Nankai University School of Medicine, Tianjin, China; State Key Laboratory of Medicinal Chemical Biology, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, The College of Life Science, Tianjin, China.
Nankai University School of Medicine, Tianjin, China.
Acta Biomater. 2017 Nov;63:190-199. doi: 10.1016/j.actbio.2017.08.037. Epub 2017 Aug 30.
Transplantation of endothelial cells (ECs) holds great promise for treating various kinds of ischemic diseases. However, the major challenge in ECs-based therapy in clinical applications is to provide high quality and enough amounts of cells. In this study, we developed a simple and efficient system to direct endothelial differentiation of mouse embryonic stem cells (ESCs) using a controllable chitosan nitric oxide (NO)-releasing hydrogel (CS-NO). ESCs were plated onto the hydrogel culture system, and the expressions of differentiation markers were measured. We found that the expression of Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) increased obviously under the controlled NO releasing environment. Moreover, the Flk-1 upregulation was accompanied by the activation of the phospho-inositide-3 kinase (PI3K)/Akt signaling. We also found that in the presence of the PI3K inhibitor (LY294002), the endothelial commitment of ESCs was abolished, indicating the importance of Akt phosphorylation in the endothelial differentiation of ESCs. Interestingly, in the absence of NO, the activation of Akt phosphorylation alone by using AKT activator (SC-79) did not profoundly promote the endothelial differentiation of ESCs, suggesting an interdependent relationship between NO and the Akt phosphorylation in driving endothelial fate specification of ESCs. Taken together, we demonstrated that NO releasing in a continuous and controlled manner is a simple and efficient method for directing the endothelial differentiation of ESCs without adding growth factors.
Fascinating data continues to show that artificial stem cell niche not only serve as a physical supporting scaffold for stem cells proliferation, but also as a novel platform for directing stem cell differentiation. Because of the lack of proper microenvironment for generating therapeutic endothelial cells (ECs) in vitro, the source of ECs for transplantation is the major limitation in ECs-based therapy to clinical applications. The current study established a feeder cell-free, 2-dimensional culture system for promoting the differentiation processes of embryonic stem cells (ESCs) committed to the endothelial lineage via using a nitric oxide (NO) controlled releasing hydrogel (CS-NO). Notably, the NO releasing from the hydrogel could selectively up-regulate Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) in the absence of growth factors, which was of crucial importance in the endothelial differentiation of ESCs. In summary, the current study proposes a simple and efficient method for directing the endothelial differentiation of ESCs without extra growth factors.
探讨内皮细胞（ECs）移植在治疗各种缺血性疾病中的应用价值。然而，在临床应用中，基于 ECs 的治疗的主要挑战是提供高质量和足够数量的细胞。本研究旨在利用可控壳聚糖一氧化氮（NO）释放水凝胶（CS-NO）开发一种简单有效的方法来指导小鼠胚胎干细胞（ESCs）向内皮细胞的分化。将 ESCs 接种到水凝胶培养系统中，测量分化标志物的表达。我们发现，在可控 NO 释放环境下，Flk-1（早期 ECs 标志物）和 VE-cadherin（成熟 ECs 标志物）的表达明显增加。此外，Flk-1 的上调伴随着磷酸肌醇-3 激酶（PI3K）/Akt 信号的激活。我们还发现，在存在 PI3K 抑制剂（LY294002）的情况下，ESCs 的内皮细胞定向分化被阻断，表明 Akt 磷酸化在 ESCs 的内皮细胞分化中很重要。有趣的是，在没有 NO 的情况下，单独使用 Akt 激活剂（SC-79）激活 Akt 磷酸化并不能显著促进 ESCs 的内皮分化，这表明 NO 和 Akt 磷酸化在驱动 ESCs 内皮命运特化方面存在相互依赖关系。总之，我们证明了以连续和可控的方式释放 NO 是一种简单有效的方法，可指导 ESCs 的内皮分化，而无需添加生长因子。
不断涌现的数据继续表明，人工干细胞龛不仅作为干细胞增殖的物理支撑支架，而且作为指导干细胞分化的新平台。由于体外生成治疗性内皮细胞（ECs）的适当微环境缺失，因此，ECs 用于移植的来源是将 ECs 为基础的治疗应用于临床的主要限制。本研究建立了一个无饲养细胞的二维培养系统，通过使用一氧化氮（NO）控制释放水凝胶（CS-NO）来促进胚胎干细胞（ESCs）向内皮谱系分化。值得注意的是，水凝胶中 NO 的释放可以在没有生长因子的情况下选择性地上调 Flk-1（早期 ECs 标志物）和 VE-cadherin（成熟 ECs 标志物），这对于 ESCs 的内皮分化至关重要。总之，本研究提出了一种无需额外生长因子即可指导 ESCs 向内皮细胞分化的简单有效的方法。
