Sepponen Kirsi, Lundin Karolina, Knuus Katri, Väyrynen Pia, Raivio Taneli, Tapanainen Juha S, Tuuri Timo
Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki 00029, Finland.
Department of Physiology, University of Helsinki, Helsinki 00014, Finland.
J Clin Endocrinol Metab. 2017 Nov 1;102(11):4303-4314. doi: 10.1210/jc.2017-01469.
Human gonads arise as a pair of epithelial ridges on the surface of intermediate mesoderm (IM)-derived mesonephros. Toxic environmental factors and mutations in various genes are known to disturb normal gonadal development, but because of a lack of suitable in vitro models, detailed studies characterizing the molecular basis of the observed defects have not been performed.
To establish an in vitro method for studying differentiation of bipotential gonadal progenitors by using human embryonic stem cells (hESCs) and to investigate the role of bone morphogenetic protein (BMP) in gonadal differentiation.
We tested 17 protocols using activin A, CHIR-99021, and varying durations of BMP-7 and the BMP inhibitor dorsomorphin. Activation of activin A, WNT, and BMP pathways was optimized to induce differentiation.
Academic research laboratory.
Cell differentiation, gene expression, and flow cytometry.
The two most efficient protocols consistently upregulated IM markers LHX1, PAX2, and OSR1 at days 2 to 4 and bipotential gonadal markers EMX2, GATA4, WT1, and LHX9 at day 8 of culture. The outcome depended on the combination of the duration, concentration, and type of BMP activation and the length of WNT signaling. Adjusting any of the parameters substantially affected the requirements for other parameters.
We have established a reproducible protocol for directed differentiation of hESCs into bipotential gonadal cells. The protocol can be used to model early gonadal development in humans and allows further differentiation to mature gonadal somatic cells.
人类性腺起源于中胚层（IM）衍生的中肾表面的一对上皮嵴。已知有毒环境因素和各种基因的突变会干扰性腺的正常发育，但由于缺乏合适的体外模型，尚未对观察到的缺陷的分子基础进行详细研究。
建立一种利用人类胚胎干细胞（hESCs）研究双潜能性腺祖细胞分化的体外方法，并研究骨形态发生蛋白（BMP）在性腺分化中的作用。
我们测试了17种使用激活素A、CHIR-99021以及不同持续时间的BMP-7和BMP抑制剂多穗柯碱的方案。优化激活素A、WNT和BMP信号通路的激活以诱导分化。
学术研究实验室。
细胞分化、基因表达和流式细胞术。
两种最有效的方案在培养第2至4天持续上调IM标志物LHX1、PAX2和OSR1，并在培养第8天上调双潜能性腺标志物EMX2、GATA4、WT1和LHX9。结果取决于BMP激活的持续时间、浓度和类型以及WNT信号传导的长度的组合。调整任何一个参数都会对其他参数的要求产生重大影响。
我们已经建立了一种可重复的方案，用于将hESCs定向分化为双潜能性腺细胞。该方案可用于模拟人类早期性腺发育，并允许进一步分化为成熟的性腺体细胞。
