Project description:Stem cell dynamics in the lung govern homeostasis, repair, and regeneration, yet there is still much unknown about the mechanisms of these processes. Furthermore, incongruencies between murine and human physiology limit the translation of some findings. In this work, we address these limitations by using a transgenic pig model to identify two populations of LGR5+ cells in the lung that are present in the human but that are absent from the mouse. Using RNA sequencing, 3D imaging, organoid models, and differentiation assays, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of developing lung bud tips. While epithelial LGR5 expression is absent from postnatal lung, it is reactivated in some organoids derived from basal airway cells. A separate population of LGR5+ cells is mesenchymal, surrounds developing and mature airways, is closely associated with nerve fibers, and acts as a multipotent progenitor cell capable of supporting the airway basal cell niche. These results point to two roles for LGR5 in orchestrating stem and progenitor cell dynamics, and provide a physiologically relevant model for further studies on the role of these populations in repair and regeneration.
Project description:Stem cell dynamics in the lung govern homeostasis, repair, and regeneration, yet there is still much unknown about the mechanisms of these processes. Furthermore, incongruencies between murine and human physiology limit the translation of some findings. In this work, we address these limitations by using a transgenic pig model to identify two populations of LGR5+ cells in the lung that are present in the human but that are absent from the mouse. Using RNA sequencing, 3D imaging, organoid models, and differentiation assays, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of developing lung bud tips. While epithelial LGR5 expression is absent from postnatal lung, it is reactivated in some organoids derived from basal airway cells. A separate population of LGR5+ cells is mesenchymal, surrounds developing and mature airways, is closely associated with nerve fibers, and acts as a multipotent progenitor cell capable of supporting the airway basal cell niche. These results point to two roles for LGR5 in orchestrating stem and progenitor cell dynamics, and provide a physiologically relevant model for further studies on the role of these populations in repair and regeneration.
Project description:Using a transgenic pig expressing H2B-GFP under the control of the endogenous LGR5 promoter, we used fluorescence activated cell sorting to isolate LGR5-high and LGR5-negative epidermal cells to generate mRNA profiles of the hair follicle stem cell population, n=2 pigs. Bulk RNAseq samples were prepared from porcine cells, at least 500ng of RNA was extracted from sorted LGR5-GFP-high or LGR5-GFP-negative populations. RNAseq was performed externally by GENEWIZ; library preparation with poly(A) selection was performed followed by paired end 150bp sequencing on Illumina HiSeq.
Project description:As transgenic INSC94Y-pigs develop a stable diabetic phenotype we were able to study the influence of elevated blood glucose levels on primary immune cells in vivo. We investigated the neutrophil proteome and compared data of transgenic animals to a control group. A total of 2371 proteins describing the whole neutrophil proteome were identified, including 396 proteins (17% of proteome) newly associated to expression in porcine neutrophils. Our studies provide novel information on the porcine granulocyte proteome and contribute to a better understanding of molecular mechanisms involved in altered immune cell function in diabetes. The data presented here is highly relevant for veterinary medicine and has translational quality for diabetes in human.
Project description:Lgr5+ crypt base columnar cells, the operational intestinal stem cells (ISCs), are thought to be dispensable for small intestinal (SI) homeostasis. Using a novel Lgr5-2A-DTR (Diphtheria Toxin Receptor) model which ablates Lgr5+ cells with near-complete efficiency and retains endogenous levels of Lgr5 expression, we show that persistent depletion of Lgr5+ ISCs in fact compromises SI epithelial integrity and reduces epithelial turnover in vivo. In vitro, Lgr5-2A-DTR SI organoids are unable to establish or survive when Lgr5+ ISCs are continuously eliminated when DT is in the media. However, transient exposure to DT at the start of culture allows organoids to form, and the rate of outgrowth reduces with increasing length of DT presence. Our results indicate that intestinal homeostasis requires a constant pool of Lgr5+ ISCs, which is supplied by rapidly reprogrammed non-Lgr5+ crypt populations when pre-existing Lgr5+ ISCs are ablated.
Project description:Mutations in APC or β-catenin that cause aberrant activation of Wnt signaling are responsible for the initiation of colorectal tumor development. LGR5 is specifically expressed in stem cells of the intestine, stomach and hair follicle, and plays essential roles in maintaining tissue homeostasis. LGR5-positive stem cells have been shown to be responsible for the intestinal adenoma initiated by some mutations in APC . Furthermore, it has recently been reported that Lgr5, which is associated with the Frizzled/Lrp Wnt receptor complex, interacts with R-spondins and thereby activates Wnt signaling. However, the function of LGR5 in colorectal tumorigenesis has been unclear. Here we show that LGR5 is required for the tumorigenicity of colorectal cancer cells. We also show that the transcription factor GATA6 directly enhances the expression of LGR5. DLD1 cells were infected with a lentivirus expressing an shRNA targeting GATA6 or LGR5.
Project description:Neonatal cochlear Lgr5+ progenitors can regenerate hair cells (HCs), and this process is regulated by several genes and signaling pathways. However, this regeneration ability is limited, and whether additional genes are involved in this process remains unknown. Serpine2 was shown to participate in regulating proliferation and differentiation of cochlear Lgr5+ progenitors in our previous in vitro study. Here, we explored the expression pattern and in vivo roles of Serpine2 in HC regeneration by constructing transgenic mice in which Serpine2 is conditionally overexpressed in cochlear Lgr5+ progenitors. We found that Serpine2 is expressed in the mouse cochlea after birth with a downward trend as the mice aged. In addition, Serpine2 conditional overexpression in vivo in Lgr5+ progenitors of neonatal mice cochlea resulted in an increase number of ectopic HCs in a dose-dependent manner. And Serpine2 knockdown ex vivo and in vivo can inhibit HC regeneration. EdU assay and lineage tracing assay demonstrated that these ectopic HCs likely originated from Lgr5+ progenitors through direct trans-differentiation rather than through mitotic regeneration. Moreover, snRNA-seq analysis and RT-qPCR results revealed that Serpine2 cOE likely induces HC regeneration via inhibiting sonic hedgehog (SHH) signal pathway and inducing Atoh1 and Pou4f3 transcription factor. In brief, our data indicate that Serpine2 plays a pivotal role in HC regeneration from Lgr5+ progenitors in the neonatal mouse cochlea, and this suggests a new avenue for future research into HC regeneration.