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Medicine

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Published: March 3rd, 2021

DOI:

10.3791/62129

1Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 2Department of Pediatrics, Jichi Medical University, 3Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 4Department of Precision Mechanics, Faculty of Science and Engineering, Chuo University
* These authors contributed equally

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins — α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) and one M-line protein Myomesin-2 (Myom2) — were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

Cardiovascular diseases are the leading cause of mortality worldwide1 and cardiomyopathy represents the third cause of cardiac-related deaths2.Cardiomyopathy is a collective group of diseases that affect cardiac muscles. The recent developments of induced pluripotent stem (iPS) cells and the directed-differentiation of iPS cells toward cardiomyocytes (PSC-CMs) have opened the door for studying cardiomyocytes with patient genome as an in vitro model of cardiomyopathy. These cells can be used to understand the pathophysiology of cardiac diseases, to elucidate their molecular mechanisms, and to test different thera....

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1. Differentiation of mouse pluripotent stem cells

  1. Maintenance of mouse ES cells
    1. Maintenance medium: Mix 50 mL of fetal bovine serum (FBS), 5 mL of L-alanine-L-glutamine, 5 mL of non-essential amino acid (NEAA), 5 mL of 100 mM Sodium Pyruvate, and 909 μl of 55 mM 2-Mercaptoethanol with 450 mL of Glasgow Minimum Essential Medium (GMEM). Supplement Leukemia inhibitory factor (LIF), CHIR-99021, and PD0325901 at a final concentration of 1000 U/mL, 1 μM, and 3 μM, respectively. Sterilize.......

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Measuring sarcomere shortening using knock-in PSC-CMs reporter lines. Sarcomere-labeled PSC-CMs were used to measure sarcomere shortening. The lines express Myom2-RFP and ACTN2-mCherry from endogenous loci. TagRFP was inserted to Myom2, coding M-proteins that localize to the M-line, while mCherry was knocked-in to ACTN2, coding α-Actinin, which localizes to the Z-line18,25. Time-lapse images.......

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PSC-CMs have great potential to be utilized as an in vitro platform to model heart disease and to test the effects of drugs. Nevertheless, an accurate, unified method to assess PSC-CMs functions must first be established. Most of functional tests work with PSC-CMs, e.g., electrophysiology, calcium transient, and metabolism26, and one of the first patient-derived PSC-CM studies was about long-QT syndrome27. However, contractility, one of the most important func.......

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We would like to acknowledge all the lab members in the Division of Regenerative Medicine at the Jichi Medical University for the helpful discussion and technical assistance. This study was supported by the grants from the Japan Agency for Medical Research and Development (AMED; JP18bm0704012 and JP20bm0804018), the Japan Society for the Promotion of Science (JSPS; JP19KK0219), and the Japanese Circulation Society (the Grant for Basic Research) to H.U.

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Name Company Catalog Number Comments
1-Thioglycerol Sigma-Aldrich M6145-25
2-Mercaptoethanol (55mM) Thermo Fisher Scientific 21985-023
2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, NOF Corp. LIPIDURE-CM5206
2-Propanol Fujifilm wako 166-04836
35-mm imaging dish with a polymer coverslip (µ-Dish 35 mm, high) ibidi 81156
AAVproR Helper Free System (AAV6)
(vectors; pHelper, pRC6, pAAV-CMV-Vector)
Takara 6651
ACTN2-mCherry (AR12, AR21) hiPSCs N.A. We inserted IRES-puromycin resistant casette to 3' UTR of TNNT2 locus and mCherry around the stop codon of ACTN2 in 610B1 hiPSC line, following a method describe elsewhere (Anzai, Methods Mol Biol, in press)
B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504-044
B-27 Supplement, minus insulin Thermo Fisher Scientific A18956-01
B27 supplement (50X), minus Vitamin A Thermo Fisher Scientific 12587-010
Benzonase (25 U/µL) Merck Millipore 70746
Blasticidin S Hydrochloride Fujifilm wako 029-18701
BMP-4, Human, Recombinant, R&D Systems, Inc. 314-BP-010
Bovine Serum Albumin Sigma-Aldrich A4503-100g
C59, Wnt Antagonist (WntC59) abcam ab142216
CAD drawing software, Robert McNeel and Associates, WA, USA Rhinoceros 6.0
Centrifugal ultrafiltration unit (100k MWCO), Vivaspin-20 Sartorius VS2042
CHIR99021 Cayman 13122
Chromium etchant Nihon Kagaku Sangyo Co., Ltd., Japan N14B
Chromium mask coated with AZP1350 Clean Surface Technology Co., Japan CBL2506Bu-AZP
Dr. GenTLE Precipitation Carrier (20mg/mL Glycogen, 3 M Sodium Acetate (pH 5.2)) Takara 9094
Dulbecco’s Modified Eagle’s Medium (DMEM) - high glucose Sigma-Aldrich D6429-500
Dulbecco’s Modified Eagle’s Medium (DMEM) - high glucose, without sodium pyruvate Sigma-Aldrich D5796
Ethanol (99.5) Fujifilm wako 057-00456
Fetal Bovine Serum Moregate 59301104
FGF-10, Human, Recombinant, R&D Systems, Inc. 345-FG-025
Fibroblast Growth Factor(basic), human, recombinant Fujifilm wako 060-04543
Gelatin from porcine skin powder Sigma-Aldrich G1890-100g
Glasgow Minimum Essential Medium (GMEM) Sigma-Aldrich G5154-500
GLASS BOTTOM culture plates MatTek P24G-1.5-13-F/H
Ham’s F-12 Thermo Fisher Scientific 11765-062
Iscove's Modified Dulbecco's Medium (IMDM) Thermo Fisher Scientific 12440-061
L-alanine-L-glutamine (GlutaMAX Supplement, 200mM) Thermo Fisher Scientific 35050-061
L(+)-Ascorbic Acid Sodium Salt Fujifilm wako 196-01252
Laminin-511 E8 fragment (LN511-E8, iMatrix-511) Nippi 892012
Mask aligner Union Optical Co., Ltd., Japan PEM-800
Maskless lithography tool NanoSystem Solutions, Inc., Japan D-Light DL-1000
MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140-050
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF (0.45-µm filter) Merck Millipore SLHVR33RS
Myom2-RFP (SMM18) N.A. Developed in our previous paper (Chanthra, Sci Rep, 2020)
N-2 Supplement (100X) Thermo Fisher Scientific 17502-048
ORCA-Flash4.0 V3 digital CMOS camera Hamamatsu C13440-20CU
PD0325901 Stemgent 04-0006-10
Penicillin-Streptomycin (10,000 U/mL) Thermo Fisher Scientific 15140-122
Petri dish Sansei medical co. Ltd 01-004
Phenol/Chloroform/Isoamyl alcohol (25:24:1) Nippon Gene 311-90151
Polydimethylsiloxane (PDMS) elastomer Dow Corning Corp., MI, USA SILPOT 184
polyethylenimine MAX (MW. 40,000) Polyscience 24765-1
Positive photoresist developer Tokyo Ohka Kogyo Co., Ltd., Japan NMD-3
PowerUp SYBR Green Master Mix Thermo Fisher Scientific A25742
Proteinase K Takara 9034
Puromycin Dihydrochloride Fujifilm wako 166-23153
Recombinant Human/Mouse/Rat Activin A Protein R&D Systems, Inc. 338-AC-050
Recombinant trypsin-like protease (rTrypsin; TrypLE express) Thermo Fisher Scientific 12604-039
RPMI1640 Medium Thermo Fisher Scientific 11875-119
Silicon wafer Matsuzaki Seisakusyo Co., Ltd., Japan N.A.
Sodium Pyruvate (100 mM) Thermo Fisher Scientific 11360-070
Spin-coater Mikasa Co., Ltd., Japan MS-A100
Spininng confocal microscopy Oxford Instruments Andor Dragonfly Spinning Disk System
StemSure LIF, Mouse, recombinant, Solution (10^6U) Fujifilm wako 195-16053
SU-8 3010 Kayaku Advanced Materials, Inc., MA, USA SU-8 3010
SU-8 developer Kayaku Advanced Materials, Inc., MA, USA SU-8 developer
Tris-EDTA Nippon Gene 314-90021
Vascular Endothelial Growth Factor-A165(VEGF), Human, recombinant Fujifilm wako 226-01781

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