Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.This work was supported by NIRM/ Clevers and Stichting Vrienden van het Hubrecht (O.B.), EU/232814-StemCellMark and Skolkovo 077 MPA (J.H.v.E.), NIH/MIT Subaward 5710002735 (to D.E.S.), KWF/PF-HUBR 2007-3956 and Stichting Vrienden van het Hubrecht (M.v.d.W.), European Research Council Advanced Grant ERC-AdG 294325-GeneNoiseControl (to K.W. and A.v.O.), and Wellcome Trust Grant 098357/Z/12/Z (to B.D.S.)

Decrypting gastrointestinal development and homeostasis one cell at a time

In this thesis, I have applied multiple single-cell techniques to advance our understanding of how tissues and organs develop and maintain homeostasis. Cells are known to have heterogeneous transcriptomes; even isogenic cells in identical culture conditions have different transcriptional profiles and exhibit distinct phenotypes. In organs, multiple cell types are exposed to diverse microenvironments, making this cellular heterogeneity even more apparent. This heterogeneity is lost when tissues are analyzed in bulk, which only yields a population average. Hence, to fully understand the complexity of an organ or tissue, single cell biology is paramount. In the first study, we developed a new method for sorting cells based on RNA abundance. By fluorescently labeling cells with dozens of probes, we are able to isolate cells by fluorescent-activated cell sorting with a resolution of 10-20 transcripts. Moreover, by preserving the quality of total RNA during probe hybridization, we are able to perform unbiased transcriptional profiling on the sorted subpopulation, e.g. by RNA-sequencing. The subsequent four studies all employ recently developed single-cell RNA sequencing to analyze single cells. By sorting and analyzing Paneth cells from the murine small intestine, we revealed the maturation trajectory of the Paneth cell from a stem cell onwards. Moreover, differential gene expression between two clusters of mature Paneth cells showed the existence of two distinct maturation states, indicated by e.g. the expression of a subset of antimicrobial defensin genes. Next, we used a similar strategy to study Lgr5+ stem cells in different gastrointestinal organs spread over three developmental time points. We isolated Lgr5+ stem cells from the stomach, small intestine and colon at E13.5, E18.5 and from adult. Our data shows large differences between stem cells in the intestine and stomach in adult, e.g. by the expression of many organ-specific differentiation markers. However, at E13.5, the transcriptional profiles of all three organs are remarkably similar, suggesting a ‘naïve’ Lgr5+ stem cell at early embryogenesis that later becomes organ specific. Preliminary data suggests a role for DNA methylation in this process. In addition to in vivo studies, we also made use of the ex vivo organoid system to study quiescence in Lgr5+ stem cells and the formation of high numbers of enteroendocrine cells. By removing EGF from the culture medium, the normally highly proliferative Lgr5+ stem cells become quiescent. Furthermore, these non-dividing stem cells demonstrate a bias towards expression of enteroendocrine markers and differentiation into the enteroendocrine lineage. Upon maturation of the cells, we found a heterogeneous population of cells, which differentially express a variety of hormones, thus mimicking the in vivo situation. In conclusion, single-cell sequencing has yielded valuable information about multiple biological processes in different organs and cell types, and with many new techniques currently being developed will continue to reveal exciting insights into cellular identity

Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells

Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into three-dimensional organoids containing all intestinal epithelial cell types at near-normal ratios. Conditions to generate the main cell types (enterocyte, goblet cells, Paneth cells, and M cells) are well established, but signals to induce the spectrum of hormone-producing enteroendocrine cells (EECs) have remained elusive. Here, we induce Lgr5+ stem cell quiescence in vitro by blocking epidermal growth factor receptor (EGFR) or mitogen-associated protein kinase (MAPK) signaling pathways in organoids and show that their quiescent state is readily reverted. Quiescent Lgr5+ stem cells acquire a distinct molecular signature biased toward EEC differentiation. Indeed, combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates a diversity of EEC hormone-expressing subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and provide an approach for the study of the elusive EECs in a defined environment

Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells

Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into three-dimensional organoids containing all intestinal epithelial cell types at near-normal ratios. Conditions to generate the main cell types (enterocyte, goblet cells, Paneth cells, and M cells) are well established, but signals to induce the spectrum of hormone-producing enteroendocrine cells (EECs) have remained elusive. Here, we induce Lgr5+ stem cell quiescence in vitro by blocking epidermal growth factor receptor (EGFR) or mitogen-associated protein kinase (MAPK) signaling pathways in organoids and show that their quiescent state is readily reverted. Quiescent Lgr5+ stem cells acquire a distinct molecular signature biased toward EEC differentiation. Indeed, combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates a diversity of EEC hormone-expressing subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and provide an approach for the study of the elusive EECs in a defined environment

Transcriptional profiling of cells sorted by RNA abundance

We have developed a quantitative technique for sorting cells on the basis of endogenous RNA abundance, with a molecular resolution of 10-20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high-fidelity transcriptome measurement of mouse induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins

Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (AlpiCreER) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi+ cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5+ stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration

De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data

Adult mitotic tissues like the intestine, skin, and blood undergo constant turnover throughout the life of an organism. Knowing the identity of the stem cell is crucial to understanding tissue homeostasis and its aberrations upon disease. Here we present a computational method for the derivation of a lineage tree from single-cell transcriptome data. By exploiting the tree topology and the transcriptome composition, we establish StemID, an algorithm for identifying stem cells among all detectable cell types within a population. We demonstrate that StemID recovers two known adult stem cell populations, Lgr5+ cells in the small intestine and hematopoietic stem cells in the bone marrow. We apply StemID to predict candidate multipotent cell populations in the human pancreas, a tissue with largely uncharacterized turnover dynamics. We hope that StemID will accelerate the search for novel stem cells by providing concrete markers for biological follow-up and validation