mESC衍生内皮祖细胞定向分化为血管内皮细胞促伤口愈合

缺血性功能障碍是重要的全球健康问题。血管内皮细胞 (vascular endothelial cell, VEC) 在血管生成和创面修复中发挥关键作用,血管重建不足可导致慢性不愈合伤口。因此,了解有效的血管内皮细胞生成策略有助于受损组织中的血管再生。胚胎干细胞 (embryonic stem cell, ESC) 在组织的内皮化研究中应用广泛。内皮祖细胞 (endothelial progenitor cell, EPC) 是血管内皮细胞发育中不可或缺的部分。本研究目的在于找到一种小鼠胚胎干细胞 (mouse embryonic stem cell, mESC) 衍生为内皮祖细胞的快速、易筛选且高重复性的方法,并从内皮祖细胞定向分化中获得存活率高和功能性好的血管内皮细胞。结果表明,胚胎干细胞通过10 ng/mL VEGF和5 ng/mL bFGF定向诱导分化为增殖能力强的“铺路石”样祖细胞。同时,差异贴壁法有助于EPC的筛选。而EPC可诱导3 d的祖细胞高表达CD133和CD34(相对表达量分别为0.88 ± 0.04和2.12 ± 0.02);采用acctuse酶消化祖细胞,并在50 ng/mL VEGF和25 ng/mL bFGF的条件下诱导7 d分化为血管内皮样细胞,该细胞不仅高表达内皮细胞标志基因CD31、CD144、LAMA5、Tek、KDR和vWF,高表达标志蛋白CD31、CD144、LAMA5(相对表达量分别为1.07 ± 0.03、0.60 ± 0.02和0.70 ± 0.02),而且具有良好的迁移、成管和Weibel Palade (W-P) 小体形成能力。随后,将PBS、EPC和VEC分别应用于大小相同的创面治疗,EPC和VEC均能加快组织愈合程度(相对愈合率分别为78.93 ± 75.35%、95.57 ± 83.73%和100.00 ± 0.00%),VEC明显增强了伤口的血管生成能力和炎症反应。该研究初步证实,mESC衍生的EPC定向诱导7 d后可分化为血管内皮细胞。此内皮细胞具有较好的组织修复功能,干细胞促进血管生成的生理途径有望成为组织重塑的新靶点。

Endothelial Progenitor Cells Derived from mESCs Were Directed to Differentiate into Vascular Endothelial Cells and Promote Wound Healing

Ischemic dysfunction is an important global health problem. Vascular endothelial cells (VECs) play a key role in angiogenesis, and insufficient vascular remodeling may lead to chronic non-healing wounds. Therefore, effective VEC generation strategies of exploration help improve angiogenesis in damaged tissues. Embryonic stem cells (ESCs) are widely used in the study of tissue endothelialization, and endothelial progenitor cells (EPCs) are indispensable parts of the development of VECs. The aims of this study were to find a rapid, easily screened and reproducible method for the derivation of EPCs from mouse embryonic stem cells (mESCs), and obtain VECs with high survival rates and strong functions from the directed differentiation of the EPCs. The results showed that mESCs were differentiated into "stepping stone"-like progenitor cells with active proliferative ability by 10 ng/mL VEGF and 5 ng/mL bFGF. At the same time, the method of differential adherence was helpful for the selection of EPCs, and EPCs induced high expression of CD133 and CD34 (The relative expression levels were 0.88 ± 0.04 and 2.12 ± 0.02, respectively) for 3 days. Then EPCs were digested with acctuse enzymes, and induced to differentiate into vascular endothelial-like cells by 50 ng/mL VEGF and 25 ng/mL bFGF for 7 days. The endothelial cells not only expressed endothelial marker genes (CD31, CD144, LAMA5, Tek, KDR and vWF),and marker proteins CD31, CD144 and LAMA5 (The relative expression levels were 1.07 ± 0.03, 0.60 ± 0.02 and 0.70 ± 0.02, respectively), but also had the good ability of migration, tubulogenesis and formation of W-P bodies. Moreover, PBS, EPC and VEC were used to treat wounds of the same size. Both EPC and VEC could accelerate the degree of tissue healing (The relative healing rates were 78.93 ± 75.35%, 95.57 ± 83.73% and 100.00 ± 0.00%, respectively), and VEC significantly enhanced the ability of wound angiogenesis and inflammatory responses. In consequence, this study preliminarily confirmed that mESC-derived EPCs could differentiate into VECs after directional induction for 7 days, which had good function of tissue repair. The physiological pathway on stem cells by stimulating angiogenesis is expected to become a new target for tissue remodeling.