RESUMEN
The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5+ stem cells or Clu+ revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.
Asunto(s)
Mucosa Intestinal/lesiones , Mucosa Intestinal/metabolismo , Organoides/lesiones , Organoides/metabolismo , Regeneración/efectos de los fármacos , Técnicas de Cultivo de Tejidos/métodos , Animales , Benzamidas/administración & dosificación , Compuestos de Bifenilo/administración & dosificación , Células Cultivadas , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Medios de Cultivo Condicionados/química , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de la radiación , Intestinos/lesiones , Intestinos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinas/administración & dosificación , Organoides/efectos de los fármacos , Organoides/efectos de la radiación , Piridonas/administración & dosificación , Traumatismos por Radiación/tratamiento farmacológico , Traumatismos por Radiación/metabolismo , Transducción de Señal/genética , Células Madre/metabolismo , Resultado del Tratamiento , Ácido Valproico/administración & dosificaciónRESUMEN
To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.