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In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.
A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.
Stem cell-based therapies have shown considerable potential as a treatment for cardiac damage caused by MI1. The use of differentiated hiPSCs provides an inexhaustible source of hiPSC-CMs2 and opens the door for the rapid development of breakthrough treatments. However, many limitations to therapeutic translation remain, including the challenge of the severely low engraftment rate of implanted cells.
Dissociating cells with trypsin initiates anoikis3, which is only accelerated once these cells are injected into harsh environments like the ischemic myocardium, where the ....
All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham and were based on the National Institutes of Health Laboratory Animal Care and Use Guidelines (NIH Publication No 85-23).
1. Preparation of Culture Media and Culture Plates
The hiPSC-CMs used in this study were derived from human origin with luciferase reporter gene; therefore, the survival rate of the transplanted cells in vivo was detected by bioluminescence imaging (BLI)17 (Figure 1A,B). For histological heart sections, human-specific cardiac troponin T (hcTnT) and human nuclear antigen (HNA) double-positive cells were classified as engrafted hiPSC-CMs (Figure 1C
The key steps of this study include obtaining pure hiPSC-CMs, improving the activity of hiPSC-CMs through Y-27632 pretreatment, and finally, transplanting a precise amount of hiPSC-CMs into a mouse MI model.
The key issues addressed here were that, first, we optimized the glucose-free purification methods19 and established a novel efficient purification system. The system procedure included applying cell-dissociation enzymes, replanting cells in gelatin-coated plates, c.......
The authors thank Dr. Joseph C. Wu (Stanford University) for kindly providing the Fluc-GFP construct and Dr. Yanwen Liu for excellent technical assistance. This study is supported by the National Institutes of Health RO1 grants HL95077, HL114120, HL131017, HL138023, UO1 HL134764 (to J.Z.), and HL121206A1 (to L.Z.), and a R56 grant HL142627 (to W.Z.), an American Heart Association Scientist Development Grant 16SDG30410018, and the University of Alabama at Birmingham Faculty Development Grant (to W.Z.).
....Name | Company | Catalog Number | Comments |
Reagent | |||
Accutase (stem cell detachment solution) | STEMCELL Technologies | #07920 | |
B27 minus insulin | Fisher Scientific | A1895601 | |
B27 Supplement | Fisher Scientific | 17-504-044 | |
CHIR99021 | Stem Cell Technologies | 72054 | |
DMEM (1x), high glucose, HEPES, no phenol red | Thermofisher | 20163029 | |
Fetal bovine serum | Atlanta Biologicals | S11150 | |
Fluo-4 AM (calcium indicator) | Invitrogen/Thermofisher | F14201 | |
Glucose-free RPMI 1640 | Fisher Scientific | 11879020 | |
IWR1 | Stem Cell Technologies | 72562 | |
Matrigel (extracellular matrix ) | Fisher Scientific | CB-40230C | |
mTeSR (human pluripotent stem cells medium) | STEMCELL Technologies | 85850 | |
Pen-strep antibiotic | Fisher Scientific | 15-140-122 | |
Pluronic F-127 (surfactant polyol) | Sigma-Aldrich | P2443 | |
Rho activator II | Cytoskeleton | CN03 | |
RPMI1640 | Fisher Scientific | 11875119 | |
Sodium DL-lactate | Sigma-Aldrich | L4263 | |
TrypLE (cell-dissociation enzymes) | Fisher Scientific | 12-605-010 | |
Verapamil | Sigma-Aldrich | V4629 | |
Y-27632 | STEMCELL Technologies | 72304 | |
Name | Company | Catalog Number | Comments |
Equipment and Supplies | |||
IVIS Lumina III Bioluminescence Instruments | PerkinElmer | CLS136334 | |
15 mm Coverslips | Warner | CS-15R15 | |
Centrifuge | Eppendorf | 5415R | |
Confocal Microscope | Olympus | IX81 | |
Cryostat | Thermo Scientific | NX50 | |
Dual Automatic Temperature Controller | Warner Instruments | TC-344B | |
Electrophoresis Power Supply | BIO-RAD | 1645050 | |
Fluoresence Microscope | Olympus | IX83 | |
High Speed Camera | pco | 1200 s | |
Laser Scan Head | Olympus | FV-1000 | |
Low Profile Open Bath Chamber (mounts into above microincubation system) | Warner Instruments | RC-42LP | |
Microincubation System | Warner Instruments | DH-40iL | |
Minivent Mouse Ventilator | Harvard Apparatus | 845 | |
NOD/SCID mice | Jackson Laboratory | 001303 | |
Precast Protein Gels | BIO-RAD | 4561033 | |
PVDF Transfer Packs | BIO-RAD | 1704156 | |
Trans-Blot System | BIO-RAD | Trans-Blot Turbo | |
Hot bead sterilizer | Fine Science Tools | 18000-45 | |
Name | Company | Catalog Number | Comments |
Antibody | |||
Anti-human Nucleolin (Alexa Fluor 647) | Abcam | ab198580 | |
Cardiac Troponin T | R&D Systems | MAB1874 | |
Cardiac Troponin C | Abcam | ab137130 | |
Cardiac Troponin I | Abcam | ab47003 | |
Cy5-donkey anti-mouse | Jackson ImmunoResearch Laboratory | 715-175-150 | |
Cy3-donkey anti-rabbit | Jackson ImmunoResearch Laboratory | 711-165-152 | |
Fitc-donkey anti-mouse | Jackson ImmunoResearch Laboratory | 715-095-150 | |
GAPDH | Abcam | ab22555 | |
Human Cardiac Troponin T | Abcam | ab91605 | |
Integrin β1 | Abcam | ab24693 | |
Ki67 | EMD Millipore | ab9260 | |
N-cadherin | Abcam | ab18203 | |
Phospho-Myosin Light Chain 2 | Cell Signaling Technology | 3671s | |
Name | Company | Catalog Number | Comments |
Software | |||
Matlab | MathWorks | R2016A | |
Image J | NIH | 1.52g |
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