FGF4 and Retinoic Acid Direct Differentiation of hESCs into PDX1-Expressing Foregut Endoderm in a Time- and Concentration-Dependent Manner

BackgroundRetinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing β cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm- the origin of pancreatic endoderm.
Methodology/Principal FindingsHere, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1+ cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1+ cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1+ cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm.
Conclusion/SignificanceIn conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1+ foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARβ through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.

研究背景
视黄酸（Retinoic acid, RA）与成纤维细胞生长因子4（fibroblast growth factor 4, FGF4）信号通路调控内胚层模式形成与胰腺诱导及扩增。基于上述研究发现，除FGF4外的RA与成纤维细胞生长因子（fibroblast growth factors, FGFs）常被应用于干细胞分化方案，以引导人类胚胎干细胞（human embryonic stem cells, hESCs）分化为内胚层与胰腺细胞类型。在体内环境中，此类信号通路的激活呈现时间与浓度依赖性特征。然而在体外，包括RA与FGFs在内的生长与分化因子（growth and differentiation factors, GDFs）的添加时机及浓度的潜在调控机制尚不明晰。因此，为建立稳定可靠的胚胎干细胞分化方案，以获得包括胰岛素分泌β细胞在内的成熟胰腺细胞类型，从机制层面阐明每一步细胞特化过程至关重要，其中便涵盖中内胚层/定型内胚层向胰腺内胚层的起源组织——前肠内胚层的分化过程。
研究方法与主要结果
本研究提供了视黄酸与FGF4单独及联合作用的相关数据，二者可引导激活素A（Activin A, AA）诱导的人类胚胎干细胞分化为表达PDX1的细胞。迄今为止，尚未有研究验证FGF4在体外调控内胚层模式形成与细胞特化的能力。本研究通过筛选FGF4与RA的最优添加浓度及作用时机，建立了一套稳定的分化方案，该方案平均可产生32%的PDX1阳性细胞。此外，本研究证实RA是将AA诱导的人类胚胎干细胞转化为PDX1阳性细胞的必需因子，其部分潜在调控机制涉及成纤维细胞生长因子受体信号通路。最后，对PDX1阳性细胞的进一步表征分析表明，此类细胞代表尚未定向分化为胰腺、胃后部或十二指肠内胚层的前肠内胚层。
结论与意义
综上，本研究证实视黄酸与FGF4可协同以稳定高效的方式引导PDX1阳性前肠内胚层的分化。通过AA/Wnt3a早期诱导RARβ所介导的RA信号通路，是PDX1表达所必需的调控途径。RA的部分活性通过成纤维细胞生长因子信号通路介导。