Name: enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity
Uploaded: 2019-07-10T12:30:00
Description: Watch this Scientific Journal Video about Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity at JoVE.com

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.).

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
<ol>
	<li>Medium preparation
	<ol>
		<li>For hiPSC medium, mix 400 mL of human pluripotent stem cell (hPSC) basal medium (Table of Materials 1</st...

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Single-Cell+Analysis+of+Transplanted+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+After+Acute+Myocardial+Infarction.">Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes After Acute Myocardial Infarction.</a> Circulation. 132 (8), 762-771 (2015).</li><li>Zhao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+Preconditioning+Enhances+Transplantation+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+in+Myocardial+Infarction+Mice.">Y-27632 Preconditioning Enhances Transplantation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Myocardial Infarction Mice.</a> Cardiovascular Research. , (2018).</li><li>Tohyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li><li>Silginer, M., Weller, M., Ziegler, U., Roth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inhibition+promotes+atypical+anoikis+in+glioma+cells.">Integrin inhibition promotes atypical anoikis in glioma cells.</a> Cell Death &amp; Disease. 5, e1012 (2014).</li><li>Lelievre, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+mediates+neuronal+cell+survival+through+Bim+down-regulation.">N-cadherin mediates neuronal cell survival through Bim down-regulation.</a> PLoS One. 7 (3), e33206 (2012).</li><li>Murata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+cell+motility+by+carbon+ion+irradiation+via+the+Rho+signaling+pathway+and+its+inhibition+by+the+ROCK+inhibitor+Y-27632+in+lung+adenocarcinoma+A549+cells.">Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells.</a> Journal of Radiation Research. 55 (4), 658-664 (2014).</li><li>Srivastava, K., Shao, B., Bayraktutan, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC-beta+exacerbates+in+vitro+brain+barrier+damage+in+hyperglycemic+settings+via+regulation+of+RhoA/Rho-kinase/MLC2+pathway.">PKC-beta exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 33 (12), 1928-1936 (2013).</li></ol>

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...

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.

<table>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase (stem cell detachment solution)</td>
			<td>STEMCELL Technologies</td>
			<td>#07920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 minus insulin</td>
			<td>Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Fisher Scientific</td>
			<td>17-504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stem Cell Technologies</td>
			<td>72054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x), high glucose, HEPES, no phenol red</td>
			<td>Thermofisher</td>
			<td>20163029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM (calcium indicator)</td>
			<td>Invitrogen/Thermofisher</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-free RPMI 1640</td>
			<td>Fisher Scientific</td>
			<td>11879020</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR1</td>
			<td>Stem Cell Technologies</td>
			<td>72562</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (extracellular matrix )</td>
			<td>Fisher Scientific</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR (human pluripotent stem cells medium)</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-strep antibiotic</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (surfactant polyol)</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho activator II</td>
			<td>Cytoskeleton</td>
			<td>CN03</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium DL-lactate</td>
			<td>Sigma-Aldrich</td>
			<td>L4263</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE (cell-dissociation enzymes)</td>
			<td>Fisher Scientific</td>
			<td>12-605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment and Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III Bioluminescence Instruments</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Coverslips</td>
			<td>Warner</td>
			<td>CS-15R15</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific</td>
			<td>NX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Automatic Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>BIO-RAD</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresence Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>pco</td>
			<td>1200 s</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scan Head</td>
			<td>Olympus</td>
			<td>FV-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Profile Open Bath Chamber (mounts into above microincubation system)</td>
			<td>Warner Instruments</td>
			<td>RC-42LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microincubation System</td>
			<td>Warner Instruments</td>
			<td>DH-40iL</td>
			<td></td>
		</tr>
		<tr>
			<td>Minivent Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>845</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD/SCID mice</td>
			<td>Jackson Laboratory</td>
			<td>001303</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast Protein Gels</td>
			<td>BIO-RAD</td>
			<td>4561033</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Transfer Packs</td>
			<td>BIO-RAD</td>
			<td>1704156</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot System</td>
			<td>BIO-RAD</td>
			<td>Trans-Blot Turbo</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human Nucleolin (Alexa Fluor 647)</td>
			<td>Abcam</td>
			<td>ab198580</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin T</td>
			<td>R&#38;D Systems</td>
			<td>MAB1874</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin C</td>
			<td>Abcam</td>
			<td>ab137130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin I</td>
			<td>Abcam</td>
			<td>ab47003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5-donkey anti-mouse</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>715-175-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-donkey anti-rabbit</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>711-165-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Fitc-donkey anti-mouse</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>715-095-150</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Abcam</td>
			<td>ab22555</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cardiac Troponin T</td>
			<td>Abcam</td>
			<td>ab91605</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin &#946;1</td>
			<td>Abcam</td>
			<td>ab24693</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>EMD Millipore</td>
			<td>ab9260</td>
			<td></td>
		</tr>
		<tr>
			<td>N-cadherin</td>
			<td>Abcam</td>
			<td>ab18203</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Myosin Light Chain 2</td>
			<td>Cell Signaling Technology</td>
			<td>3671s</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>R2016A</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td>1.52g</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity

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.

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</...

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Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

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. ...

Our data demonstrates that transient inhibition of Rho-kinase activity enhances adhesion, survival, and engraftment of hiPSC-derived cardiomyocytes after being transplanted into the infarcted animal hearts. We provide a simple, low-cost, while highly efficient method to increase engraftment of transplanted hiPSC-derived cardiomyocytes in the injured animal hearts. As this technique can improve cell engraftment rates safely and efficiently in acute cardiac infarction and heart failure, it contributes for the resulting improvements in cardiac function, vascularities, and apoptosis.Although we only tested our protocols in hiPSCs, these methods can be applied to other types of cells, such as embryonic stem cells, mesenchymal stem cells, et cetera. The dose and duration of Y-27632 treatment is critical. The visual demonstration can demonstrate in great detail all key steps in the protocol, which is beneficial for someone who has never performed this technique before.On day 21 after hiPSC-CM differentiation, aspirate the medium and wash the cells with sterile PBS once. Incubate the cells with 0.5 milliliters of cell-dissociation enzymes per well at 37 degrees Celsius for five to seven minutes. After the incubation time, repeatedly pipette the cell suspension using a one-milliliter pipette in order to thoroughly dissociate the cells.Then, add one milliliter of neutralizing solution to each well, collect the cell mixture in a 15-milliliter centrifuge tube, and centrifuge at 200 times g for three minutes. After the centrifugation, discard the supernatant and resuspend the cells in the neutralizing solution. Then, replant the cells onto gelatin-coated, six-well plates at a density of two million cells per well.After 24 to 48 hours, replace the culture medium with the purification medium for three to five days. Prior to transplantation, culture the cells in the treatment group for 12 hours in RB-plus medium supplemented with 10-micromolar Y-27632. Similarly, perform verapamil treatment on the cells in the RB-plus medium with one micromole of verapamil for 12 hours.After the treatment, wash the hiPSC-CMs with PBS once, and incubate the cells with 0.5 microliters of cell-dissociation enzymes per well for a maximum of two minutes. Repeatedly pipette the cell suspension using a one-milliliter pipette to thoroughly dissociate the cells. Next, neutralize the cells with neutralizing solution, collect the cell mixture in a 15-milliliter centrifuge tube, and centrifuge at 200 times g for three minutes.After centrifugation, resuspend the cells in PBS at a concentration of 0.1 million cells per five microliters in preparation for injection. Immediately following myocardial infarction induction, inject five microliters of control hiPSC-CMs, Y-27632-pretreated hiPSC-CMs, verapamil-treated hiPSC-CMs, or an equal volume of PBS into the mouse's myocardium at each site, one in the infarct area and two in the areas around the infarct. 12 hours before performing calcium transient and contractility detection, add 10-micromolar Y-27632, 100-nanomolar RA, one-micromolar verapamil, or an equal volume of PBS into the culture medium of hiPSC-CMs.Next, discard the medium, and wash the plate with PBS once. Change the medium to a phenol red-free DMEM containing 0.02 weight per volume percent of surfactant polyol, five-micromolar calcium indicator, and the corresponding Y-27632, RA, verapamil, or an equal volume of PBS, and incubate the plate at 37 degrees Celsius for 30 minutes. After the incubation, discard the supernatant, wash the cells with the dye-free DMEM three times, and let the medium rest for 30 minutes to de-esterify the mapping dye.Place the cell-seeded coverslip in an open bath chamber, and insert the chamber into a microincubation system equilibrated to 37 degrees Celsius with an automatic temperature controller. Perfuse it with Tyrode's solution, and continuously add RI, RA, or PBS to the perfusion solution. Use an inverted fluorescence microscope and a laser scanning head to record spontaneous calcium transient by employing an x-t line scan.Record the spontaneous contraction of hiPSC-CMs using the differential interference contrast functionality of the confocal microscope. The survival rate of the transplanted cells was confirmed by the increased luciferase activity. The increased human cardiac troponin T and human nuclear antigen expression showed that Y-27632 pretreatment could significantly improve the cell engraftment rate.Y-27632 pretreated cells exhibited a larger and more defined rod-shaped cytoskeletal structure. Also, Y-27632 pretreatment decreased TUNEL-positive cells, indicating the reduced transplanted hiPSC-CM apoptosis in vivo. Western blot suggested that Y-27632 pretreatment increased expression of integrin beta-1 and N-cadherin and decreased the expression of phospho-myosin light chain 2.This change was normalized 72 hours after Y-27632 withdrawal. In vitro mouse HL-1 cell attachment experiments indicated that Y-27632 pretreatment significantly increased hiPSC-CM adherence relative to HL-1. Y-27632 pretreatment reduced the contractile force, the peak calcium transient fluorescence, and the calcium transient duration.Y-27632 pretreatment regulated cardiomyocyte contraction by significantly reducing the expression of cTnI and cTnT. Similar to Y-27632, verapamil pretreatment increased the luciferase activity and numbers of hcTnT/HNA double-positive cells. The most important step is to culture cells in the treatment group for 12 hours in Rb-plus medium supplemented with 10-micromolar Y-27632.Based on this method, slow release of Y-27632 to further increase the cell engraftment rate is a promising strategy. This technique paves the way for researchers to study the physiology and function of hiPSC of CMs in vivo. The small molecule CHIR99021 used for cardiomyocytes differentiation is hazardous, which may cause respiratory irritation.Please don't intake or inhale by any way.

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