Johannesson Martina, Ståhlberg Anders, Ameri Jacqueline, Sand Fredrik Wolfhagen, Norrman Karin, Semb Henrik
Lund Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
PLoS One. 2009;4(3):e4794. doi: 10.1371/journal.pone.0004794. Epub 2009 Mar 11.
Retinoic 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 beta 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 FINDINGS: Here, 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/SIGNIFICANCE: In 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 RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.
视黄酸（RA）和成纤维细胞生长因子4（FGF4）信号通路控制内胚层模式形成以及胰腺诱导/扩张。基于这些发现，除FGF4外，RA和其他成纤维细胞生长因子（FGFs）经常被用于分化方案中，以指导人胚胎干细胞（hESCs）向内胚层和胰腺细胞类型分化。在体内，这些信号通路以时间和浓度依赖性方式发挥作用。然而，在体外，包括RA和FGFs在内的生长和分化因子（GDFs）添加时间以及浓度的潜在依据尚不明确。因此，为了开发出将胚胎干细胞（ESCs）可靠且稳健地分化为成熟胰腺细胞类型（包括产生胰岛素的β细胞）的方案，从机制上理解每个分化步骤将非常重要。这包括中内胚层/定形内胚层向前肠内胚层的分化——胰腺内胚层的起源。
方法/主要发现：在此，我们提供了关于RA和FGF4在指导激活素A（AA）诱导的hESCs分化为表达PDX1的细胞过程中的单独作用及联合作用的数据。迄今为止，FGF4在体外影响内胚层模式形成和分化的能力尚未得到测试。通过测试FGF4和RA添加的最佳浓度和时间，我们提出了一种稳健的分化方案，平均可产生32%的PDX1(+)细胞。此外，我们表明RA是将AA诱导的hESCs转化为PDX1(+)细胞所必需的，其部分潜在机制涉及FGF受体信号传导。最后，对PDX1(+)细胞的进一步表征表明，它们代表尚未定向分化为胰腺、后胃或十二指肠内胚层的前肠内胚层。
结论/意义：总之，我们表明RA和FGF4以稳健且高效的方式共同指导PDX(+)前肠内胚层的分化。通过AA/Wnt3a早期诱导RARβ介导的RA信号传导是PDX1表达所必需的。RA的部分活性由FGF信号传导介导。
