Gata4, Mef2c 및 Tbx5의 최적 비율을 표현하는 폴리 시스 트론 구조를 사용하여 유도 된 심근의 향상 발전
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
유도 된 심근 세포로 심장 섬유 아세포 (CFS)의 직접 변환 (ICMS)는 심장 질환의 치료를위한 대안 전략을 제공함으로써 재생 의학을위한 큰 잠재력을 보유하고있다. 이 변환은 Gata4 (G), Mef2c (M) 및 Tbx5 (T)로 정의 요인의 강제 식으로 이루어졌다. 전통적으로, ICMS는 이러한 개별 요소를 표현 바이러스의 칵테일에 의해 생성됩니다. 그러나, 효율을 재 프로그래밍하는 것은 상대적으로 낮고 시험관 대부분 G, M, 섬유 아세포가 곤란 리 프로그래밍 메커니즘을 연구 할 수있게, 충분히 재 프로그램되지 않은 T-형질. 우리는 최근 G, M의 화학 양론이, T 효율적 ICM의 프로그래밍에 대한 중요 것으로 나타났습니다. M하고 MGT 폴리 시스 트론 벡터 (이하 MGT라고 함) 상당히 증가 리 프로그래밍 효율을 이용함으로써 달성 G 및 T의 낮은 수준의 상대 수준이 높은 G, M, T 최적의 화학 양론과 시험 관내에서 향상된 품질 ICM. 여기서 우리는 심근 아세포에서 MGT 구성체 ICMS를 생성하기 위해 사용 된 방법의 상세한 설명을 제공한다. 심근 아세포, 프로그래밍 및 리 프로그래밍 프로세스 평가 용 바이러스의 생성은 분리 ICMS의 효율적이고 재생 가능한 생성을위한 플랫폼을 제공하기 위해 포함된다.
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
이 프로토콜을 사용하는 경우 성공적인 ICM 생성을위한, 전체 효율에 영향을 미치는 중요한 요인은 몇 가지가있다. 특히 섬유 아세포의 개시 조건으로 레트로 바이러스 부호화 MGT의 품질이 크게 리 프로그래밍의 효율에 영향을 미칠 수있다.
그것은 가능한 한 신선하고 건강한 섬유 아 세포를 생성하는 것이 중요하다. 이식편 후 7 일이 접시에 플레이 팅 하였다 전에 이식편 배…
Declarações
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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