Jaramillo Maria, Banerjee Ipsita
Department of Bioengineering, University of Pittsburgh, USA.
J Vis Exp. 2012 Mar 27(61):3759. doi: 10.3791/3759.
Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A in combination with several growth factors. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation. The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.
胚胎干细胞（ESC）具有两个主要特征：它们能够在体外以未分化状态无限增殖，并且具有多能性，因此有潜力分化为多种细胞谱系。这些特性使得胚胎干细胞在基于细胞的治疗和再生治疗应用中极具吸引力。然而，要充分实现其潜力，必须将这些细胞分化为成熟且具有功能的表型，这是一项艰巨的任务。诱导细胞分化的一种有前景的方法是在体外环境中紧密模拟器官发生的过程。已知胰腺发育发生在特定阶段，始于内胚层，内胚层可发育成包括肝脏和胰腺在内的多个器官。通过添加激活素A并结合多种生长因子来调节节点信号通路，可以实现内胚层诱导。确定的内胚层细胞随后通过抑制音猬因子信号来进行胰腺定向分化，这在体外可通过添加环杷明来实现。胰腺成熟由几个并行事件介导，包括抑制Notch信号；胰腺祖细胞聚集形成三维簇；诱导血管生成等等。到目前为止，通过补充DAPT抑制Notch信号，已实现了胚胎干细胞来源的胰腺祖细胞最成功的体外成熟。尽管取得了成功，但这导致成熟表型的产量较低且功能降低。一个研究较少的领域是内皮细胞信号在胰腺成熟中的作用，内皮细胞信号作为体内胰岛成熟的一个重要促成因素，其作用越来越受到重视。当前的研究探讨了内皮细胞信号在人胚胎干细胞来源的胰腺祖细胞成熟为产生胰岛素的胰岛样细胞过程中的这种作用。我们报告了一种多阶段定向分化方案，其中首先通过激活素A诱导人胚胎干细胞向内胚层分化，并抑制PI3K信号通路。通过环杷明抑制音猬因子信号并添加视黄酸诱导视黄酸信号，实现内胚层细胞的胰腺定向分化。成熟的最后阶段通过共培养配置实现的内皮细胞信号诱导。虽然在共培养中测试了几种内皮细胞，但在此我们展示了与大鼠心脏微血管内皮细胞（RHMVEC）相关的数据，主要是为了便于分析。
