5 research outputs found
Analysis of <i>Runx1</i> Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an <i>In Vivo</i> Approach
Summary: Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis. : The authors found that Cre-mediated Runx1 ablation induced in vivo at pre-liver stages resulted in appearance of Runx1-null HSCs in the fetal liver. By contrast, deletion of Runx1 in cultured AGM region fully blocked HSC development. Appearance of Runx1-null HSCs in the liver is explained by presence of uncontrolled long-lasting (at least 3 days) Cre-inducing tamoxifen activity in vivo. Keywords: Runx1, hematopoietic stem cells, AGM, development, hematopoiesis, conditional knockout, tamoxife
Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus
Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders
Studying the cell cycle status during haematopoietic stem cell development
In adults blood stem cells, called haematopoietic stem cells (HSC), give rise to all blood cells
throughout life. The origin and biology of HSCs during embryo development has been an
intensely studied topic. Definitive HSCs are generated intra-embryonically in the aorta-gonad-mesonephros
(AGM) region of the mid-gestation embryo. Recent research revealed that HSCs
emerge through multistep maturation of precursors: proHSC → preHSC I → preHSC II →
definitive HSC (dHSC). A hallmark of the HSC emergence is the appearance of intra-aortic
haematopoietic clusters that are considered to be sites of haematopoiesis. It was shown in vitro
that the E11.5 HSCs are slowly cycling compared to progenitor cells. However, cell cycle
status and its role during early HSC development remain unclear. Here I used Fucci transgenic
mice that enable in vivo visualisation of the cell cycle. Functional and phenotypic analysis
showed that in the early embryo the proHSC precursors cycle slowly, whereas committed
progenitors are actively cycling. Meanwhile the preHSC I precursors arising in the E10.5
AGM region become more rapidly cycling. They are located closer to the luminal cavity of
the dorsal aorta, while their ancestors, the proHSCs, are slowly cycling and are located at base
of the clusters. Furthermore, in the mid-gestation embryo the preHSC I become slowly cycling
and are closer to the endothelial lining of the aorta, while they give rise to the actively cycling
preHSC II that are located to the luminal area of the artery. Finally, definitive HSCs are mainly
slowly cycling at this stage like their foetal liver counterparts. As expected, HSCs in adult
bone marrow are mainly dormant. The data suggest that transition from one precursor type to
another is accompanied by distinct changes in cell cycle profile and that HSCs become
progressively quiescent during development. To test the role of cell cycle in HSC maturation,
we used inhibitors against signalling pathways known to play important roles in HSC
development. Notch inhibitor affected the cell cycle status of haematopoietic precursors, by
possibly promoting them to rapidly proliferate and potentially blocking the maturation from
preHSC I to preHSC II precursors. Shh antagonist had the opposite effect and enhanced the
HSC activity from the preHSC I precursors. Altogether these results suggest that the cell cycle
status plays an important role in the HSC development. A better understanding of the
molecules that control this process will allow us to optimize the culture condition for
generation of functional HSCs in the laboratory
Ectopic Humanized Mesenchymal Niche in Mice Enables Robust Engraftment of Myelodysplastic Stem Cells
Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus
Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders