Summary

GATA4、MEF2C及びTBX5の最適な比率を表現ポリシストロン構築物を用いて誘導心筋細胞の改善された世代

Published: November 13, 2015
doi:

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

誘導された心筋細胞へ心臓線維芽細胞(CFS)の直接変換(ICMS)は、心臓病の治療のための代替戦略を提供することにより、再生医療のための大きな可能性を保持しています。この変換は、GATA4(G)、MEF2C(M)及びTBX5(T)として定義された要素の強制発現によって達成されています。伝統的には、ICMSは、これらの個々の因子を発現するウイルスの混合により生成されます。しかし、効率性を再プログラミングすることは比較的低く、 インビトロ G、M、T-形質導入線維芽細胞のほとんどは、それが困難な再プログラミングのメカニズムを研究すること、完全に再プログラムさになりません。我々は最近、G、Mの化学量論は、Tは、効率的なICMの再プログラミングのために重要であることを示しています。 M、我々のポリシストロンMGTベクトル(以下、MGTと呼ばれる)有意に増加した再プログラミング効率を使用することによって達成GとTの低レベルの相対的な高レベルのG、M、Tの最適な化学量論およびインビトロでの改善されたICMの品質。ここでは、心臓線維芽細胞からMGT構築物ICMSを生成するために使用される方法の詳細な説明を提供します。心臓線維芽細胞の単離、再プログラミングおよび再プログラミングのプロセスの評価のためのウイルスの生成はまた、ICMSの効率的かつ再現可能な生成のためのプラットフォームを提供するために含まれます。

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

ここで説明するプロトコルは、新生児マウスを使用しています。動物のケアと実験は、ノースカロライナ大学チャペルヒルでの実験動物医学(DLAM)の部門によって確立されたガイドラインに従って行われています。 バッファとメディアの作製線維芽細胞(FB)培地を準備します100ミリリットルウシ胎児血清(FBS)、6 mlの?…

Representative Results

再プログラミングステップは、MGTの導入後、細胞を再プログラミングにおけるGFP発現は、形質導入した細胞の3ピューロマイシン選択が3日目から始まり、PMX-puroの場合、最初の2週間の間、維持され、早ければ一日として検出することができた。 図1の模式によって要約されています-MGT構築物が使用されます。 14日目に10日目では、cTnTのとαActininなどの心臓マーカーの発現が開始線…

Discussion

このプロトコルを使用するときに成功したICMの世代のために、全体的な効率に影響を与えるいくつかの重要な要因があります。特に繊維芽細胞を開始する条件及びレトロウイルスをコードするMGTの品質が大幅に再プログラミングの効率に影響を与えることができます。

それは可能な限り新鮮で健康的な線維芽細胞を生成することが重要です。外植片が皿上にプレーティ?…

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

  1. Mozaffarian, D., et al. Heart Disease and Stroke Statistics–2015 Update: A Report From the American Heart Association. Circulation. , (2014).
  2. Whelan, R. S., Kaplinskiy, V., Kitsis, R. N. Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu Rev Physiol. 72, (2010).
  3. Senyo, S. E., et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature. 493, 433-436 (2013).
  4. 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).
  5. Hosoda, T., et al. Clonality of mouse and human cardiomyogenesis in vivo. Proc Natl Acad Sci U S A. 106, 17169-17174 (2009).
  6. Choi, W. Y., Poss, K. D. Cardiac regeneration. Curr Top Dev Biol. 100, 319-344 (2012).
  7. Souders, C. A., Bowers, S. L., Baudino, T. A. Cardiac fibroblast: the renaissance cell. Circ Res. 105, 1164-1176 (2009).
  8. Ieda, M., et al. Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell. 16, 233-244 (2009).
  9. Moore-Morris, T., et al. Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest. 124, 2921-2934 (2014).
  10. 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).
  11. Chen, J. X., et al. Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5. Circ Res. 111, 50-55 (2012).
  12. 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).
  13. Fu, J. D., et al. Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem Cell Reports. 1, 235-247 (2013).
  14. 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).
  15. Ieda, M., et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 142, 375-386 (2010).
  16. 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).
  17. 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).
  18. 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).
  19. Muraoka, N., et al. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures. Embo J. 33, 1565-1581 (2014).
  20. Nam, Y. J., et al. Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors. Development. 141, 4267-4278 (2014).
  21. Nam, Y. J., et al. Reprogramming of human fibroblasts toward a cardiac fate. Proc Natl Acad Sci USA. 110, 5588-5593 (2013).
  22. 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).
  23. 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).
  24. Wang, H., et al. Small Molecules Enable Cardiac Reprogramming of Mouse Fibroblasts with a Single Factor, Oct4. Cell Rep. , (2014).
  25. Wang, L., et al. Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming. Circ Res. , (2014).
  26. Song, K., et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 485, 599-604 (2012).
  27. Qian, L., et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 485, 593-598 (2012).
  28. 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).
  29. 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).
  30. Muraoka, N., Ieda, M. Stoichiometry of transcription factors is critical for cardiac reprogramming. Circ Res. , 116-216 (2015).
  31. Srivastava, D., Ieda, M. Critical factors for cardiac reprogramming. Circ Res. 111, 5-8 (2012).

Play Video

Cite This Article
Wang, L., Liu, Z., Yin, C., Zhou, Y., Liu, J., Qian, L. Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5. J. Vis. Exp. (105), e53426, doi:10.3791/53426 (2015).

View Video