Ben-Reuven Lihi, Reiner Orly
Weizmann Institute of Science, Rehovot 7610001, Israel.
Bioengineering (Basel). 2020 Dec 18;7(4):164. doi: 10.3390/bioengineering7040164.
Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids). These organoids exhibit remarkable self-organization that resembles key aspects of in vivo organ development. However, organoids have an unpredictable anatomy, and poorly reflect the topography of the dorsoventral, mediolateral, and anteroposterior axes. In vivo the temporal and the spatial patterning of the developing tissue is orchestrated by signaling molecules called morphogens. Here, we used morphogen-soaked beads to influence the spatial identities within hESC-derived brain organoids. The morphogen- and synthetic molecules-soaked beads were interpreted as local organizers, and key transcription factor expression levels within the organoids were affected as a function of the distance from the bead. We used an on-chip imaging device that we have developed, that allows live imaging of the developing hESC-derived organoids. This platform enabled studying the effect of changes in WNT/BMP gradients on the expression of key landmark genes in the on-chip human brain organoids. Titration of CHIR99201 (WNT agonist) and BMP4 directed the expression of telencephalon and medial pallium genes; dorsal and ventral midbrain markers; and isthmus-related genes. Overall, our protocol provides an opportunity to study phenotypes of altered regional specification and defected connectivity, which are found in neurodevelopmental diseases.
干细胞技术的最新进展包括将人类胚胎干细胞（hESC）分化为类器官结构（类器官）。这些类器官展现出显著的自我组织能力，类似于体内器官发育的关键方面。然而，类器官的解剖结构不可预测，并且不能很好地反映背腹、内外侧和前后轴的地形。在体内，发育中组织的时空模式由称为形态发生素的信号分子精心编排。在此，我们使用浸泡有形态发生素的珠子来影响hESC来源的脑类器官内的空间身份。浸泡有形态发生素和合成分子的珠子被视为局部组织者，类器官内关键转录因子的表达水平受距珠子距离的影响。我们使用了自己开发的芯片成像设备，该设备能够对发育中的hESC来源的类器官进行实时成像。这个平台使得研究WNT/BMP梯度变化对芯片上人类脑类器官中关键标志性基因表达的影响成为可能。CHIR99201（WNT激动剂）和BMP4的滴定指导了端脑和内侧 pallium 基因、背侧和腹侧中脑标记物以及峡部相关基因的表达。总体而言，我们的方案提供了一个研究神经发育疾病中发现的区域特化改变和连接缺陷表型的机会。
