The Effect Of Topography And Component Of Cellular Microenvironment On ESC Behaviors

Embryonic stem cells(ESCs)are pluripotent cells with the ability to differentiate into all somatic cell types.Due to this distinct property,ESCs hold great promise for regenerative medicine and tissue engineering.Although most scientists agree on the potential of ESCs,it has also become clear that pluripotency is a double-edged sword:the same plasticity that permits ESCs to generate hundreds of different cell types also makes them difficult to control.The challenge of controlling ESC behavior has therefore attracted considerable interest among researchers.This work focuses on the effect of physical property of biomaterials and the chemical component of cellular microenvironment,as well as their combined effect in regulating ESC behaviors.We commenced by investigating the effect of surface topography-roughness,one of the most basic properties of biomaterials,on the pluripotency maintenance and osteogenic differentiation of mouse ESCs(mESCs).The results provide useful information for the design of biomaterials used for stem cell culture and site-specific differentiation.In addition,considering the importance of heparin in controlling ESC differentiation,a heparin-mimicking biomolecule-β-CD sulfonate(CD-S)was designed and utilized to change the chemical composition of cellular microenvironments in order to guide neural differentiation of mESCs.These steps led to the development of a surface-mediated photoporation strategy that combined the effect of topography and chemical component of cellular microenvironment on ESC behavior.We were able to achieve high-efficiency delivery of different macromolecules into various cell types.This platform has the potential to be used for regulating ESC properties by macromolecular delivery and the preparation of induced pluripotent stem(iPS)cells.Detailed research contents are as follows:(Ⅰ)The effect of surface topography-surface roughness on long-term pluripotency maintenance of mESCs:Gold nanoparticle layer(GNPL)with micro/nano structures was deposited onto gold surface via a chemical plating method.The surface roughness of GNPL was tuned from nano-to microscale by simply changing the volume of the gold plating solution.SEM and AFM were used to characterize the surface morphology and roughness of GNPL.It was found that GNPL with nanoscale and low sub-microscale surface roughnesses(Rq ≤ 392 nm)provided good support for the long-term maintenance of mESC pluripotency.However,GNPL with high sub-microscale and microscale surface roughnesses(Rq ≥ 573 nm)decreased cell pluripotency from day 3,and at day 7,the loss of pluripotency was more significant.The Oct-4 expression level in mESCs on microrough GNPL after 7 days decreased by approximately 50%compared with cells grown on smooth gold surface(p<0.001).E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions are engaged in the topological sensing of mESCs.Similar to the effect on smooth gold surface,the expression of E-cadherin(a cell-cell interaction protein)and vinculin(a focal adhesion protein)on nanorough GNPL was much stronger than on microrough GNPL,indicating that strong cell-cell and cell-matrix interactions on nanorough GNPL are responsible for the long-term pluripotency maintenance of mESCs.(Ⅱ)The effect of surface topography-surface roughness on the proliferation and osteogenic differentiation of mESCs:The proliferation results from mESCs grown on GNPL with surface roughness ranging from nano-to microscale showed that even though surface roughness decreased cell attachment,GNPLs with nanoscale and low sub-microscale surface roughnesses(Rq≤392 nm)provided better support for cell proliferation than did the smooth Au and GNPLs with high sub-microscale and microscale surface roughnesses(Rq ≥ 573 nm).In addition,we found that osteogenic differentiation of mESCs can happen on GNPL with surface roughness lower than 392 nm.After induction for 7 days,cells on these surfaces exhibited significant morphological changes.As the cell colonies disappeared,cells migrated from the colonies and began to grow in the form of a monolayer.These cells contained visible nuclei and irregular protruding shapes,such as spindles and polygons.In contrast,no obvious morphological change was observed in the cells cultured on GNPL with surface roughnesses greater than 573 nm.Furthermore the ALP(alkaline phosphatase)activity of cells on these surfaces was much lower than on smooth gold surface and GNPL with surface roughness lower than 392 nm.(Ⅲ)The effect of chemical component of cellular microenvironment on the differentiation of mESCs:Heparin-mimicking molecule-β-CD sulfonate(CD-S)was synthesized using a click reaction.Results from NMR,FTIR and MALDI-ToF proved the successful synthesis of CD-S.We found that CD-S showed a similar effect to heparin on the proliferation and neural induction of mESCs.In addition,CD-S can serve as a carrier for all trans retinoic acid(RA,commonly used drug for neural differentiation of stem cells)by forming inclusion complexes that improved the bioactivity of RA in inducing neural differentiation of mESCs.The promotion effect of the CD-S/RA inclusion complex was even greater than that of heparin and RA,presumably due to the synergism between CD-S and RA on stem cell differentiation.(IV)By combing the effect of topography and the chemical components of the cellular microenvironment on ESC behaviors,a GNPL-mediated photoporation strategy was developed for macromolecular delivery into cells.Upon irradiation with an 808 nm continuous-wave laser light,the GNPL surface showed such good photothermal properties that absorption of the laser energy caused a rapid increase in surface temperature.At a powder density of 5.1 W/cm2,the surface temperature of GNPL increased to over 100 ℃after only 30 s,while the temperature of smooth gold surface only stayed at about 40 ℃.The dramatic temperature increase of the GNPL surface under laser irradiation led to enhanced membrane permeability of the cultured cells and the diffusion of macromolecules into the cytosol from the surrounding medium.The high-efficiency delivery of different macromolecules such as dextran and plasmid DNA into different cell types was achieved.For plasmid DNA transfection towards easy-to-transfect cell type such as Hela cells,the transduction efficiency was almost 100%,which is comparable to that obtained by commercially available transfection reagent-Lipofectamine 2000(Lipo2000).More importantly,the platform vastly outperformed Lipo2000 in transfecting hard-to-transfect cell types including mouse embryonic fibroblasts(mEFs)and human umbilical vein endothelial cells(HUVECs).The plasmid transfection efficiency was ≈53%vs ≈19%for mEFs and ≈44%vs ≈8%for HUVECs.More importantly,as the gold nanoparticles(GNPs)constituting the GNPL are firmly immobilized together,the potential cytotoxicity caused by endocytosis of GNPs is effectively avoided.This platform opens up an innovative avenue for high-efficiency intracellular delivery and has the potential to be used for the regulation of ESC behaviors by macromolecular delivery and preparation of iPS.