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Atoh1⁺ secretory progenitors possess renewal capacity independent of Lgr5⁺ cells during colonic regeneration.

ABSTRACT: During homeostasis, the colonic epithelium is replenished every 3-5 days by rapidly cycling Lgr5 ⁺ stem cells. However, various insults can lead to depletion of Lgr5 ⁺ stem cells, and colonic epithelium can be regenerated from Lgr5-negative cells. While studies in the small intestine have addressed the lineage identity of the Lgr5-negative regenerative cell population, in the colon this question has remained unanswered. Here, we set out to identify which cell(s) contribute to colonic regeneration by performing genetic fate-mapping studies of progenitor populations in mice. First, using keratin-19 (Krt19) to mark a heterogeneous population of cells, we found that Lgr5-negative cells can regenerate colonic crypts and give rise to Lgr5 ⁺ stem cells. Notch1 ⁺ absorptive progenitor cells did not contribute to epithelial repair after injury, whereas Atoh1 ⁺ secretory progenitors did contribute to this process. Additionally, while colonic Atoh1 ⁺ cells contributed minimally to other lineages during homeostasis, they displayed plasticity and contributed to epithelial repair during injury, independent of Lgr5 ⁺ cells. Our findings suggest that promotion of secretory progenitor plasticity could enable gut healing in colitis.

SUBMITTER: Castillo-Azofeifa D

PROVIDER: S-EPMC6376326 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration.

Castillo-Azofeifa David D Fazio Elena N EN Nattiv Roy R Good Hayley J HJ Wald Tomas T Pest Michael A MA de Sauvage Frederic J FJ Klein Ophir D OD Asfaha Samuel S

The EMBO journal 20190111 4

During homeostasis, the colonic epithelium is replenished every 3-5 days by rapidly cycling Lgr5+ stem cells. However, various insults can lead to depletion of Lgr5+ stem cells, and colonic epithelium can be regenerated from Lgr5-negative cells. While studies in the small intestine have addressed the lineage identity of the Lgr5-negative regenerative cell population, in the colon this question has remained unanswered. Here, we set out to identify ...[more]

PMID: 30635334

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Project description:BACKGROUND & AIMS:Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells. METHODS:We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. RESULTS:Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a ?-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased. CONCLUSIONS:cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

Project description:The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)-derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model.VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31(+)CD146(+) VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion-injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell- and CB-iPSC-derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days.VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

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Atoh1⁺ secretory progenitors possess renewal capacity independent of Lgr5⁺ cells during colonic regeneration.

Publications

Atoh1<sup>+</sup> secretory progenitors possess renewal capacity independent of Lgr5<sup>+</sup> cells during colonic regeneration.

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