An Efficient and Footprint-Free Protocol for the Transdifferentiation of Hepatocytes Into Insulin-Producing Cells With IVT mRNAs
Notice bibliographique
Résumé
BACKGROUND: Direct transdifferentiation of adult somatic cells into insulin-producing cells (IPCs) is a promising approach for cell-based therapies for type 1 diabetes mellitus. Liver cells are an ideal source for generating IPCs because they have regenerative ability and a developmental process similar to that of the pancreas. Pancreas versus liver fate is regulated by TALE homeoprotein (TGIF2) during development. Here, we wanted to investigate whether TGIF2 could enhance the efficiency of transdifferentiation of hepatocytes into IPCs induced by three pancreatic transcription factors (pTFs), i.e., Pdx1, NeuroD, and Mafa, which are crucial for pancreatic development in the embryo. METHODS: and sequentially supplemented in hepatocytes. On day 6, the expression of transcription factors was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and insulin expression was detected by immunofluorescence. Glucose-stimulated insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). The key genes controlling cell polarity and the Wnt/PCP signaling pathway were assayed by qRT-PCR, and the level of JNK protein phosphorylation, which regulates the Wnt/PCP signaling pathway, was detected by western blotting. RESULTS: IVT mRNAs could be efficiently transfected into hepatocytes. Quantitative real-time polymerase chain reaction results revealed that compared with ectopic expression of the three pTFs alone, ectopic expression of the three pTFs plus TGIF2 could strongly reduce hepatic gene expression and subsequently improve the induction of a set of pancreatic genes. Immunofluorescence analysis showed that TGIF2 expression could double the transdifferentiation yield; 30% of the cells were insulin positive if induced by TGIF2 plus the 3 pTFs, while only 15% of the cells were insulin positive if induced by the three pTFs alone. ELISA analysis confirmed that glucose-stimulated insulin secretion was less efficient after transfection with the three pTFs alone. The differentiated cells derived from the addition of TGIF2 mRNA could form islet-like clusters. By contrast, the cells differentiated with the three pTFs did not form clusters under the same conditions. Tgif2 induced transdifferentiation more efficiently by remodeling the expression of genes in the Wnt/PCP pathway. Overexpression of TGIF2 in hepatocytes could activate the expression of key genes controlling cell polarity and genes in the Wnt/PCP signaling pathway, increasing the level of JNK protein phosphorylation. CONCLUSIONS: Our study established a novel footprint-free protocol for efficient transdifferentiation of hepatocytes into IPCs using IVT mRNAs of TGIF2 and 3 pTFs, which paved the way toward a clinical application.
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Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».