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During human forebrain development, neural progenitor cells (NPCs) in the ventricular zone (VZ) undergo asymmetric cell divisions to produce a self-renewed progenitor cell, maintaining the potential to go through additional rounds of cell divisions, and differentiating daughter cells, populating the developing cortex. Previous work in the embryonic rodent brain suggested that the preferential inheritance of the pre-existing (older) centrosome to the self-renewed progenitor cell is required to maintain stem cell properties, ensuring proper neurogenesis. If asymmetric segregation of centrosomes occurs in NPCs of the developing human brain, which depends on unique molecular regulators and species-specific cellular composition, remains unknown. Using a novel, recombination-induced tag exchange (RITE)-based genetic tool to birthdate and track the segregation of centrosomes over multiple cell divisions in human embryonic stem cell (hESC)-derived regionalized forebrain organoids, we show the preferential inheritance of the older mother centrosome towards self-renewed NPCs. Aberration of asymmetric segregation of centrosomes by genetic manipulation of the centrosomal, microtubule-associated protein Ninein alters fate decisions of NPCs and their maintenance in the VZ of human cortical organoids. Thus, the data described here use a novel genetic approach to birthdate centrosomes in human cells and identify asymmetric inheritance of centrosomes as a mechanism to maintain self-renewal properties and to ensure proper neurogenesis in human NPCs.Impact StatementGenetic birthdating in forebrain organoids shows asymmetric inheritance of centrosomes in human neural progenitor cells, required for proper human neurogenesis.

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.

Asymmetric segregation of cellular components regulates the fate and behavior of somatic stem cells. Similar to dividing budding yeast and precursor cells in C. 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 polyubiquitinated, 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.SummaryHuman neural progenitors (NPCs) establish a diffusion barrier during cell division in the membrane of the endoplasmic reticulum, allowing for asymmetric segregation of cellular components, which may contribute to the fate and behavior of human NPCs.

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.

SummaryThe division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. We developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analysing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT allows for novel insights into stem cell behaviour. Further, we used single cell RNA-sequencing (scRNA-seq) of iCOUNT-labelled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a novel 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.HighlightsiCOUNT reports previous cell divisions in mouse and human cells in vitroiCOUNT detects cell division biographies in complex mouse tissues in vivoiCOUNT allows for the analysis of human neural stem/progenitor cells in human forebrain organoidsSingle cell RNA-sequencing of iCOUNT cells derived from the mouse developing cortex and human forebrain organoids identifies molecular consequences of previous rounds of cell divisionsGraphical abstract

Iris neural stem/progenitor cells from mature porcine eyes were investigated using a new protocol for tissue culture, which consists of dispase treatment and Matrigel embedding. We used a number of culture conditions and found an intense differentiation of neuronal cells from both the iris pigmented epithelial (IPE) cells and the stroma tissue cells. Rod photoreceptor-like cells were also observed but mostly in a later stage of culture. Neuronal differentiation does not require any additives such as fetal bovine serum or FGF2, although FGF2 and IGF2 appeared to promote neural differentiation in the IPE cultures. Furthermore, the stroma-derived cells were able to be maintained in vitro indefinitely. The evolutionary similarity between humans and domestic pigs highlight the potential for this methodology in the modeling of human diseases and characterizing human ocular stem cells.