Vurdering cardiomyocyte undertyper Efter Transcription Factor-medieret Omprogrammering af Mouse Embryonale fibroblaster
Summary
This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.
Abstract
Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs).
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.
Introduction
Hjertet er den første funktionelle organ til at udvikle i embryonet 1, 2. I forbindelse med kredsløbssystemet, det leverer ilt, næringsstoffer, og en mekanisme affaldsbortskaffelse under udviklingen. Tre uger efter befrugtningen, det menneskelige hjerte slår for første gang og den korrekte regulering vedligeholdes af cardiomyocytter (CMS). Den uoprettelige tab af disse specialiserede celler er derfor det grundlæggende spørgsmål bag progressiv hjerteinsufficiens. Mens nogle organismer såsom zebrafisk og Xenopus har potentiale for hjerte- regenerering, det voksne mammale hjerte er mere begrænset 3, 5, 6. I betragtning af den kritiske funktion af hjertet, er således ikke overraskende, at hjertesygdom er den hyppigste årsag til dødsfald i verden, og at 600.000 dødsfald i USA alene 7. DetDerfor indgår, cellebaserede behandlingsformer til effektivt reparere eller erstatte den skadede myokardiet er af stor klinisk interesse.
Den skelsættende undersøgelse af Yamanaka og kolleger 8 viste, at tvungen ekspression af fire transkriptionsfaktorer er tilstrækkelig til at omdanne fuldstændigt differentierede fibroblastceller pluripotente stamceller. Imidlertid har den tumorigen kapacitet for alle pluripotente stamceller strategier været et kritisk problem i deres anvendelse til terapeutiske formål. Dette motiverede det videnskabelige område for at søge efter alternative metoder til at transdifferentiate celler samtidig undgå en pluripotent stadie. For nylig har flere grupper vist muligheden for denne strategi ved at vise direkte omdannelse af musefibroblaster til inducerede cardiomyocyte-lignende celler (iCLMs) med ektopisk udtryk for transskription faktorer Gata4, Mef2c, Tbx5, og senere, Hand2 (GMT og GHMT henholdsvis) 9, 10. Furthermore, kan den samme strategi udføres in vivo og in human-afledte væv 9, 11, 12. Nylige undersøgelser har peget yderligere faktorer eller signalveje, som kan moduleres til yderligere at forbedre hjertefunktionen omprogrammering effektivitet 13, 14, 15. Tilsammen disse undersøgelser viser potentialet i dirigeret transdifferentiering for regenerative behandlinger. Men den lave effektivitet CM omprogrammering, de ukendte molekylære mekanismer, inkonsekvent reproducerbarhed på grund af metodologiske forskelle 16, og den heterogene karakter af iCLMs forblive adresseløse.
For at direkte evaluere iCLM heterogenitet, vi designet en diskret og robust single-celleassay til identifikation af sarkomeret udvikling og kardial afstamning specification-to nødvendige karakteristika funktionelle cardiomyocytter. Der er mindst tre hovedtyper af CM i hjertet som defineret ved deres placering og unikke elektriske egenskaber: atrial (AM), ventrikulær (VM) og pacemaker (PM) 17, 18, 19, 20. I en organiseret kombination, de tillader en korrekt pumpning af blod. Under hjerte skade, måske en eller alle undertyper blive berørt, og typen af celleterapi vil skulle løses fra sag til sag. I øjeblikket er de fleste strategier fokuserer på den samlede generation af cardiomyocytter, mens lidt arbejde der gøres for at studere de molekylære mekanismer, der regulerer undertype specifikation.
Den følgende undersøgelse beskriver, hvordan man korrekt kvantificere velorganiserede sarkomerer og identificere et bredt sæt af cardiomyocyte undertyper. Ved hjælp af en pacemaker (PM) -specifik reporter mus, er vi i stand til at anvende en immunocytochemical tilgang til at skelne inducerede atrial-lignende myocytter (IAM), inducerede ventrikulære-lignende myocytter (IVM) og inducerede PM-lignende myocytter (IPMS) 21. Baseret på vores observationer, kun celler, der udviser sarkomer organisation er i stand til spontant bankende. Denne unikke omprogrammering platform giver mulighed for at vurdere, hvilken rolle af visse parametre i sarkomeret organisation, undertype specifikation, og effektiviteten af CM omprogrammering på encellede opløsning.
Protocol
Representative Results
Discussion
Den foreliggende undersøgelse giver en direkte-omprogrammering strategi for konvertering af MEF'er til et bredt sæt af hjerte-undertyper via retrovirus-medieret udtryk for hjertets transskription faktorer Gata4, Mef2c, Tbx5, og Hand2 (GHMT). Ved hjælp af en multiplex immunfarvning fremgangsmåde i kombination med en PM-specifik reporter mus, er vi i stand til at identificere IAM, IVMS, og IPMS på enkelt celle opløsning. En sådan analyse giver mulighed for en eksperimentel in vitro system, der kan isol…
Divulgations
The authors have nothing to disclose.
Acknowledgements
A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.
Materials
| DMEM | Sigma | D5796 | Component of iCLM media, Plat-E media, fibroblast, and Transfection media |
| Medium 199 | Thermo Fisher Scientific | 11150059 | Component of iCLM media |
| Fetal bovine serrum (FBS) | Sigma | F2442 | Component of iCLM media, Plat-E media, fibroblast, and Transfection media |
| Insulin-Transferrin-Selenium G | Thermo Fisher Scientific | 41400-045 | Component of iCLM media |
| MEM vitamin solution | Thermo Fisher Scientific | 11120-052 | Component of iCLM media |
| MEM amino acids | Thermo Fisher Scientific | 1601149 | Component of iCLM media |
| Non-Essential amino acids | Thermo Fisher Scientific | 11140-050 | Component of iCLM media |
| Antibiotic-Antimycotics | Thermo Fisher Scientific | 15240062 | Component of iCLM media |
| B-27 supplement | Thermo Fisher Scientific | 17504044 | Component of iCLM media |
| Heat-Inactivated Horse Serum | Thermo Fisher Scientific | 26050-088 | Component of iCLM media |
| NaPyruvate | Thermo Fisher Scientific | 11360-70 | Component of iCLM media |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 1514022 | Component of Plat-E media and fibroblast media |
| Puromycin | Thermo Fisher Scientific | A11139-03 | Component of Plat-E media |
| Blasticidin | Gemini Bio-Products | 400-128P | Component of Plat-E media |
| Glutamax | Thermo Fisher Scientific | 35050-061 | Component of Fibroblast media |
| Confocal laser scanning LSM700 | Zeiss | For confocal analysis | |
| FuGENE 6 transfection Reagent | Promega | E2692 | Transfection reagent |
| Opti-MEM Reduced Serum Medium | Thermo Fisher Scientific | 31985-070 | Transfection reagent |
| Polybrene | Millipore | TR-1003-G | Induction reagent. Use at a final concentration of 8um/mL |
| Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic | CellBiolabs | RV-101 | Retroviral pacaking cell line |
| Trypsin 0.25% EDTA | Thermo Fisher Scientific | For MEFs and Plat-E dissociation | |
| Mouse anti α-Actinin (Clone EA-53) | Sigma | A7811 | Antibody for confocal analysis. Use at 1:200 |
| Chicken anti-GFP IgY | Thermo Fisher Scientific | A10262 | Antibody for confocal analysis. Use at 1:200 |
| Rabbit Pab anti-NPPA | Abgent | AP8534A | Antibody for confocal analysis. Use at 1:400 |
| Rabbit Pab anti Myl2 IgG | ProteinTech | 10906-1-AP | Antibody for confocal analysis. Use at 1:200 |
| Vectashield solution with DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Vector Labs | H-1500 | Dye for confocal analysis |
| Superfrost Plus Microscope slides | Thermo Fisher Scientific | 12-550-15 | 25 x 75 x 1.0 mm |
| BioCoat Fibronectin 12mm coverslips | NeuVitro Corp | GG-12-1.5 | Coverslips for confocal analysis |
| 100um cell strainer | Thermo Fisher Scientific | 08-771-19 | |
| 0.45um Syringes filters SFCA 25MM | Thermo Fisher Scientific | 09-740-106 | For virus filtration |
| 6ml Syringes | Covidien | 8881516937 | For virus filtration |
| Goat anti-Chicken IgY (H&L) A488 | Abcam | AB150169 | Secondary antibody for confocal analysis. Use at 1:400 |
| Donkey anti-rabbit A647 IgG(H+L) | Thermo Fisher Scientific | A31573 | Secondary antibody for confocal analysis. Use at 1:400 |
| Goat anti-mouse IgG(H+L) A555 | Thermo Fisher Scientific | A21422 | Secondary antibody for confocal analysis. Use at 1:400 |
| Triton X-100 | Sigma | 93443-100ml | For cell permeabilization |
| Dulbecco's PBS without CaCl2 and MgCl2 (D-PBS) | Sigma | D8537 | |
| Power Block 10X Universal Blocking reagent | Thermo Fisher Scientific | NC9495720 | Dilute to 1X in H20 |
| 16% Paraformaldehyde aqueous solution (PFA) | Electro Microscopy Sciences | 15710 | Use at 4% diluted in dH20 |
| 6 cm plates | Olympus | 25-260 | |
| 6-well plates | Genesee Scientific | 25-105 | |
| 24-well plates | Genesee Scientific | 25-107 | |
| 10 cm Tissue culture dishes | Corning | 4239 | |
| 15 cm Tissue culture dishes | Thermo Fisher Scientific | 5442 | |
| 15 ml Conical tubes | Corning | 4308 | |
| 50 ml Conical tubes | Corning | 4249 | |
| 0.4% Trypan blue solution | Sigma | T8154 | For viability |
| Ethyl Alcohol 200 proof | Thermo Fisher Scientific | 7005 | |
| Bleach | Thermo Fisher Scientific | 6009 |
References
- Sissman, N. J. Developmental landmarks in cardiac morphogenesis: comparative chronology. Am J Cardiol. 25 (2), 141-148 (1970).
- Buckingham, M., Meilhac, S., Zaffran, S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 6 (11), 826-835 (2005).
- Ali, S. R., et al. Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice. Proc Natl Acad Sci U S A. 111 (24), 8850-8855 (2014).
- Bergmann, O., et al. Evidence for cardiomyocyte renewal in humans. Science. 324 (5923), 98-102 (2009).
- Senyo, S. E., et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature. 493 (7432), 433-436 (2013).
- Lin, Z., Pu, W. T. Strategies for cardiac regeneration and repair. Sci Transl Med. 6 (239), 239rv231 (2014).
- Writing Group, M., et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 133 (4), e38-e360 (2016).
- Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126 (4), 663-676 (2006).
- Song, K., et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 485 (7400), 599-604 (2012).
- Ieda, M., et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 142 (3), 375-386 (2010).
- Qian, L., et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 485 (7400), 593-598 (2012).
- Fu, J. D., et al. Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem Cell Reports. 1 (3), 235-247 (2013).
- Zhou, H., Dickson, M. E., Kim, M. S., Bassel-Duby, R., Olson, E. N. Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes. Proc Natl Acad Sci U S A. 112 (38), 11864-11869 (2015).
- Zhou, Y., et al. Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming. Cell Stem Cell. 18 (3), 382-395 (2016).
- Zhao, Y., et al. High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling. Nat Commun. 6, 8243 (2015).
- Miki, K., Yoshida, Y., Yamanaka, S. Making steady progress on direct cardiac reprogramming toward clinical application. Circ Res. 113 (1), 13-15 (2013).
- Atkinson, A., et al. Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks. J Mol Cell Cardiol. 51 (5), 689-701 (2011).
- Bootman, M. D., Smyrnias, I., Thul, R., Coombes, S., Roderick, H. L. Atrial cardiomyocyte calcium signalling. Biochim Biophys Acta. 1813 (5), 922-934 (2011).
- Miquerol, L., Beyer, S., Kelly, R. G. Establishment of the mouse ventricular conduction system. Cardiovasc Res. 91 (2), 232-242 (2011).
- Später, D., Hansson, E. M., Zangi, L., Chien, K. R. How to make a cardiomyocyte. Development. 141 (23), 4418-4431 (2014).
- Nam, Y. J., et al. Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors. Development. 141 (22), 4267-4278 (2014).
- Conner, D. A. . Current Protocols in Molecular Biology. , (2001).
- Jozefczuk, J., Drews, K., Adjaye, J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J Vis Exp. (64), (2012).
- Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A. 90 (17), 8033-8037 (1993).
- Ichim, C. V., Wells, R. A. Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation. J Transl Med. 9, 137 (2011).
- 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 (6), 1204-1215 (2013).
- Yang, R., et al. Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors. Nat Commun. 5, 5807 (2014).
- Muraoka, N., Ieda, M. Direct reprogramming of fibroblasts into myocytes to reverse fibrosis. Annu Rev Physiol. 76, 21-37 (2014).
- Coffin, J. M., Hughes, S. H., Varmus, H. E., Coffin, J. M., Hughes, S. H., Varmus, H. E. . Retroviruses. , (1997).
- Bass, G. T., et al. Automated image analysis identifies signaling pathways regulating distinct signatures of cardiac myocyte hypertrophy. J Mol Cell Cardiol. 52 (5), 923-930 (2012).
