Melhoria Geração de Induced Cardiomyocytes utilizando uma construção policistrónico Expressando proporção ideal de GATA4, MEF2C e 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
Conversão directa de fibroblastos cardíacos (CFS) em cardiomiócitos induzida (ICMS) tem um grande potencial para a medicina regenerativa, oferecendo estratégias alternativas para o tratamento de doenças cardíacas. Esta conversão foi conseguida através da expressão forçada de factores tais como definidos GATA4 (L), MEF2C (M) e Tbx5 (T). Tradicionalmente, iCMS são gerados por um cocktail de vírus que expressam esses factores individuais. No entanto, a eficiência da reprogramação é relativamente baixa e a maior parte do in vitro, G, M, T transduzidas fibroblastos não se tornem totalmente reprogramado, o que torna difícil estudar os mecanismos de reprogramação. Recentemente, têm mostrado que a estequiometria de L, M, T, é crucial para a eficiente reprogramação MIC. Um estequiometria óptima de G, M, T com alto nível relativo de M e baixos níveis de G e T obtidos usando nosso vetor MGT policistrónico (doravante referida como MGT) aumentou significativamente a eficiência de reprogramação e melhoria da qualidade iCM in vitro. Aqui nós fornecemos uma descrição detalhada da metodologia utilizada para gerar ICMS com MGT construção de fibroblastos cardíacos. Isolamento de fibroblastos cardíacos, geração de vírus de reprogramação e avaliação do processo de reprogramação também estão incluídos para fornecer uma plataforma para a geração eficiente e reprodutível de ICMS.
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
Para a geração iCM bem sucedido ao usar este protocolo, existem vários fatores importantes que têm um impacto sobre a eficiência global. Particularmente as condições de fibroblastos de partida e da qualidade de codificação retrovírus MGT pode afetar consideravelmente a eficiência de reprogramação.
É importante para gerar fibroblastos tão fresco e saudável possível. Para o método de cultura de explante, os fibroblastos podem ser usadas antes de sete dias depois de os explant…
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 |
References
- Mozaffarian, D., et al. Heart Disease and Stroke Statistics–2015 Update: A Report From the American Heart Association. Circulation. , (2014).
- Whelan, R. S., Kaplinskiy, V., Kitsis, R. N. Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu Rev Physiol. 72, (2010).
- Senyo, S. E., et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature. 493, 433-436 (2013).
- Soonpaa, M. H., Field, L. J. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol. 272, 220-226 (1997).
- Hosoda, T., et al. Clonality of mouse and human cardiomyogenesis in vivo. Proc Natl Acad Sci U S A. 106, 17169-17174 (2009).
- Choi, W. Y., Poss, K. D. Cardiac regeneration. Curr Top Dev Biol. 100, 319-344 (2012).
- Souders, C. A., Bowers, S. L., Baudino, T. A. Cardiac fibroblast: the renaissance cell. Circ Res. 105, 1164-1176 (2009).
- Ieda, M., et al. Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell. 16, 233-244 (2009).
- Moore-Morris, T., et al. Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest. 124, 2921-2934 (2014).
- Addis, R. C., et al. Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success. J Mol Cell Cardiol. 60, 97-106 (2013).
- Chen, J. X., et al. Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5. Circ Res. 111, 50-55 (2012).
- Christoforou, N., et al. Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming. PLoS One. 8, e63577 (2013).
- Fu, J. D., et al. Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem Cell Reports. 1, 235-247 (2013).
- Hirai, H., Katoku-Kikyo, N., Keirstead, S. A., Kikyo, N. Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain. Cardiovasc Res. 100, 105-113 (2013).
- Ieda, M., et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 142, 375-386 (2010).
- Inagawa, K., et al. Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5. Circ Res. 111, 1147-1156 (2012).
- Islas, J. F., et al. Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors. Proc Natl Acad Sci U.S.A. 109, 13016-13021 (2012).
- Jayawardena, T. M., et al. MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes. Circ Res. 110, 1465-1473 (2012).
- Muraoka, N., et al. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures. Embo J. 33, 1565-1581 (2014).
- Nam, Y. J., et al. Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors. Development. 141, 4267-4278 (2014).
- Nam, Y. J., et al. Reprogramming of human fibroblasts toward a cardiac fate. Proc Natl Acad Sci USA. 110, 5588-5593 (2013).
- Protze, S., et al. A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells. J Mol Cell Cardiol. 53, 323-332 (2012).
- Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro. Nat Protoc. 8, 1204-1215 (2013).
- Wang, H., et al. Small Molecules Enable Cardiac Reprogramming of Mouse Fibroblasts with a Single Factor, Oct4. Cell Rep. , (2014).
- Wang, L., et al. Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming. Circ Res. , (2014).
- Song, K., et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 485, 599-604 (2012).
- Qian, L., et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 485, 593-598 (2012).
- Mathison, M., et al. In vivo cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the infarcted myocardium with vascular endothelial growth factor. J Am Heart Assoc. 1, e005652 (2012).
- Srivastava, D., Ieda, M., Fu, J., Qian, L. Cardiac repair with thymosin beta4 and cardiac reprogramming factors. Ann NY Acad Sci. 1270, 66-72 (2012).
- Muraoka, N., Ieda, M. Stoichiometry of transcription factors is critical for cardiac reprogramming. Circ Res. , 116-216 (2015).
- Srivastava, D., Ieda, M. Critical factors for cardiac reprogramming. Circ Res. 111, 5-8 (2012).
