Peter Manuel, Shipman Seth, Macklis Jeffrey D
Department of Stem Cell and Regenerative Biology, and Center for Brain Science, Harvard University, Cambridge, MA, USA.
Current Address: Gladstone Institute of Data Science and Biotechnology, and Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, USA.
bioRxiv. 2023 Aug 26:2023.08.04.552012. doi: 10.1101/2023.08.04.552012.
Differentiation of human pluripotent stem cells (hPSC) into distinct neuronal populations holds substantial potential for disease modeling , toward both elucidation of pathobiological mechanisms and screening of potential therapeutic agents. For successful differentiation of hPSCs into subtype-specific neurons using protocols, detailed understanding of the transcriptional networks and their dynamic programs regulating endogenous cell fate decisions is critical. One major roadblock is the heterochronic nature of neurodevelopment, during which distinct cells and cell types in the brain and during differentiation mature and acquire their fates in an unsynchronized manner, hindering pooled transcriptional comparisons. One potential approach is to "translate" chronologic time into linear developmental and maturational time. Attempts to partially achieve this using simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells have not been able to achieve this goal, due to asynchrony of promotor onset in individual cells. Toward solving this, we generated and tested a range of knock-in hPSC lines that express five distinct dual FP timer systems or single time-resolved fluorescent timer (FT) molecules, either in 293T cells or in human hPSCs driving expression from the endogenous paired box 6 (PAX6) promoter of cerebral cortex progenitors. While each of these dual FP or FT systems faithfully reported chronologic time when expressed from a strong inducible promoter in 293T cells, none of the tested FP/FT constructs followed the same fluorescence kinetics in developing human neural progenitor cells, and were unsuccessful in identification and isolation of distinct, developmentally synchronized cortical progenitor populations based on ratiometric fluorescence. This work highlights unique and often surprising expression kinetics and regulation in specific cell types differentiating from hPSCs.
将人类多能干细胞（hPSC）分化为不同的神经元群体，在疾病建模方面具有巨大潜力，有助于阐明病理生物学机制和筛选潜在治疗药物。要使用相关方案成功地将hPSC分化为亚型特异性神经元，详细了解调节内源性细胞命运决定的转录网络及其动态程序至关重要。一个主要障碍是神经发育的异时性，在此过程中，大脑中以及分化过程中的不同细胞和细胞类型以不同步的方式成熟并获得其命运，这阻碍了汇总转录比较。一种潜在的方法是将时间顺序转化为线性发育和成熟时间。此前试图通过使用简单的二元启动子驱动荧光蛋白（FP）来汇集相似细胞以部分实现这一目标，但由于单个细胞中启动子起始的异步性，未能实现这一目标。为了解决这个问题，我们构建并测试了一系列敲入hPSC系，这些细胞系在293T细胞或人类hPSC中表达五种不同的双FP定时器系统或单个时间分辨荧光定时器（FT）分子，由大脑皮层祖细胞的内源性配对盒6（PAX6）启动子驱动表达。虽然这些双FP或FT系统中的每一个在从293T细胞中的强诱导型启动子表达时都忠实地报告了时间顺序，但在发育中的人类神经祖细胞中，没有一个测试的FP/FT构建体遵循相同的荧光动力学，并且在基于比率荧光识别和分离不同的、发育同步的皮层祖细胞群体方面也未成功。这项工作突出了从hPSC分化而来的特定细胞类型中独特且往往令人惊讶的表达动力学和调控。
