Keratinocyte Growth Factor and Dexamethasone Plus Elevated cAMP Levels Synergistically Support Pluripotent Stem Cell Differentiation into Alveolar Epithelial Type II Cells.. Mus musculus

Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application. Overall design: 10 samples in total. mESCs at d8 of differentiation (Control) mESCs at d8 of differentiation with KGF treatment mESCs at d17 of differentiation (Control) mESCs at d17 of differentiation with KGF treatment mESCs at d17 of differentiation with DCI treatment mESCs at d17 of differentiation with KGF and DCI treatment mESCs at d24 of differentiation (Control) mESCs at d24 of differentiation with KGF treatment mESCs at d24 of differentiation with DCI treatment mESCs at d24 of differentiation with KGF and DCI treatment

肺泡上皮II型（Alveolar epithelial type II, ATII）样细胞可由小鼠胚胎干细胞（murine embryonic stem cells, ESCs）诱导生成，但迄今为止尚未建立起应用特异性分化因子的稳定高效诱导方案。我们提出假说：角质细胞生长因子（keratinocyte growth factor, KGF）作为肺器官发生、原发性ATII细胞成熟与增殖的重要介导因子，联合地塞米松、8-溴腺苷-cAMP与异丁基甲基黄嘌呤（以下简称DCI）——该组合可诱导原代胎儿ATII细胞成熟——同样能够支持小鼠ESCs的肺泡分化进程。本研究证实，上述刺激因子可协同增强ESCs的肺泡分化能力，具体表现为表面活性蛋白（surfactant proteins, SP）-C与SP-B的表达水平显著升高。若KGF不仅在分化晚期施加，同时至少覆盖分化中期，该促进效应最为显著。研究结果表明，KGF大概率并非促进（中）内胚层或NK2同源框1（NK2 homeobox 1, Nkx2.1）阳性祖细胞的生成，而是在DCI的协同作用下，于分化中期加速呼吸系祖细胞的进一步分化与成熟，并在分化晚期促进新生ATII细胞的成熟与增殖。超微结构分析证实，培养体系中存在携带细胞内复合结构与板层小体的ATII样细胞。最后，我们从携带ATII细胞特异性lacZ报告基因的转基因小鼠中诱导生成了诱导多能干细胞（induced pluripotent stem cells, iPSCs）。实验结果同样显示，KGF与DCI可协同提升iPSC培养体系中SP-C与SP-B的表达水平，并诱导出表达lacZ的ATII样细胞。综上，ATII细胞特异性报告基因的表达首次为小鼠iPSC来源的ATII细胞生成提供了可靠的验证依据。此外，本研究证实KGF与DCI可协同支持ESCs与iPSCs向ATII样细胞的分化生成。联合应用这两种因子将有助于更高效地获取干细胞来源的ATII细胞，为后续基础研究与潜在治疗应用提供有力支撑。总体实验设计：共包含10个样本，具体分组如下：
1. 分化第8天的小鼠ESCs（对照组）
2. 分化第8天且经KGF处理的小鼠ESCs
3. 分化第17天的小鼠ESCs（对照组）
4. 分化第17天且经KGF处理的小鼠ESCs
5. 分化第17天且经DCI处理的小鼠ESCs
6. 分化第17天且经KGF与DCI联合处理的小鼠ESCs
7. 分化第24天的小鼠ESCs（对照组）
8. 分化第24天且经KGF处理的小鼠ESCs
9. 分化第24天且经DCI处理的小鼠ESCs
10. 分化第24天且经KGF与DCI联合处理的小鼠ESCs