。胚胎干细胞（ESCs）是可以在体内外分化为内、中、外三个胚层的几乎所有类型细胞的多潜能干细胞，既可作为细胞分化和发育调控机制的体外研究模型，亦可用作细胞治疗及组织器官构建的种子细胞，使其成为研究的热点。模拟体内ESCs 生长微环境中化学、物理因素，可探究其对ESCs 定向分化的影响，提升分化效率和细胞成熟度。无机纳米粒子硅酸钙（CS），已被证实能够促进间充质干细胞的成骨向分化，但其对人ESCs（hESCs）的分化（尤其是肝向分化）是否具有调控作用还鲜有研究；基底硬度作为典型的生理力学调控因素，对hESCs 分化调控的作用亦不明朗。因此本文从细胞生长的生物化学因素（CS 浸出液）及基于生理病理的力学微环境（基底硬度）两方面分别探究hESCs 的定向分化，分述如下：

1. CS 浸出液对hESCs 肝向分化的影响

在优化的hESCs（H9细胞系）分化诱导方案，即干性维持阶段（STEM）→定型内胚层阶段（DE）→前体肝细胞阶段（Pre-H）→成熟肝细胞阶段（M-H），的不同阶段，分别加入高（1/64）、低（1/256）两种不同稀释浓度CS浸出液，研究hESCs不同分化阶段对CS添加顺序和浓度的依赖性。结果表明，分化的前两个阶段，高浓度浸出液可迅速启动分化，而低浓度浸出液则有利于持续性的DE 向分化。基于此，筛选出hESCs 肝向分化的四个阶段最适的CS 添加方式及浓度组合条件，所得到的肝细胞能显著改善CCl₄ 注射小鼠的早期肝纤维化症状。上述研究揭示了CS 浸出液浓度与作用阶段对hESCs 肝向分化的贡献，确定CS 促分化的最适添加条件，验证了所获得的肝细胞对疾病模型的治疗作用。

2. 基底-细胞间粘附影响hESCs 的DE 向分化机制

基底-细胞间粘附是hESCs（H1 细胞系）维持干性和启动分化的重要力学因素。使用硬度可调的聚丙烯酰胺水凝胶、制备了可模拟生理/病理条件下肝脏硬度的三种基底（0.14 kPa、6.1 kPa、46.7 kPa），将接种于其上的H1细胞系定向诱导分化为DE 细胞。结果表明，随着硬度的增加，DE 分化标志物增加，基底-细胞间β₁-integrin、粘着斑组成蛋白vinculin、磷酸化FAK 的表达量增加、细胞受到基底的牵引力变大，力学敏感蛋白YAP 的活化入核比例升高。阻断β₁-integrin 后DE 分化减弱、解聚F-actin 纤维、或使用YAP小分子抑制剂，均可促进YAP 活化、增强hESCs 的DE 向分化。硬基底可增强YAP 活化并入核、进而促进H1分化为DE 细胞；在ESCs 的DE 向分化过程中不同硬度基底上细胞所受牵引力与YAP 活化呈现正相关。上述研究深化了对基底硬度调控ESCs 向DE 细胞分化的生物力学机制的认识。

综上，本文通过CS 浸出液和基底硬度对胚胎干细胞H9/H1 细胞系肝向分化进行调控的研究，证实了CS 能够促进ESCs 体外肝向分化，验证了基底硬度通过β₁-整合素调控细胞牵引力、并影响干细胞分化这一作用机制，揭示了YAP 活化对ESCs 向DE 细胞分化的促进作用及YAP 活化与牵引力之间的正相关关系。以上研究丰富了对ESCs 肝向分化的力学-化学-生物学耦合规律的认识。

Stem cells are early undifferentiated cells with self-renewal, high proliferation and multidirectional differentiation potential. They can differentiate into various functional cells and form different tissues and organs under specific conditions. Embryonic stem cells (ESCs) are a kind of specialized stem cells, which can differentiate into almost all types of cells in endoderm, mesoderm, ectoderm in vivo and in vitro. ESCs can not only be used as an in vitro model of understanding cell differentiation and development regulation mechanism, but also serve as seed cells for cell therapy, tissue engineering and organ construction. Elucidating the biological features of ESCs attract attentions mainly due to their multidirectional differentiation and application potential in tissue engineering and regenerative medicine. Various chemical and physical factors have critical effects on the basic study and application of ESCs differentiation. Therefore, it is crucial to simulate the chemical and physical factors in the growth microenvironment of ESCs in vivo to explore their effects on the directional differentiation of ESCs, so as to improve the differentiation efficiency and cell maturity. On a hand, a new type of inorganic nano particle calcium silicate (CS) has been proved to promote the osteogenic differentiation of mesenchymal stem cells, but it is unknown whether it can regulate the differentiation of human ESCs (hESCs) (especially directed to liver differentiation). On the other hand, substrate stiffness serves as a typical physiological and mechanical regulatory factor, but its effect on the directed differentiation of hESCs is still unclear. Therefore, this dissertation explores the directional differentiation of hESCs from two aspects: a biochemical view based on CS extracts and a mechanical view based on substrate stiffness mimicking pathophysiological microenvironment in vivo, as follows.

1.    Effect of calcium silicate extracts on differentiation of human embryonic stem cells.

An optimized four-stage liver differentiation induction scheme of hESCs (H9 cell line) was used, including those of stemness maintenance (STEM), definitive endoderm (DE), hepatocyte commitment (pre-H) and hepatocyte maturation (M-H). Two different concentrations of CS extracts, high (1/64) and low (1/256), were added to the culture medium at different stages to systematically test the dependences of CS concentration and addition order. In the first two stages of differentiation, high concentration of CS extracts could quickly initiate DE differentiation, but low concentration of CS extracts was conducive to sustained DE differentiation. This CS addition protocol was then optimized from the first two stages, serving as the starting point for screening the addition concentrations of CS extracts at the latter two stages to establish most conducive protocol in hepatocyte differentiation of hESCs. In vivo data indicated that these induced hepatocytes could alleviate the early symptoms of liver fibrosis in mice injured by CCl₄ injection. This study first explored the promoting effect of CS extracts on the differentiation of hESCs, optimized the hepatocytes differentiation scheme of H9 with CS extracts, and verified the in vivo therapeutic potential of these induced hepatocytes.

2.    Regulation of cell-substrate adhesion on DE differentiation of hESCs.

Cell-substrate adhesion is a key physical factor for hESCs (H1 cell line) to maintain their stemness and induced the directed differentiation. A polyacrylamide hydrogel with three stiffnesses (0.14 kPa, 6.1 kPa, or 46.7 kPa) was applied as the substrate for H1 differentiation to mimic the liver stiffnesses under varied pathophysiological conditions. After inducing H1 cells into DE cells, the expressions of DE-differentiating markers, β₁-integrin and adhesion plaque associated proteins (vinculin and phosphorylated FAK), increased with the increase of stiffness. Stronger traction force and higher activation ratio of mechanosensitive protein YAP were also observed at high stiffness of 46.7 kPa, compared to those at low ones. Blocking β₁-integrin reduced the capacity of DE differentiation, while de-polymerizing F-actin and promoting YAP activation increased the DE differentiation. These results confirmed that YAP is highly activated when H1 differentiates into DE cells in vitro. Positive correlation was also found between cell traction force and YAP activation in DE differentiation of H1 cells. This work deepened the understanding of substrate stiffness in regulating DE differentiation of hESCs from a biomechanical viewpoint.

In conclusion, based on the above results of CS extract and substrate stiffness regulating the differentiation of embryonic stem cells (H9/H1), this dissertation deciphers the roles of chemical inorganic molecules on the induction of liver differentiation of stem cells in vitro, and further identifies the contributions of substrates stiffness in regulating cell traction force and affecting stem cell DE differentiation through β₁-integrin. It also uncovers the promoting effect of YAP activation on DE differentiation of H1 cells and the positive correlation between YAP activation and cell traction force. This work is conducive to understand the mechano-chemo-biological coupling mechanisms on stem cell differentiation.