Lineage tracing has become the method of choice to study the fate and dynamics of stem cells (SCs) during development, homeostasis, and regeneration. However, transgenic and knock-in Cre drivers used ...to perform lineage tracing experiments are often dynamically, temporally, and heterogeneously expressed, leading to the initial labeling of different cell types and thereby complicating their interpretation. Here, we developed two methods: the first one based on statistical analysis of multicolor lineage tracing, allowing the definition of multipotency potential to be achieved with high confidence, and the second one based on lineage tracing at saturation to assess the fate of all SCs within a given lineage and the "flux" of cells between different lineages. Our analysis clearly shows that, whereas the prostate develops from multipotent SCs, only unipotent SCs mediate mammary gland (MG) development and adult tissue remodeling. These methods offer a rigorous framework to assess the lineage relationship and SC fate in different organs and tissues.
The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage ...restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.
Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over ...slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.
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•Clonal analysis for determining homeostatic tracheal epithelial cell hierarchy•Basal cells comprise two subpopulations: stem cells and luminal precursors•Luminal secretory cells are short-lived and self-renewing•Secretory cells are the major steady-state source of new ciliated cells
Using clonal analysis, mathematical modeling, and single-cell qRT-PCR, Watson et al. define the homeostatic tracheal epithelial lineage. The epithelium contains two major, equally distributed subpopulations of basal cells: stem cells and long-lived precursors that are already committed to differentiation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes. PIK3CA and P53 (also known as ...TP53) are the two most frequently mutated genes and are associated with different types of human breast cancers. The cellular origin and the mechanisms leading to PIK3CA-induced tumour heterogeneity remain unknown. Here we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and the impact of mutations in this gene on tumour heterogeneity. Surprisingly, oncogenic Pik3ca(H1047R) mutant expression at physiological levels in basal cells using keratin (K)5-CreER(T2) mice induced the formation of luminal oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive tumours, while its expression in luminal cells using K8-CReER(T2) mice gave rise to luminal ER(+)PR(+) tumours or basal-like ER(-)PR(-) tumours. Concomitant deletion of p53 and expression of Pik3ca(H1047R) accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca(H1047R) in unipotent basal cells gave rise to luminal-like cells, while its expression in unipotent luminal cells gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that underwent cell fate transition upon Pik3ca(H1047R) expression in unipotent progenitors demonstrated a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures characteristic of the different cell fate switches that occur upon Pik3ca(H1047R) expression in basal and luminal cells, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca(H1047R) activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Glandular epithelia, including the mammary and prostate glands, are composed of basal cells (BCs) and luminal cells (LCs)
. Many glandular epithelia develop from multipotent basal stem cells (BSCs) ...that are replaced in adult life by distinct pools of unipotent stem cells
. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression
. This suggests that an active mechanism restricts BSC multipotency under normal physiological conditions, although the nature of this mechanism is unknown. Here we show that the ablation of LCs reactivates the multipotency of BSCs from multiple epithelia both in vivo in mice and in vitro in organoids. Bulk and single-cell RNA sequencing revealed that, after LC ablation, BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs-reminiscent of the genetic program that regulates multipotency during embryonic development
. By predicting ligand-receptor pairs from single-cell data
, we find that TNF-which is secreted by LCs-restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways were activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating the TNF pathway, inhibited regeneration-induced BC multipotency. Our study demonstrates that heterotypic communication between LCs and BCs is essential to maintain lineage fidelity in glandular epithelial stem cells.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Les cellules souches assurent le développement des tissus, leur renouvellement ainsique leur réparation suite à des blessures. L’une des questions clés du domaine de labiologie des cellules souches ...est l’identification des différents types cellulaires qu’unecellule souche peut donner. Les cellules souches peuvent être multipotentes, c’est-à-direcapables de donner naissance à plusieurs types cellulaires différents, ou unipotentes,c’est-à-dire qu’elles ne peuvent alors se différencier qu’en un seul type cellulaire. Lesexpériences de traçage cellulaire sont réalisées quotidiennement en biologie dudéveloppement et en biologie des cellules souches afin d’évaluer le devenir des cellulessouches in vivo. Cependant, il n’existe à ce jour aucune méthode rigoureuse permettantd’interpréter les résultats d’expériences de traçage cellulaire de manière non ambigüe etde déterminer la multipotence ou l’unipotence des cellules souches avec grande précisionet de manière statistiquement fiable. Nous avons développé de nouvelles méthodes afind’évaluer avec une très grande précision le caractère unipotent ou multipotent descellules souches du sein et de la prostate. Ces nouvelles découvertes démontrent demanière non ambigüe que la prostate provient de cellules souches multipotentes, alorsque seules des cellules souches unipotentes contribuent au développement et auremodelage de la glande mammaire au stade adulte. D’autre part, nous montrons que cesont des cellules souches multipotentes qui sont responsables des phases précoces dudéveloppement embryonnaire de la glande mammaire, et que ces cellules deviennentunipotentes peu avant la naissance. Nous avons étudié les mécanismes régulant le passagede l’état multipotent à l’état unipotent et démontrons que le facteur de transcription p63joue un rôle crucial dans la restriction du potentiel de différenciation des cellules souchesmammaires embryonnaires. Enfin, nous montrons que les cellules souches mammairesadultes, normalement unipotentes, peuvent redevenir multipotentes en conditionsphysiopathologiques telles que l’ablation spécifique d’une lignée cellulaire mammaire ouau cours de l’initiation tumorale. Nous essayons donc de comprendre de manière généraleles mécanismes impliqués dans le passage de l’état unipotent à l’état multipotent descellules souches mammaires adultes, et d’élucider les similarités existant entre lesdifférentes conditions dans lesquelles des cellules souches mammaires multipotentessont observées.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
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