Regulation Of Growth And Differentiation In Human Embryonic Stem Cells(hESCs)

Embryonic stem cells(ESCs) are pluripotent stem cells derived from the totipotent cells of preimplantation embryos, and are capable of unlimited, self-renewing proliferation in vitro when cultured under appropriate conditions. Researches found that the self-renewal and differentiation of human embryonic stem cell(hESCs) are regulated by extracellular microenvironments, including physical and mechanical environments surrounding cells, soluble molecules, cell-cell interactions and exogenous gene interferences. Mechanical microenvironment impacts the growth and differentiation of embryonic stem cells and other stem cells. Previous studies have shown that substrate stiffness and topology can independently affect early differentiation of embryonic stem cells, cell spreading morphology, proliferation and functional maintenance. However, cooperative regulation of substrate stiffness and microtopography on ESC growth and differentiation into specific cell lineage remains unknown. Exogenous gene interference to embryonic stem cells can alter their normal growth and differentiation, elucidate underlying mechanisms of ESCs growth and differentiation, and uncover unknown genes or propose new functions to known genes. Efficiency of gene transfection to ESCs, however, is quite low and optimization of gene transfection methodology is required.Upon the hypothesis that substrate mechanics and gene transfection can affect ESCs fate, the impact of stiffness coupled with topographical substrate and gene transfection on hESCs growth and differentiation was investigated extensively in this work. Outcomes are expected to be applicable, in combination with biochemical and matrix-encoded signals, in regulating differentiation of hESCs for therapeutic purposes. Two major specific aims were addressed:First, to explore the potential roles of substrate stiffness coupled with topography on directed differentiation of hESCs to hepatocytes, microfibrication technique was used to prepare polyacrylamide hydrogel substrate in the respective elasticity of 6.1 kPa and 46.7 kPa with four of square, groove, hexagonal and planar configurations. After the substrate hydrophilization, hESCs were seeded on each of the eight substrates for three days and then induced through a series stages with different differentiation-inducing medium to direct the hESCs differentiation into lined hepatocyte-like cells. It was indicated, from morphological changes, biomarker expressions at protein and gene levels, and hepatic function test, that substrate stiffness and microtopography play different roles in different stages of cell growth and differentiation. At the stage of stemness maintenance, both soft and stiff substrate are able to retain hESCs stemness where Nanog and Oct-4 genes present higher expressions on soft substrate but groove topography is not favorable for stemness maintenance. At the stage of defined endodermal lineage, stiff substrate is favorable for hESCs differentiation and endodermal biomarkers are highly expressed on hexagonal or square pillar topography. At the stage of hepatic progenitor cells, hepatocyte-like phenotype is promoted on soft substrate and high albumin production and CK18 expression are observed on planar or or square pillar topography. At the stage of mature hepatocytes, soft substrate is much better than stiff one in maintaining the function of hepatocytes while no significant differences in hepatocyte-like functions were found in four topographies. Collectively, substrate stiffness governs directed hepatic differentiation of hESCs cooperated with distinct contribution of substrate topography, implying that coupling of substrate stiffness and topography is required for enhancing the differentiating efficiency and maturity in hESCs in vitro culture and differentiation.Second, the influences of size and surface group as well as cytotoxicity and endocytosis on hESCs gene transfection was tested systematically using poly(amidoamine)(PAMAM) dendrimers ended with amine, hydroxyl, or carboxyl as model. It was found that in culture medium of mTeSR 1 the particle sizes of G5, G7, G4.5COOH, and G5 OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential, respectively, whereas G1 was slightly negative, and G4.5COOH and G5 OH were significantly negative. Only amine-terminated dendrimers were able to accomplish gene transfection in hESCs, which is greater than that from Lipofectamine 2000 transfection. Ten ?M G5 had the greatest efficiency and was better than 1000 ?M G1. Low concentration(0.5 and 1 ?M) of G7 could realize gene delivery. Amine-ended dendrimers, especially with higher generations, were detrimental to the growth and pluripotent maintenance of hESCs. In contrast, similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity, in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. Endocytosis into and significant exocytosis from hESCs were also confirmed using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size, concentration, and zeta potential, particularly the identity and position of groups, as well as minimized exocytosis are required in designing a vector for hESCs gene delivery, which helps to optimize an effective way in hESCs gene transduction.