Epigenetisk Konvertering som en sikker og enkel metode til at opnå insulinproducerende celler fra voksne hud fibroblaster
Summary
Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.
Abstract
Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a “highly permissive” state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated “epigenetic cell conversion”. It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.
Introduction
Et grundlæggende mål for regenerativ medicin er dannelsen af nye, funktionelle celler, der kan bruges til at reparere eller udskifte beskadigede, degenereret væv. Genskabe let tilgængelige voksne celler til nye, ved at konvertere dem fra en celle til en anden, er en særlig tiltalende tilgang, især når den krævede cellepopulation er ikke rigelige eller svært tilgængelige. Men voksne celler er bemærkelsesværdigt stabil. De får deres differentierede tilstand gennem en gradvis begrænsning i deres muligheder, og når de når den modne terminal specialisering de stabilt beholde det en.
I de seneste år er der udviklet en række protokoller, der gør det muligt for omprogrammering til pluripotens af en somatisk celle (IPS) opnås gennem den tvungne udtryk for et sæt af transskription faktorer 2,3. Alternativt kan celle konvertering opnås ved direkte afstamning transdifferentiering, at indføre en enkelt 4 </sup> eller en kombination af transkriptionsfaktorer 5-7. Denne strategi indebærer ikke overgangen gennem en de-differentieret tilstand, men kræver høj ekspression af specifikke transkriptionsfaktorer 8.
Vi har for nylig udviklet en konvertering protokol baseret på den korte eksponering af voksne celler til demethylering egenskaber af cytidinanalog 5-azacytidin (5-aza-CR), en velkarakteriseret DNA methyltransferase inhibitor. Demethyleringen trin efterfølges umiddelbart af en specifik differentiering protokol 9-11 gør det muligt at opnå den ønskede terminal fænotype. Denne metode kan konvertere modne, differentierede celler til celler af en anden slægt og har den væsentlige fordel at undgå både anvendelsen af virale vektorer og transfektion af eventuelle exogene transkriptionsfaktorer. Købet af en stabil pluripotent tilstand, og den relaterede øget modtagelighed til celle ustabilitet er også undgået.
<p class="Jove_content"> Den detaljerede protokol, der tillader omdannelsen af voksne humane hudfibroblaster til fuldt funktionelle insulinproducerende celler præsenteres her. Men det er værd at bemærke, at teknikken er blevet anvendt på forskellige celletyper og har skabt positive resultater, når adressering celler mod forskellige differentieringsveje. Desuden er epigenetisk konvertering været anvendt med succes i de menneskelige og svin 9-13 samt i hunden (manuskript indsendt) antyder en bred effekt og robusthed tilgang.Protocol
Representative Results
Discussion
Den foreliggende manuskript beskriver en fremgangsmåde, som tillader omdannelsen af humane hudfibroblaster i insulinproducerende celler, gennem en forbigående og kortvarig udsættelse for 5-aza-CR, efterfulgt af en vævsspecifik induktionsprotokol. Denne fremgangsmåde giver mulighed for et skift fra mesoderm til endoderm relaterede celler, uden tvungen udtryk for transkriptionsfaktorer eller microRNA eller erhvervelsen af en stabil pluripotent tilstand, der gør cellerne mere ustabil og udsat for fejl 14.<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev finansieret af Carraresi Foundation og Det Europæiske Institut for Studiet af Diabetes (EFSD). GP er understøttet af en post-doc stipendium af universitetet i Milano. Forfatterne er medlemmer af COST Action FA1201 Epiconcept: Epigenetik og Periconception miljø og COST Action BM1308 Deling forskud på store dyremodeller (SALAAM). TALB er medlem af COST Action CM1406 Epigenetisk Chemical Biology (EPICHEM).
Materials
| Dulbecco's Phosphate Buffered Saline | Sigma | D5652 | PBS; for cell wash and solution preparation |
| Antibiotic Antimycotic Solution | Sigma | A5955 | Component of Fibroblast, HP and Pancreatic media |
| 100 mm petri dish | Sarstedt | 83.3902 | For Fibroblast isolation |
| Porcine Gelatin | Sigma | G1890 | For dish coating |
| Water | Sigma | W3500 | For solution preparation |
| 35 mm petri dishes | Sarstedt | 83.39 | For Fibroblast isolation |
| DMEM, high glucose, pyruvate | Life Technologies | 41966052 | For Fibroblast culture medium |
| Fetal Bovine Serum | Life Technologies | 10500064 | FBS; Component of Fibroblast and HP media |
| L-Glutamine solution | Sigma | G7513 | Component of Fibroblast, HP and Pancreatic media |
| Trypsin-EDTA solution | Sigma | T3924 | For Fibroblast dissociation |
| KOVA GLASSTIC SLIDE 10 WITH GRIDS | Hycor Biomedical | 87144 | Cell counting |
| 5-Azacytidine | Sigma | A2385 | 5-aza-CR, for increrase cell plasticity in fibroblasts |
| Ham's F-10 Nutrient Mix | Life Technologies | 31550031 | For HP medium |
| DMEM, low glucose, pyruvate | Life Technologies | 31885023 | For HP medium |
| KnockOut Serum Replacement | Life Technologies | 10828028 | Component of HP medium |
| MEM Non-Essential Amino Acids Solution | Life Technologies | 11140035 | Component of HP and Pancreatic Basal media |
| 2-Mercaptoethanol | Sigma | M7522 | Component of HP and Pancreatic Basal media |
| Guanosine | Sigma | G6264 | Nucleoside mix stock component of HP medium |
| Adenosine | Sigma | A4036 | Nucleoside mix stock component of HP medium |
| Cytidine | Sigma | C4654 | Nucleoside mix stock component of HP medium |
| Uridine | Sigma | U3003 | Nucleoside mix stock component of HP medium |
| Thymidine | Sigma | T1895 | Nucleoside mix stock component of HP medium |
| Millex-GS 0,22 µm | Millipore | SLGS033SB | For sterilizing of solution |
| FGF-Basic (AA 1-155) Recombinant Human Protein | Life Technologies | PHG0261 | bFGF; Component of HP and Pancreatic Basal medium |
| Bovine Serum Albumin | Sigma | A3311 | BSA; Component of Pancreatic Basal medium |
| DMEM/F-12 | Life Technologies | 11320074 | For Pancreatic Basal medium |
| B-27 Supplement Minus Vitamin A | Life Technologies | 12587010 | Component of Pancreatic medium |
| N-2 Supplement | Life Technologies | 17502048 | Component of Pancreatic Basal medium |
| Activin A Recombinant Human Protein | Life Technologies | PHG9014 | For Pancreatic medium |
| Retinoic Acid | Sigma | R2625 | For Pancreatic medium |
| Dimethyl sulfoxide | Sigma | D2650 | DMSO; for Retinoic Acid stock preparation |
| Insulin-Transferrin-Selenium | Life Technologies | 41400045 | ITS; for Pancreatic Final medium |
| Anti-Vimentin antibody | Abcam | ab8069 | For immunocytochemical analisys. Working dilution 1:100 |
| 4′,6-Diamidino-2-phenylindole dihydrochloride | Sigma | 32670 | DAPI. For immunocytochemical analisys. Working dilution 1µg/ml |
| 5-Methylcytidine | Eurogentec | MMS-900P-B | For immunocytochemical analisys. Working dilution 1:500 |
| Anti-C Peptide antibody | Abcam | ab14181 | For immunocytochemical analisys. Working dilution 1:100 |
| Anti-PDX1 antibody | Abcam | ab47267 | For immunocytochemical analisys. Working dilution 1:500 |
| Mercodia Insulin ELISA | Mercodia | 10-1113-10 | For insulin release detection |
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