Génération améliorée de cardiomyocytes induite en utilisant une construction polycistroniques Exprimant rapport optimal de Gata4, MEF2C et Tbx5
Summary
We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.
Abstract
La conversion directe des fibroblastes cardiaques (SCF) en cardiomyocytes induites (ICMS) détient un grand potentiel pour la médecine régénérative en offrant des stratégies alternatives pour le traitement de la maladie de coeur. Cette conversion a été obtenue par expression forcée de facteurs tels que définis Gata4 (G), MEF2C (M) et Tbx5 (T). Traditionnellement, ICMS sont générés par un cocktail de virus exprimant ces facteurs individuels. Cependant, la reprogrammation efficacité est relativement faible et la plupart de l'in vitro G, M, T-fibroblastes transduits ne deviennent pas entièrement reprogrammés, ce qui rend difficile l'étude des mécanismes de reprogrammation. Nous avons récemment montré que la stoechiométrie de G, M, T est cruciale pour l'efficacité de reprogrammation ICM. Une stoechiométrie optimale de G, M, T avec relativement haut niveau de M et de faibles niveaux de G et T obtenus à l'aide de notre vecteur de MGT polycistronique (ci-après dénommé MGT) a augmenté de manière significative l'efficacité de la reprogrammation et amélioration de la qualité iCM in vitro. Ici nous fournissons une description détaillée de la méthodologie utilisée pour générer ICMS avec MGT construction à partir de fibroblastes cardiaques. Isolement des fibroblastes cardiaques, la génération du virus pour la reprogrammation et l'évaluation du processus de reprogrammation sont également inclus afin de fournir une plate-forme pour la production efficace et reproductible du SGCI.
Introduction
Cardiovascular disease remains the leading cause of death worldwide, accounting for 17.3 million deaths per year1. Loss of cardiomyocytes resulting from myocardial infarction (MI) or progressive heart failure is a major cause of morbidity and mortality2. Due to limited regenerative capacity, adult mammalian hearts usually suffer from impaired pump function and heart failure following injury3-6. As such, efficient (re)generation of cardiomyocytes in vivo and in vitro for treatment of heart disease and for disease modeling is a critical issue needing to be addressed.
Recent development of direct reprogramming, which directly reprograms cells from one differentiated phenotype to another without transitioning through the pluripotent state, offers a promising alternative approach for regenerative medicine. The mammalian heart contains abundant cardiac fibroblasts (CFs), which account for approximately half of the cells in heart and massively proliferate upon injury7-9. Thus, the vast pool of CFs could serve as an endogenous source of new CMs for regenerative therapy if they could be directly reprogrammed into functional CMs. It has been shown that a combination of transcription factors, such as Gata4 (G), Mef2c (M) and Tbx5 (T), with or without microRNAs or small molecules can reprogram fibroblasts into iCMs10-26. Importantly, this conversion can also be induced in vivo, and results in an improvement in cardiac function and a reduction in scar size in an infarcted heart16,27-29. These studies indicate that direct cardiac reprogramming may be a potential avenue to heal an injured heart. However, the low efficiency of iCM reprogramming has become a major hurdle for further mechanistic studies. In addition, the reproducibility of cardiac reprogramming is another controversial issue of this technology11,30,31.
Very recently, we generated a complete set of polycistronic constructs encoding G,M,T in all possible splicing orders with identical 2A sequences in a single mRNA. These polycistronic constructs yielded varied G, M and T protein expression levels, which led to significantly different reprogramming efficiency25. The most efficient construct, named MGT, which showed a relatively high Mef2c and low Gata4 and Tbx5 expression, significantly improved reprogramming efficiency and produced large amounts of iCMs with CM markers expression, robust calcium oscillation and spontaneous beating25. Moreover, by using MGT polycistronic construct, our study avoided the use of multiple vectors and generated cells with homogenous expression ratio of G,M,T, thus providing an improved platform for cardiac reprogramming research. To increase experimental reproducibility, here we describe in detail how to isolate fibroblasts, produce retrovirus carrying MGT cassette, generate iCMs and evaluate the reprogramming efficiency.
Protocol
Representative Results
Discussion
Pour réussir une génération iCM lors de l'utilisation de ce protocole, il ya plusieurs facteurs importants qui ont une incidence sur l'efficacité globale. Notamment les conditions de fibroblastes de départ et la qualité de l'encodage de rétrovirus MGT peuvent grandement affecter l'efficacité de reprogrammation.
Il est important de générer des fibroblastes aussi frais et sain que possible. Pour la méthode de explant, les fibroblastes peuvent être utilisés avant le…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful for expert technical assistance from the UNC Flow Cytometry Core and UNC Microscopy Core. We thank members of the Qian lab and the Liu lab for helpful discussions and critical reviews of the manuscript. This study was supported by NIH/NHLBI R00 HL109079 grant to Dr. Liu and American Heart Association (AHA) Scientist Development Grant 13SDG17060010 and the Ellison Medical Foundation (EMF) New Scholar Grant AG-NS-1064-13 to Dr. Qian.
Materials
| anti-cardiac troponin T | Thermo Scientific | MS-295-PO | 1:200 for FACS and 1:400 for ICC |
| anti-GFP | Life Technologies | A11122 | 1:500 for both FACS and ICC |
| anti- aActinin | Sigma-Aldrich | A7811 | 1:500 for both FACS and ICC |
| anti-Connexin43 | Sigma-Aldrich | C6219 | 1:500 for ICC |
| anit-Mef2c | Abcam | ab64644 | 1:1000 for ICC |
| anti-Gata4 | Santa Cruz Biotechnology | sc-1237 | 1:200 for ICC |
| anti-Tbx5 | Santa Cruz Biotechnology | sc-17866 | 1:200 for ICC |
| Alexa Fluor 488–conjugated donkey anti-rabbit IgG | Jackson ImmunoResearch Inc | 711-545-152 | 1:500 for both FACS and ICC |
| Alexa Fluor 647–conjugated donkey anti-mouse IgG | Jackson ImmunoResearch Inc | 715-605-150 | 1:500 for both FACS and ICC |
| Cytofix/Cytoperm kit for intracellular staining | BD Biosciences | 554722 | |
| Rhod-3 Calcium Imaging Kit | Life Technologies | R10145 | |
| Thy1.2 microbeads | Miltenyi Biotec | 130-049-101 | |
| Vectashield solution with DAPI | Vector labs | H-1500 | |
| FBS | Sigma-Aldrich | F-2442 | |
| Trypsin-EDTA (0.05%) | Corning | 25-052 | |
| PRMI1640 medium | Life Technologies | 11875-093 | |
| B27 supplement | Life Technologies | 17504-044 | |
| IMDM | Life Technologies | 12440-053 | |
| Opti-MEM Reduced Serum Medium | Life Technologies | 31985-070 | |
| M199 medium | Life Technologies | 10-060 | |
| DMEM, high glucose | Life Technologies | 10-013 | |
| Penicillin-streptomycin | Corning | 30-002 | |
| Non-essential amino acids | Life Technologies | 11130-050 | |
| Lipofectamine 2000 | Life Technologies | 11668500 | |
| blasticidin | Life Technologies | A11139-03 | |
| puromycin | Life Technologies | A11138-03 | |
| Collagenase II | Worthington | LS004176 | |
| polybrene | Millipore | TR-1003-G | |
| Triton X-100 | Fisher | BP151-100 | |
| CaCl2 | Sigma-Aldrich | C7902 | |
| HEPES | Sigma-Aldrich | H4034 | |
| NaCl | Sigma-Aldrich | BP358-212 | |
| KCl | Sigma-Aldrich | PX1405 | |
| Na2HPO4 | Sigma-Aldrich | S7907 | |
| Glucose | Sigma-Aldrich | G6152 | |
| Bovine serum albumin | Fisher | 9048-46-8 | |
| paraformaldehyde | EMS | 15714 | |
| Retrovirus Precipitation Solution | ALSTEM | VC-200 | |
| 0.4%Trypan blue solution | Sigma-Aldrich | T8154 | |
| gelatin | Sigma-Aldrich | G1393 | |
| Dulbecco's PBS without CaCl2 and MgCl2 (D-PBS, 1x) | Sigma-Aldrich | D8537 | |
| HBSS (Hanks Balanced Salt Solution) | Corning | 21022 | |
| LS column | Miltenyi Biotec | 130-042-401 | |
| 0.45 μm cellulose acetate filter | Thermo Scientific | 190-2545 | |
| 24-well plates | Corning | 3524 | |
| 10cm Tissue culture dishes | Thermo Scientific | 172958 | |
| 60mm center well culture dish | Corning | 3260 | |
| 96 Well Clear V-Bottom 2mL Polypropylene Deep Well Plate | Denville Scientific | P9639 | |
| Polystyrene round-bottom tubes with cell-strainer cap | BD Biosciences | 352235 | |
| Centrifuge | Eppendorf | 5810R | |
| Vortexer MINI | VWR | 58816-121 | |
| EVOS® FL Auto Cell Imaging System | Life Technologies | AMAFD1000 | |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
| MidiMACS Separator | Miltenyi Biotec | 130-042-302 | |
| Round glass cover slip | Electron Microscopy Sciences | 72195-15 |
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