Graduate Program in Quantitative and Systems Biology, University of California, Merced, California, USA.
Stem Cells Dev. 2011 Dec;20(12):2153-61. doi: 10.1089/scd.2010.0432. Epub 2011 May 13.
Endothelial cells (ECs) are desired for their therapeutic potential in a variety of areas including gene therapy, cardiac regeneration, development of tissue-engineered vascular grafts, and prevascularized tissue transplants. Pluripotent embryonic stem cells (ESCs) can be induced to differentiate into ECs in vitro using embryoid bodies, monolayer cultures, or by genetic manipulation and immortalization. However, obtaining homogeneous cultures of proliferating ESC-derived ECs without genetic manipulation is a challenging undertaking and often requires optimization of protocols and rigorous purification techniques. Moreover, current differentiation methods that use medium containing fetal calf or bovine serum components introduce additional challenges because of our limited ability to control the differentiation signals and batch-to-batch variations of serum. We have explored the development of new medium formulations for deriving ECs from murine embryonic stem cells (mESCs) using only chemically defined reagents. We present 2 different medium formulations along with the detailed methodologies, including the optimization of extracellular matrix-derived substrates known to play a role in cell attachment and proliferation as well as cell differentiation. Characterization of the ESC-derived ECs indicate that (1) chemically defined medium formulations reproducibly generate superior ECs compared with previous serum-containing formulations, (2) fibronectin, and not collagen type-IV, is the optimal substrate for EC induction in our chemically defined medium formulations, (3) without additional activation of Notch-signaling, ESC-ECs develop predominantly into venous ECs, and (4) using these medium formulations, a second rigorous selection step is not required to generate proliferating ECs from ESCs, but it does enhance the final purity of the ECs.
内皮细胞 (ECs) 因其在多种领域的治疗潜力而备受关注，包括基因治疗、心脏再生、组织工程血管移植物的开发以及预血管化组织移植。多能胚胎干细胞 (ESCs) 可以通过胚状体、单层培养或通过遗传操作和永生化在体外诱导分化为 ECs。然而，在没有遗传操作的情况下获得增殖的 ESC 来源的 ECs 的同质培养是一项具有挑战性的任务，通常需要优化方案和严格的纯化技术。此外，目前使用含有胎牛或牛血清成分的培养基的分化方法由于我们控制分化信号和批次间血清变化的能力有限而带来了额外的挑战。我们已经探索了使用仅化学定义的试剂从鼠胚胎干细胞 (mESCs) 中获得 EC 的新培养基配方。我们提出了 2 种不同的培养基配方以及详细的方法学，包括对细胞附着和增殖以及细胞分化起作用的细胞外基质衍生底物的优化。对 ESC 来源的 ECs 的特征分析表明：(1) 化学定义的培养基配方可重复性地产生优于以前含血清配方的 ECs；(2) 纤连蛋白而不是 IV 型胶原是我们化学定义的培养基配方中诱导 EC 的最佳底物；(3) 在没有 Notch 信号通路的额外激活下，ESC-EC 主要发育成静脉 EC；(4) 使用这些培养基配方，从 ESCs 生成增殖的 EC 不需要进行第二次严格的选择步骤，但确实可以提高 EC 的最终纯度。
