Vurdere kardiomyocytt Subtyper Etter transkripsjonsfaktor-mediert omprogrammering av mus 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 funksjonelle organ for å utvikle seg i embryo 1, 2. I forbindelse med sirkulasjonssystemet, leverer den oksygen, næringsstoffer, og en avfalls mekanisme under utvikling. Tre uker etter befruktning, slår det menneskelige hjertet for første gang og riktig regulering opprettholdes av cardiomyocytes (CMS). Den irreversible tap av disse spesialiserte celler er derfor det grunnleggende problemet som ligger til grunn progressiv hjertesvikt. Mens noen organismer som sebrafisk og Xenopus har potensial for hjerte-regenerering, er den voksne pattedyr-hjerte mer begrensede 3, 5, 6. Således, gitt den kritiske funksjon av hjertet, er det forbausende at hjertesykdom er den ledende årsak til død i verden, og utgjør 600.000 dødsfall i USA alene 7. Derefore, cellebasert terapi for å effektivt reparere eller erstatte den skadde hjertemuskelen er av stor klinisk interesse.
Den banebrytende studie av Yamanaka og kolleger 8 viste at tvangs uttrykk av fire transkripsjonsfaktorer er tilstrekkelig til å konvertere fullt differensierte fibroblast celler til pluripotente stamceller. Imidlertid har den tumorgene kapasiteten til alle pluripotent stamcelle strategier har vært et kritisk problem i deres bruk for terapeutiske formål. Dette motiverte fagfeltet for å søke etter alternative metoder for å transdifferentiate celler samtidig unngå en pluripotent scenen. Nylig har flere grupper vist gjennomførbarheten av denne strategien ved å vise direkte konvertering av musefibroblastere til indusert cardiomyocyte lignende celler (iCLMs) med ektopisk uttrykk av transkripsjonsfaktorene Gata4, Mef2c, Tbx5, og senere, Hand2 (GMT og GHMT , henholdsvis) 9, 10. furthermore, kan den samme strategi bli utført in vivo og in human-deriverte vev 9, 11, 12. Nyere studier har markert tilleggsfaktorer eller signalveier som kan være modulert for å ytterligere forbedre hjerte omprogrammering effektivitet 13, 14, 15. Samlet utgjør disse studiene viser potensialet rettet transdifferensiering for regenerativ behandling. Men den lave effektiviteten i CM omprogrammering, de ukjente molekylære mekanismer, inkonsekvent reproduserbarhet på grunn av metodiske forskjeller 16, og den heterogene natur av iCLMs forbli uadressert.
For å evaluere direkte iCLM heterogeniteten, har vi utviklet en diskret og robust enkeltcelleanalyse for identifisering av sarcomere utvikling og kardial avstamning spesifikasjonern-to nødvendige egenskapene til funksjonelle cardiomyocytes. Det er minst tre hovedtyper av CM i hjertet som definert av deres plassering og unike elektriske egenskaper: atrial (AM), ventrikkel (VM) og pacemaker (PM) 17, 18, 19, 20. I en orkestrert kombinasjon, de tillater riktig pumping av blod. Under hjerte skade, kan en eller alle undergrupper bli berørt, og den type celleterapi må tas opp fra sak til sak. Foreløpig fleste strategier fokusere på den totale generasjonen av cardiomyocytes, mens lite arbeid som gjøres for å studere molekylære mekanismer som regulerer subtype spesifikasjonen.
Følgende studie viser hvordan du skal kvantifisere velorganiserte sarcomeres og identifisere et mangfoldig sett av cardiomyocyte subtyper. Ved hjelp av en pacemaker (PM) -spesifikk reporter mus, er vi i stand til å anvende en jegmmunocytochemical tilnærming for å skille indusert atrial lignende muskelceller (IAM), induserte ventrikulære lignende muskelceller (IVM), og indusert PM-lignende muskelceller (IPMS) 21. Basert på våre observasjoner, bare celler som viser sarcomere organisasjonen er i stand til spontan juling. Denne unike omprogrammering plattform åpner for å vurdere rollen av visse parametre i sarcomere organisasjon, subtype spesifikasjon, og effektiviteten av CM omprogrammering på enkeltcelle oppløsning.
Protocol
Representative Results
Discussion
Denne studien gir en direkte omprogrammering strategi for konvertering av MEFs til et mangfoldig sett av hjerte subtyper via retrovirus-mediert uttrykk for hjertetranskripsjonsfaktorer Gata4, Mef2c, Tbx5, og Hand2 (GHMT). Ved hjelp av en multiplex farging tilnærming i kombinasjon med en PM-spesifikke reporter mus, er vi i stand til å identifisere IAM, iVMs, og IPMS på enkelt celle oppløsning. En slik analyse gjør det mulig for en eksperimentell in vitro-system er i stand til å isolere de individuelle bidr…
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 |
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