The Study On Differentiation Of Neural Crest Stem Cells Modulated By Mechanical Strain

Neural crest stem cells (NCSCs) are multiponent neural stem cells and have the potential to differentiate into a wide variety of cell types, including vascular smooth muscle cells (SMCs), osteoblasts, chondrocyte, adipocytes, schwann cells and Neuron. So NCSCs are a valuable source for the construction of tissue-enginnered vascular grafts. However, it's not yet well understood how NCSCs are regulated by vascular microenvironmental factors, particularly in vivo mechanical stimuli within blood vessel wall.Blood vessel wall is constantly subjected to cyclic mechanical strain in the circumferential direction due to the pulsatile nature of the blood flow, which may play an important role in NCSC differentiation into a vascular SMC phenotype. To simulate the vascular cell alignment, soft lithography was used to create elastomeric membranes with parallel microgrooves. And its topographic pattern kept NCSCs aligned parallel/ perpendicular to the strain axis. NCSCs were derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) as a model. Cyclic uniaxial strain (5%, 1 Hz) was applied by the custom-built uniaxial stretch system. We investigated the anisotropic mechanical sensing by NCSCs, and how uniaxial mechanical strain modulates the differentiation of NCSCs into SMCs on micropatterned surfaces.This study was classified into 3 groups: Unpatterned group that NCSCs on unpatterned membranes were subjected to strain; Parrallel-patterned group that NCSCs on micropatterned membranes were subjected to strain in the direction parallel to the microgrooves; Perpendicular-patterned group that NCSCs on micropatterned membranes were subjected to strain in the direction perpendicular to the microgrooves. The three groups were examined from cell morphology, cell proliferation, gene expression, protein analysis, et al. The results were summarized below:1. Anisotropic effects of mechanical stain on NCSCs.NCSCs are multipotent and play an important role in the development and tissue regeneration. However, the anisotropic effects of mechanical strain on NCSCs are not well known. To investigate the anisotropic mechanosensing, NCSCs were cultured on micropatterned membranes, and subjected to cyclic uniaxial strain in the direction parallel or perpendicular to the microgrooves. Cell and nuclear shape were both regulated by micropatterning and mechanical strain. Among the unpatterned, parallel-patterned and perpendicular-patterned groups, mechanical strain in the parallel-patterned group caused an increase in HDAC activity, accompanied by the increase of cell proliferation. Mechanical strain in the unpatterned and parallel-patterned groups increased the expression of contractile marker calponin-1 (CNN1) and SMCs mid-stage marker smoothelin (SMTN) but not other differentiation markers. These results demonstrated that NCSCs responded differently to the anisotropic mechanical environment. In addition, Mechanical strain in the direction parallel to the microgrooves better minic vascular microenvironment, and drive the differentiation of NCSCs into SMCs.2. Uniaxial Mechanical Strain Modulates the Differentiation of Neural Crest Stem Cells into Smooth Muscle Lineage on Micropatterned Surfaces.To determine whether vascular mechanical strain modulates the differentiation of NCSCs into SMCs, the study focused on the parrallel-patterned group. NCSCs were cultured on micropatterned membranes to mimic the organization of SMCs, and subjected to cyclic uniaxial strain in the direction parallel to the microgrooves. Mechanical strain changed cell morphology, and enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 and SMCs mid-stage marker SMTN within 2 days and slightly induced mature SMC marker myosin heavy chain (MHC), but not other differentiation markers On the other hand, mechanical strain suppressed the differentiation into Schwann cells. These results suggested that mechanical strain in the direction parallel to the microgrooves drived the special differentiation of NCSCs into SMCs. The induction of CNN1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β1. For NCSCs pre-treated with TGF-β1, mechanical strain induced the gene expression of both CNN1 and MHC. These results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs.Understanding the mechanical regulation of NCSC differentiation will reveal the role of mechanical factors in NCSC differentiation during development, shed light on the remodeling of vascular tissues, and provide a basis for using NCSCs for tissue engineering.