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Abstract：
Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However, the substantial variability between existing protocols for generating IM cells compromises their efficiency, reproducibility, and overall success, potentially hindering the utility of urogenital system organoids. Here, we examined the role of high levels of Nodal signaling and BMP activity, as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 μM CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 μM CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence, this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro, with potential applications in regenerative medicine, drug discovery, and disease modeling.
摘要：
几种分化方案已经使得能够从人多能干细胞(hPSC)产生中间中胚层(IM)来源的细胞。然而,用于生成IM细胞的现有协议之间的实质性可变性损害了它们的效率，再现性，和整体成功，可能阻碍泌尿生殖系统类器官的效用。这里,我们检查了高水平的Nodal信号和BMP活性的作用，以及来自UCSD167i-99-1人诱导多能干细胞（hiPSC）系的IM细胞的规范中的WNT信号传导。我们证明了WNT和BMP信号传导的精确调节显着增强了IM分化效率。在分化48小时后,用3μΜCHIR99021处理hPSC诱导TBXT+/MIXL1+中胚层祖细胞(MP)。用3μMCHIR99021和4ng/mLBMP4的组合进一步处理导致在随后的48小时内产生OSR1+/GATA3+/PAX2+IM细胞。通过免疫荧光染色和RT-qPCR确认分化细胞的分子表征。因此，这项研究为hiPSC分化为IM细胞建立了一个一致且可重复的方案,该方案忠实地概括了IM发育的分子特征.该协议有望改善旨在在体外产生泌尿生殖系统类器官的协议的成功。在再生医学中的潜在应用，药物发现,和疾病建模。
