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 hypoxic environment accelerates the course toward cell death. Of the remaining cells, a large proportion is washed out from the implantation site into the bloodstream and spread throughout the periphery. One of the key apoptotic pathways is the RhoA/ROCK pathway4. Based on previous research, the RhoA/ROCK pathway regulates the actin cytoskeletal organization5,6, which is responsible for cell dysfunction7,8. The ROCK inhibitor Y-27632 is widely used during somatic and stem cell dissociation and passaging, to increase cell adhesion and reduce cell apoptosis9,10,11. In this study, Y-27632 is used to treat hiPSC-CMs prior to transplantation in an attempt to increase the cell engraftment rate.
Several methods aimed at improving the cell engraftment rate, such as heat shock and basement membrane matrix coating12, have been established. Aside from these methods, genetic technology can also promote cardiomyocyte proliferation13 or reverse nonmyocardial cells into cardiomyocytes14. From the bioengineering perspective, cardiomyocytes are seeded onto a biomaterial scaffold to improve the transplantation efficiency15. Unfortunately, the majority of these methods are complicated and costly. On the contrary, the method proposed here is simple, cost-efficient, and effective, and it can be used as a basal treatment before transplantation, as well as in conjugation with other technologies.
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
2. hiPSC maintenance and Cardiomyocyte Ddifferentiation
3. hiPSC-CMs Purification and Small Molecule Pre-treatment
NOTE: Highly purified, recombinant cell-dissociation enzymes (Table of Materials 1) were used to dissociate hiPSC-CMs.
4. Myocardial Infarction and Cell Transplantation
NOTE: All surgical instruments are presterilized with autoclave and are maintained in aseptic condition during multiple surgeries via a hot bead sterilizer (Table of Materials 2).
5. Calcium Transient and Contractility Recording
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). Both results indicated that Y-27632 pretreatment significantly improved the cell engraftment rate. The luciferase activity of the hiPSC-CM+RI group was increased roughly sixfold on days 3, 7, and 28 after the transplantation, compared to that of the hiPSC-CM-RI group (Figure 1B). Moreover, the hcTnT/HNA expression increased close to sevenfold in the hiPSC-CM+RI group, relative to that in the hiPSC-CM-RI group (Figure 1C).
The results also indicated that Y-27632 pretreatment regulated cytoskeletal changes in transplanted cells. On days 7 and 28 of transplantation, hiPSC-CMs+RI exhibited a larger and more defined rod-shaped cytoskeletal structure compared to hiPSC-CMs-RI (Figure 1D).
Moreover, Y-27632 pretreatment has the potential to reduce transplanted hiPSC-CM apoptosis in vivo. TUNEL staining showed that the number of TUNEL-positive cells was significantly decreased in the hiPSC-CM+RI group relative to that in the hiPSC-CM-RI group on day 2 after transplantation (Figure 1E).
ROCK inhibition promoted the adhesion of transplanted cells and had the potential to further increase the retention of implanted cells at administration sites. Western blot and cardiac tissue immunostaining suggested that Y-27632 pretreatment reversibly promoted the increased expression of integrin β1 and N-cadherin and decreased the expression of phospho-myosin light chain 2 (p-MLC2) (Figure 2A,B).
The mouse cell line HL-1 was selected as the control CMs for in vitro cell attachment experiments. The results indicated that Y-27632 pretreatment significantly increased hiPSC-CM adherence relative to HL-1. To further confirm these findings, hiPSC-CMs+RI were incubated with N-cadherin or integrin β1 neutralizing antibody for 1 h, resulting in a vanishing of the improved adhesion seen previously (Figure 2C).
Compared with the hiPSC-CM-RI group, the contractile force in the hiPSC-CM+RI group was reduced by 32%, and in the hiPSC-CM+RA group (hiPSC-CMs pretreated with ROCK activator, Table of Materials 1), it was increased by 42% (Figure 3A). Meanwhile, compared with the hiPSC-CM-RI group, peak calcium transient fluorescence (Peak ΔF/F0) for the hiPSC-CM+RI group was reduced by 41%, and the calcium transient duration (CaTD50) was reduced by 11% (Figure 3B,C). In contrast, Peak ΔF/F0 for the hiPSC-CM+RA group was increased by 48%, and CaTD50 was increased by 13% (Figure 3B,C).
In addition, a pretreatment of the cardiomyocytes with Y-27632 for 12 h prior to the transplantation significantly reduced the expression of cTnI and cTnT (Figure 3D), both of which are troponin (Tn) subunits that regulate cardiomyocyte contraction.
Similar to Y-27632, a verapamil pretreatment (1 μM, 12 h) significantly increased the engraftment rate of hiPSC-CMs in induced MI mice. The hypothesis was confirmed through the observation of an increased luciferase signal in the bioluminescence assay (Figure 4A) and increased numbers of hcTnT/HNA double-positive cells on days 7 and 28 after cell transplantation (Figure 4B).
Figure 1: Y-27632 pretreatment increased the engraftment rate of hiPSC-CMs in MI mice hearts. (A) Standard curve of BLI measurements. (B) Luciferin signal in NOD/scid mice treated with PBS, hiPSC-CMs-RI, or hiPSC-CMs+RI on days 3, 7, and 28 after surgery. n = 6-9 mice per group. *P < 0.05 vs. PBS; †P < 0.05 vs. hiPSC-CMs-RI. (C) Immunostaining of heart sections for hcTnT and HNA. Scale bars = 100 µm (10x images); 20 µm (40x images). n = 5 mice per group. *P < 0.05 vs. hiPSC-CMs-RI. (D) Representative images of heart sections stained with phalloidin and hcTnT. Scale bar = 20 µm. n = 5 mice per group. *P < 0.05 vs. hiPSC-CMs-RI. (E) Representative images of heart sections for TUNEL staining. Scale bar = 20 µm. n = 4-6 mice per group. *P < 0.05 vs. hiPSC-CMs-RI. This figure has been modified from Zhao et al.18. Please click here to view a larger version of this figure.
Figure 2: Y-27632 enhanced the adhesion of hiPSC-CMs by maintaining the expression of adhesion proteins. (A and B) Western blot analysis of the expression of integrin ß1, N-cadherin, and phosphorylation of MLC2 (p-MLC2) in hiPSC-CMs treated with RI and in nontreated hiPSC-CMs. n = 5 replicates per group. (C) hcTnT immunofluorescence staining analysis of -RI and +RI hiPSC-CMs with and without a pretreatment of anti-N-cadherin (N-Cad) and anti-integrin ß1 (Integ) antibodies. Scale bars = 100 µm. *P < 0.05 vs. hiPSC-CMs-RI; †P < 0.05 vs. hiPSC-CMs+RI. This figure has been modified from Zhao et al.18. Please click here to view a larger version of this figure.
Figure 3: Pretreatment with Y-27632 reduced the contractility of hiPSC-CMs and down-regulated the expression of cardiac troponin subunits. (A) Quantification of the percentage of shortening of hiPSC-CMs exposed to RI and RA treatment. *P < 0.05 vs. hiPSC-CMs-RI; †P < 0.05 vs. hiPSC-CMs+RI. (B and C) Representative images and quantification of calcium transient measurements of hiPSC-CMs treated with RI and RA and of a nontreated group. *P < 0.05 vs. hiPSC-CMs-RI; †P < 0.05 vs. hiPSC-CMs+RI. (D) Western blot analysis of the expression of cardiac troponin subunits (cTnC, cTnT, and cTnI) in hiPSC-CMs treated with RI and RA and in a nontreated group. This figure has been modified from Zhao et al.18. Please click here to view a larger version of this figure.
Figure 4: Verapamil pretreatment improved the engraftment rate of hiPSC-CMs in MI mice. (A) Luciferin signal in NOD/scid mice treated with PBS, hiPSC-CMs-VER, or hiPSC-CMs+VER on days 3, 7, and 28 after surgery. n = 8 mice per group. *P < 0.05 vs. PBS; †P < 0.05 vs. hiPSC-CMs-VER. (B) Immunostaining of heart sections for hcTnT and HNA. For 10x images, Scale bars = 100 µm (10x images); 20 µm (40x images). n = 5 mice per group. *P < 0.05 vs. hiPSC-CMs-VER. This figure has been modified from Zhao et al.18. Please click here to view a larger version of this figure.
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, culturing the cells in the neutralization medium for 24 h after replating, and minimizing the digestion time of the cells before transplantation, all of which were performed to achieve the highest activity of the cells while obtaining pure cardiomyocytes.
Second, we elaborated on the pretreatment of hiPSC-CMs with Y-27632 at 12 h before cell injection. The anoikis is usually induced by the modification of cells’ adhesion proteins, such as integrins4,20 and N-cadherin21, that lead to the activation of the apoptotic pathway. We demonstrated that Y-27632 pretreatment could promote the adhesion of hiPSC-CMs to the transplantation site through maintaining the expression of integrin β1 and N-cadherin, suppressing the expression of p-MLC2, which is related to changes in the cytoskeletal architecture22,23. Seven days after hiPSC-CM transplantation, hiPSC-CMs+RI resulted in a greater cell engraftment area, finer defined rod shapes, and a more complete cytoskeletal organization compared to hiPSC-CMs-RI (Figure 1B-D).
Third, we established a novel intracellular injection system in mouse MI models, with 5 μL per injection point to accurately inject the required number of cells, while avoiding a decrease in the mouse survival rate due to excessive injection volume.
Fourth, we elaborated on how to determine the changes in cell contraction and calcium transient in hiPSC-CMs after pretreatment with RI or RA. We found that Y-27632 can reversibly reduce the cell contractility and calcium transient. The hypothesis here is that due to the lower energy requirements of the transplanted cells, the engraftment rate increased. Similar effects can be achieved using the calcium channel inhibitor verapamil. The proposed mechanism resulting in the contractility inhibition is that Y-27632 reduces the expression of troponin subunits cTnT and cTnI in hiPSC-CMs.
The main limitation is that Y-27632 only played a transient role during the transplantation. How to inhibit Rho kinase for a longer period, thus further improving the transplantation efficiency of hiPSC-CMs, is a problem to be solved in the future. As for more applications of this method, ROCK inhibitor can not only improve the activity of cardiomyocytes during transplantation but also enhance the activity of other cells; therefore, it can also be used during the pretreatment in the transplantation processes of other cells. Moreover, the approach presented here lays a good experimental foundation for more research on heart disease.
The authors have nothing to disclose.
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.).
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 |