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Summary Pluripotent stem cell-derived human cortical organoids allow for the analysis of stem cell behavior and neurogenesis in neural tissues. Delivery of plasmid DNA into organoids permits visualization of individual cells, characterization of cellular components, and manipulation of gene expression. We describe a protocol to transfect cells inside organoids with plasmid DNA using micro-injection and electroporation, allowing for DNA delivery to cells within cortical units. This protocol was optimized for cortical organoids; however, it may be adapted to other organoid models. For complete details on the use and execution of this protocol, please refer to Denoth-Lippuner et al. (2021), Graphical abstract, Highlights • Plasmid injection of pluripotent stem cell-derived human cortical organoids • Electroporation of human cortical organoids • Genetic manipulation of progenitors and their progeny in cortical organoids • Protocol enables robust DNA transfection of human cortical organoids, Pluripotent stem cell-derived human cortical organoids allow for the analysis of stem cell behavior and neurogenesis in neural tissues. Delivery of plasmid DNA into organoids permits visualization of individual cells, characterization of cellular components, and manipulation of gene expression. We describe a protocol to transfect cells inside organoids with plasmid DNA using micro-injection and electroporation, allowing for DNA delivery to cells within cortical units. This protocol was optimized for cortical organoids; however, it may be adapted to other organoid models.

Asymmetric segregation of cellular components regulates the fate and behavior of somatic stem cells. Similar to dividing budding yeast and precursor cells in Caenorhabditis elegans, it has been shown that mouse neural progenitors establish a diffusion barrier in the membrane of the endoplasmic reticulum (ER), which has been associated with asymmetric partitioning of damaged proteins and cellular age. However, the existence of an ER diffusion barrier in human cells remains unknown. Here, we used fluorescence loss in photobleaching (FLIP) imaging to show that human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells establish an ER diffusion barrier during cell division. The human ER diffusion barrier is regulated via lamin-dependent mechanisms and is associated with asymmetric segregation of mono- and polyubiquitylated damaged proteins. Further, forebrain regionalized organoids derived from hESCs were used to show the establishment of an ER membrane diffusion barrier in more naturalistic tissues, mimicking early steps of human brain development. Thus, the data provided here show that human neural progenitors establish a diffusion barrier during cell division in the membrane of the ER, which may allow for asymmetric segregation of cellular components, contributing to the fate and behavior of human neural progenitor cells.

Summary The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair., Graphical abstract, Highlights • iCOUNT reports previous cell divisions in mouse and human cells in vitro • iCOUNT detects cell division biographies in complex mouse tissues in vivo • iCOUNT allows for the analysis of human progenitors in forebrain organoids • scRNA-seq of iCOUNT cells identifies molecular consequences of previous cell divisions, In this study, Denoth-Lippuner and colleagues developed an inducible cell division counter (iCOUNT) that reports the number of previous divisions of stem cells in vitro and in vivo. Using the iCOUNT, they identify molecular changes occurring with increased cell division history in the mouse developing brain and in human brain organoids.

Somatic stem cells are required for tissue development, homeostasis, and repair. Recent data suggested that previous biographical experiences of individual stem cells influence their behavior in the context of tissue formation and govern stem cell responses to external stimuli. Here we provide a concise review how a cell's biography, for example, previous rounds of cell divisions or the age-dependent accumulation of cellular damage, is remembered in stem cells and how previous experiences affect the segregation of cellular components, thus guiding cellular behavior in vertebrate stem cells. Further, we suggest future directions of research that may help to unravel the molecular underpinnings of how past experiences guide future cellular behavior.