三维垂体基板类器官的构建和鉴定

王胜杰; 姜得悦; 秦巧臻; 江小霞; 郭清华

doi:10.12435/j.issn.2095-5227.2024.060

三维垂体基板类器官的构建和鉴定

Construction and identification of three-dimensional pituitary placode organoids

摘要

摘要:
背景垂体类器官具有广泛的应用前景，但三维垂体基板类器官相关研究十分有限。
目的将人诱导多能干细胞(human induced pluripotent stem cells，hiPSCs)在体外三维环境中诱导为三维垂体基板类器官。
方法 hiPSCs在体外经过悬浮培养形成球状体，并在一系列细胞因子、激活剂或抑制剂的作用下逐步发育成三维垂体基板类器官。定量聚合酶链式反应(quantitative polymerase chain reaction，qPCR)检测hiPSCs或类器官中Nanog同源框(Nanog homeobox，NANOG)、SIX同源框1(SIX homeobox 1，SIX1)、配对样同源域1(paired like homeodomain 1，PITX1)、LIM同源框3(LIM homeobox 3，LHX3) mRNA的表达水平，免疫荧光染色检测三维垂体基板类器官中PITX1、LHX3的表达情况。
结果 hiPSCs在体外经过悬浮培养形成球状体，随着培养时间的延长，类器官形状也从规则的球形变为不规则形。qPCR和免疫荧光染色结果显示类器官在第30、40天分别成功表达垂体基板标志物PITX1和LHX3，这标志着三维垂体基板类器官的成功构建。
结论本研究初步构建了三维垂体基板类器官，为垂体发育研究、药物开发和再生医学研究提供了实验室模型。

Abstract:
Background The three-dimensional pituitary placode organoids hold extensive potential applications, yet current research in this field remains limited.
Objective To induce human induced pluripotent stem cells (hiPSCs) into pituitary placode organoids within an in vitro three-dimensional environment.
Methods hiPSCs were cultivated in suspension to form spheroids and gradually differentiated into pituitary placode organoids through supplement with a series of cell factors, agonists, or inhibitors. qPCR was employed to measure the mRNA expression levels of NANOG, SIX1, PITX1, and LHX3 in the organoids. Immunofluorescence staining was used to detect the expression of PITX1 and LHX3 in the organoids.
Results hiPSCs formed spheroids in vitro through suspension culture. As the culture time extended, the shape of the organoids changed from regular spherical to irregular. qPCR and immunofluorescence staining revealed successful expression of the pituitary placode markers PITX1 and LHX3 in the organoids at 30 and 40 days, respectively, indicating the successful construction of pituitary placode organoids.
Conclusion This study preliminarily establishes pituitary placode organoids, providing a laboratory-based model for pituitary development research, drug development, and regenerative medicine therapies.

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