Résumé
BACKGROUND:The quantity and quality of seed cel s is a critical bottleneck of the development of vascular tissue engineering. To address this issue, stem cel-derived endothelial cel s have been a hot spot in this field due to their potential in providing the ideal seed cel s. OBJECTIVE:To elucidate the effect of vascular endothelial growth factor (VEGF) supplementation combined with hypoxic culture condition on the lineage-specific differentiation of embryonic stem cel s into endothelial cel s. METHODS:Serum-free medium mTeSR?1 was applied to cultivate H9 cel s in vitro. A conditioned medium containing 50μg/L vascular endothelial growth factor was utilized to induce H9 cel s to differentiate into endothelial cel s under the hypoxic culture condition (5%O2). The cel under normal condition (5%CO2) with or without vascular endothelial growth factor served as controls. The phenotype and function of human embryonic stem cel s-derived endothelial cel s were assayed by immunofluorescence staining, quantitative RT-PCR, and low-density lipoprotein uptake experiment. RESULTS AND CONCLUSION:Compared with the control group, the H9 cel s were induced to be differentiated into endothelial-like cel s more efficiently when they were cultivated under a conditioned medium with vascular endothelial growth factor supplementation under the hypoxic condition. These differentiated cel s not only expressed some important surface markers of endothelia cel s, including kdr, pecam, but also took in low-density lipoprotein to form microvessle-like structures. This culture system supports a synergy effect of vascular endothelial growth factor and hypoxic environment that can efficiently promotes the lineage-specific differentiation of embryonic stem cel s into endothelial cel s with good phenotype and functionality.
Résumé
BACKGROUND:Constructing a three-dimensional tissue-like structure in vitro plays a critical role in modern tissue engineering and regenerative medicine. Several advances have been made in the past decade. However, it is stil a chal enge to promote microvascular-like structure formation and improve limited nutritional transportation, thereby promoting cel viability. OBJECTIVE:To explore the feasibility of constructing a three-dimensional microvascular-like structure through the co-culture technique. METHODS:Human bone marrow mesenchymal stem cel s and human endothelial cel s were co-cultured on a three-dimensional porous silk scaffold. Cel proliferation was analyzed by Pico-green DNA assay. Their growth profiles were evaluated by scanning electron microscope and laser scanning confocal microscopy, respectively. The mRNA levels of von Wil ebrand factor and CD31, two key functional markers of endothelial cel s, in the co-cultured endothelial cel s was assayed by real-time quantitative reverse transcription-PCR. RESULTS AND CONCLUSION:The three-dimensional culture system constructed by the silk scaffold and bone marrow mesenchymal stem cel s provided an ideal microenvironment for cel growth and proliferation in vitro. Moreover, this microenvironment was capable of promoting endothelial cel differentiation evidenced by their significantly improved mRNA levels of von Wil ebrand factor and CD31. Premicrovascular-like structure was also observed in the co-cultures under the confocal microscope. Thus, al the data supported that the unique co-culture system could promote endothelial cel differentiation and self-assembling in vitro. This culture system provides a robust tool for the studies addressing microvessel-based tissue engineering.
Résumé
Objective To simulate the three-dimensional(3-D) growth pattern of stem cells in vivo by a 3-D culture system in vitro constructed by rat tail collagen scaffold.Methods Circular strands were prepared by mixing suspended murine embryonic stem cells(mESCs) with rat tail collagen.Growth profile of mESCs within the collagen strand was observed with phase contrast microscopy.Their metabolic activity was evaluated by glucose/lactic acid contents.To evaluate the effect of 3D culture system on ESCs differentiation,ES-derived cardiomyocytes were detected by immunohistochemistry,RT-PCR and transmission electron microscopy respectively.Results ESCs grew well in the 3-D culture system constructed by rat tail collagen.Cell connections can be found in those cell clusters formed within collagen stands,which indicated that tissue-like cultures should be produced during the process of 3-D culture in vitro.ESCs cultured by 3-D collagen strand differentiated into cardiomyocytes spontaneously.Conclusion Collagen provides an ideal growth matrix for ESCs proliferation in vitro and promotes ESCs differentiation towards tissue-like structures.Thus,the three dimensional culture system constructed by rat tail collagen can be applied to study ESCs differentiation in vivo.