The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.
SummaryPancreatic duct epithelial cells have been suggested as a source of progenitors for pancreatic growth and regeneration. However, genetic lineage-tracing experiments with pancreatic duct-specific Cre expression have given conflicting results. Using immunofluorescence and flow cytometry, we show heterogeneous expression of both HNF1β and SOX9 in adult human and murine ductal epithelium. Their expression was dynamic and diminished significantly after induced replication. Purified pancreatic duct cells formed organoid structures in 3D culture, and heterogeneity of expression of Hnf1β and Sox9 was maintained even after passaging. Using antibodies against a second cell surface molecule CD51 (human) or CD24 (mouse), we could isolate living subpopulations of duct cells enriched for high or low expression of HNF1β and SOX9. Only the CD24high (Hnfβhigh/Sox9high) subpopulation was able to form organoids.
Diabetes can be caused by an insufficiency in β-cell mass. Here, we performed a genetic screen in a zebrafish model of β-cell loss to identify pathways promoting β-cell regeneration. We found that both folate receptor 1 (folr1) overexpression and treatment with folinic acid, stimulated β-cell differentiation in zebrafish. Treatment with folinic acid also stimulated β-cell differentiation in cultures of neonatal pig islets, showing that the effect could be translated to a mammalian system. In both zebrafish and neonatal pig islets, the increased β-cell differentiation originated from ductal cells. Mechanistically, comparative metabolomic analysis of zebrafish with/without β-cell ablation and with/without folinic acid treatment indicated β-cell regeneration could be attributed to changes in the pyrimidine, carnitine, and serine pathways. Overall, our results suggest evolutionarily conserved and previously unknown roles for folic acid and one-carbon metabolism in the generation of β-cells.
Previously, we showed that thyroid hormone (TH) triiodothyronine (T) enhanced β-cell functional maturation through induction of High levels of T have been linked to decreased life span in mammals and low levels to lengthened life span, suggesting a relationship between TH and aging. Here, we show that T increased (a β-cell senescence marker and effector) mRNA in rodent and human β-cells. The kinetics of and induction suggested both genes as targets of TH via TH receptors (THRs) binding to specific response elements. Using specific agonists CO23 and GC1, we showed that expression was controlled by THRA and by THRB. Using chromatin immunoprecipitation and a transient transfection yielding biotinylated THRB1 or THRA isoforms to achieve specificity, we determined that THRA isoform bound to , whereas THRB1 bound to but not to On a cellular level, T treatment accelerated cell senescence as shown by increased number of β-cells with acidic β-galactosidase activity. Our data show that T can simultaneously induce both maturation () and aging ( ) effectors and that these dichotomous effects are mediated through different THR isoforms. These findings may be important for further improving stem cell differentiation protocols to produce functional β-cells for replacement therapies in diabetes.
Regenerating pancreatic β-cells is a potential curative approach for diabetes. We previously identified the small molecule CID661578 as a potent inducer of β-cell regeneration, but its target and mechanism of action have remained unknown. We now screened 257 million yeast clones and determined that CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an interaction we genetically validated in vivo. CID661578 increased β-cell neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and human pancreatic ductal organoids. Mechanistically, we found that CID661578 boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in the translation initiation complex at the mRNA cap. Unexpectedly, this blocking activity augmented eIF4E phosphorylation depending on MNK1 and bolstered the interaction between eIF4E and eIF4G, which is necessary for both hypertranslation and β-cell regeneration. Taken together, our findings demonstrate a targetable role of MNK2-controlled translation in β-cell regeneration, a role that warrants further investigation in diabetes.
Traditionally, studies of cells and tissues have been performed on isolated primary cells or immortalized cell lines by culturing them in 2D culture dishes or flasks. However, a caveat regarding 2D culture is that the cells poorly recapitulate their in vivo counterparts, mainly due to a lack of 3D cell‐cell and cell–extracellular matrix interactions. In recent years, the development of in vitro organoids as 3D culture has gained substantial attention as a model to study different tissues. In adults, pancreatic ductal cells are considered as a source of stem or progenitor cells, so developing new methods to study ductal cells would be useful. Here, we provide a protocol to isolate mouse pancreatic ductal cells and a cost‐effective protocol to generate 3D organoid structures from such ductal cells. Additionally, we have devised a protocol for immunostaining of intact whole organoids without sectioning. © 2018 by John Wiley & Sons, Inc.
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