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) and 100 mL of hPSC 5x supplement; store the mixture at 4 &#176;C.</li>
		<li>For RPMI 1640/B27 minus insulin (RB-) medium, mix 500 mL of RPMI 1640 and 10 mL of B27 supplement minus insulin.</li>
		<li>For RPMI 1640/B27 (RB+) medium, mix 500 mL of RPMI 1640, 10 mL of B27 supplement, and 5 mL of penicillin-streptomycin (pen-strep) antibiotic.</li>
		<li>For purification medium16, mix 500 mL of no-glucose RPMI 1640, 10 mL of B27 supplement, 5 mL of pen-strep antibiotic, and 1,000 &#956;L of sodium DL-lactate solution (at a final concentration of 4 mM).</li>
		<li>For neutralizing solution, mix 200 mL of RPMI 1640, 50 mL of fetal bovine serum (FBS), 2.5 mL of pen-strep antibiotic, and 50 &#956;L of Y-27632 (at a final concentration of 10 &#956;M).</li>
		<li>For freezing medium, mix 9 mL of FBS, 1 mL of dimethyl sulfoxide (DMSO), and 10 &#956;L of Y-27632 (at a final concentration of 10 &#956;M).</li>
		<li>For extracellular matrix solution (EMS), thaw concentrated extracellular matrix (Table of Materials 1) at 4 &#176;C until it has reached an evenly consistent liquid state. Precool pipette tips and tubes prior to making matrix aliquots of 350 &#181;L each on ice, and store the aliquots at -20 &#176;C. Before coating the plates, dilute one thawed aliquot into 24 mL of ice-cold DMEM/F12 medium (EMS); keep the EMS in 4 &#176;C for up to 2 weeks. 
		NOTE: Perform all coating steps on ice to prevent basement membrane matrix solidification.</li>
		<li>For Tyrode&#8217;s solution, take 90% of the final required volume of tissue culture grade water, add the appropriate amount of the following reagents, and stir until dissolved. Then, use 1 N NaOH to adjust the pH to 7.4. Add extra water to a final concentration of 140 mmol/L sodium chloride, 1 mmol/L magnesium chloride, 10 mmol/L HEPES, 5 mmol/L potassium chloride, 10 mmol/L glucose, and 1.8 mmol/L calcium chloride. Sterilize by filtration, using a 0.22 &#181;m membrane.</li>
	</ol>
	</li>
	<li>Cell culture plate coating
	<ol>
		<li>For hiPSC culture plates, apply 1 mL of EMS to each well of a 6-well plate and incubate the plate for at least 1 h at 37 &#176;C. Aspirate DMEM/F12 solution before seeding cells.</li>
		<li>For gelatin coating, dissolve 0.2 g of gelatin powder in tissue culture grade water to make a 0.2% (w/v) solution. Sterilize by autoclaving at 121 &#176;C, 15 psi for 30 min. Coat each well of a 6-well plate with 2 mL of the solution and incubate for 1 h at 37 &#176;C. Before passaging the cells, aspirate the solution and allow the plate to dry for at least 1 h at room temperature inside the tissue culture hood.</li>
	</ol>
	</li>
</ol>
2. hiPSC maintenance and Cardiomyocyte Ddifferentiation
<ol>
	<li>Culture the hiPSCs in hiPSC medium (see step 1.1.1) with a daily medium change until the cells reach 80%&#8211;90% confluence per well. 
	NOTE: For maintenance purposes, hiPSCs are generally ready for passage every 4 days.</li>
	<li>To passage hiPSCs, aspirate the medium and wash the well 1x with 1x phosphate-buffered saline (PBS). Add 0.5 mL of stem cell detachment solution (Table of Materials 1) to each well and incubate at 37 &#176;C for 5&#8211;7 min. 
	NOTE: The incubation time depends on the density of the cells and the rate of dissociation, which can be observed under a microscope.</li>
	<li>Neutralize the stem cell detachment solution with 1 mL of hiPSC medium supplemented with 5 &#956;M Y-27632. Collect the mixture in a 15 mL centrifuge tube. Centrifuge the tube for 5 min at 200 x g, aspirate the supernatant without disturbing the cell pellet, and then, resuspend the cells in 1 mL of hiPSC medium containing 5 &#956;M Y-27632.</li>
	<li>Seed the hiPSCs onto a new EMS-coated 6-well plate at a ratio between 1:6 to 1:18. Change the medium to hiPSC medium without Y27632 after 24 h.</li>
	<li>To freeze the cells, resuspend the dissociated hiPSCs in freezing medium at a concentration of 0.5&#8211;1 million/500 &#956;L, place the suspension in cryovials, and store them in a -80 &#176;C freezer. Transfer the frozen vials to liquid nitrogen after 24 h.</li>
	<li>To differentiate, replace the hiPSC medium with 2 mL of RB- medium supplemented with 10 &#956;M GSK-3&#946; inhibitor CHIR99021 (CHIR) at 80%&#8211;90% cell-confluence. Replace the medium with 3 mL of RB- medium after 24 h and incubate for an additional 48 h (day 3).</li>
	<li>On day 3, change the medium to 3 mL of RB- medium with 10 &#956;M Wnt inhibitor IWR1 for 48 h (day 5), and then replace the medium with 3 mL of RB- medium and maintain for 48 h (day 7).</li>
	<li>On day 7, replace the medium with 3 mL of RB medium and change the medium every 3 days going forward. Spontaneous beating of hiPSC-CMs should be observed between days 7&#8211;10.</li>
</ol>
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.
<ol>
	<li>On day 21 after hiPSC-CM differentiation, aspirate the medium and wash the cells 1x with sterile PBS. Incubate the cells with 0.5 mL of cell-dissociation enzymes per well for 5&#8211;7 min at 37 &#176;C. Repeatedly pipette the cell suspension using a 1 mL pipette in order to thoroughly dissociate the cells.</li>
	<li>After the cells are dissociated, add 1 mL of neutralizing solution to each well, collect the cell mixture into a 15 mL centrifuge tube and centrifuge at 200 x g for 3 min. Discard the supernatant and resuspend the cells in neutralizing solution.</li>
	<li>Replant the cells onto gelatin-coated 6-well plates at a density of 2 million cells per well.</li>
	<li>After 24&#8211;48 h, replace the culture medium with purification medium for 3&#8211;5 days. 
	NOTE: Purification is complete when more than 90% of the cells in each microscope view field are beating. To prevent further damage to the cardiomyocytes, do not extend the purification duration beyond 5 days. If the first round does not yield the necessary purity, replace the purification medium with RB+ medium for 1 day, and then complete a second round of purification.</li>
	<li>Prior to transplantation, culture the cells in the treatment group for 12 h in RB+ medium supplemented with 10 &#956;M Y-27632. Similarly, perform verapamil treatment on the cells in the RB+ medium with 1 &#956;M verapamil for 12 h (hiPSC-CMs+VER).</li>
	<li>After treatment, wash the hiPSC-CMs 1x with PBS and incubate the cells with 0.5 mL of cell-dissociation enzymes per well for a maximum of 2 min. Repeatedly pipette the cell suspension, using a 1 mL pipette, to thoroughly dissociate the cells. Neutralize the cells with neutralizing solution, collect the cell mixture in a 15 mL centrifuge tube, and centrifuge at 200 x g for 3 min. Resuspend the cells in PBS at a concentration of 0.1 million cells/5 &#956;L in preparation for injection.</li>
</ol>
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).
<ol>
	<li>Anesthetize NOD/scid mice (Table of Materials 2) with inhaled isoflurane (1.5%&#8211;2%). Monitor the anesthesia levels by the mice&#8217;s responses to a toe pinch. Place vet optical ointment onto the eyes to prevent them from drying during surgery.</li>
	<li>Supply 0.1 mg/kg buprenorphine subcutaneously for pain management prior to surgery.</li>
	<li>Position and secure the mouse in a supine position on a heated operating table, remove the hair from the ventral neck region and the left thorax using a depilatory cream, expose the skin, and disinfect it with 70% alcohol.</li>
	<li>Cut a 0.5 cm incision at the center of the neck using surgical scissors, separate the subcutaneous fat with sterile forceps, and expose the trachea. Introduce orally the intubation cannula into the trachea, connect the cannula to a small animal ventilator, and adjust the ventilation settings (set the tidal volume at 100&#8211;150 &#956;L and the respiration rate at 100x&#8211;150x per min).</li>
	<li>After the mouse stabilizes, cut a 0.5 cm incision in the middle of the left chest skin. Use forceps to bluntly separate the muscle layer and make a small incision in the fifth intercostal space to expose the chest cavity. Place the retractor in the incision to open the thoracic cavity and locate the left descending coronary artery (LAD). Under a dissecting surgical microscope, ligate the LAD with an 8-0 nonabsorbable suture.</li>
	<li>Immediately following MI induction, inject 5 &#956;L of hiPSC-CMs-RI, hiPSC-CMs+RI, hiPSC-CMs-VER, hiPSC-CMs+VER, or an equal volume of PBS into the mouse&#8217;s myocardium at each site (3 x 105 cells/animal, 1 x 105 cells/site), one in the infarct area and two in the areas around the infarct. 
	NOTE: Since 5 &#956;L is very small volume, it is not easy to accurately control the volume by directly sucking it with a syringe. The lid of a sterile Petri dish can be turned over, and 5 &#956;L of the cells can be first deposited on the lid with a pipette. Due to the surface tension, it will not spread but will condense into a small liquid mass. This can be easily absorbed and injected into the mouse&#8217;s myocardium.</li>
	<li>Eliminate residual air in the thoracic cavity by filling it up with warm isotonic saline solution. Stitch the ribs, muscles, and skin in sequence with a 6-0 absorbable suture. Turn off the isoflurane anesthesia injection. Keep the surgery animals on the heating pad and closely observe them while they regain full consciousness. Then, place the animals in a clean cage. Do not return the animals to the company of other animals until they are fully recovered.</li>
	<li>Following surgery, inject the mice intraperitoneally with buprenorphine (0.1 mg/kg) every 12 h for 3 consecutive days and inject them with ibuprofen (5 mg/kg) every 12 h for one day. Perform subsequent analysis studies at specified time points. 
	NOTE: Follow your local animal care committee guidelines for analgesia.</li>
</ol>
5. Calcium Transient and Contractility Recording
<ol>
	<li>Autoclave 15 mm-diameter coverslips (Table of Materials 2) and tweezers in a small glass beaker at 121 &#176;C, 15 psi for 15 min. Precool the coverslips and tweezers at 4 &#176;C.</li>
	<li>Using the tweezers, place the coverslip into a 12-well plate and pipette 300 &#956;L of extracellular matrix onto each coverslip. 
	NOTE: Do not let the matrix exceed the edge of the coverslip to prevent reducing the matrix coating amount. Incubate the 12-well plate in a 37 &#176;C cell culture incubator for 1 h.</li>
	<li>Aspirate the medium in the gelatin-coated 6-well plate with purified hiPSC-CMs, wash it 1x with PBS, and then digest it with 0.5 mL of cell-dissociation enzymes for 1.5 min. Add 1 mL of neutralizing solution, pipette repeatedly for no more than 5x&#8211;10x with a 1 mL pipette, and centrifuge the mixture at 200 x g for 3 min.</li>
	<li>Aspirate the coated extracellular matrix from the coverslips. Count the cell number and resuspend the cells in the neutralizing solution to a concentration of 40,000/300 &#956;L, and then add 300 &#956;L of the cell suspension onto each coverslip. Culture the plate in a 37 &#176;C incubator.</li>
	<li>After 24 h, gently aspirate the neutralized solution, add 500 &#956;L of RB+ medium to each well of the plate, and continue to culture for 2 days.</li>
	<li>Add Y-27632 (10 &#956;M), Rho kinase activator (RA, 100 nM), verapamil (1 &#956;M), or an equal volume of PBS into the culture medium of hiPSC-CMs, 12 h before calcium transient and contractility detection. 
	NOTE: In addition to the detection of the cells&#8217; calcium transient and contractility after 12 h of chemical treatment, the detection of these parameters was also tested at 12, 24, 48, and 72 h after chemical withdrawal.</li>
	<li>Take out the plate, discard the medium, and wash the plate 1x with PBS. Change the medium to a phenol-red-free DMEM containing 0.02% (w/v) of surfactant polyol (Table of Materials 1), 5 &#956;M calcium indicator (Table of Materials 1), and the corresponding Y-27632 (10 &#956;M), RA (100 nM), verapamil (1 &#956;M), or an equal volume of PBS, and incubate the plate for 30 min at 37 &#176;C.</li>
	<li>Discard the supernatant, wash the cells 3x with dye-free DMEM medium, and let the medium rest for 30 min to de-esterify the mapping dye.</li>
	<li>Place the cell-seeded coverslip in an open bath chamber and insert the chamber into a microincubation system equilibrated to 37 &#176;C with an automatic temperature controller (Table of Materials 2), perfuse it with Tyrode&#8217;s solution, and continuously add Rho kinase inhibitor (RI), RA, or PBS to the perfusion solution.</li>
	<li>Use an inverted fluorescence microscope (Table of Materials 2) and a laser scanning head (Table of Materials 2) to record spontaneous calcium transient by employing an x-t line scan, using a 488 nm argon laser for dye excitation and a 515 &#177; 10 nm barrier filter to collect emission light. Record the spontaneous contraction of hiPSC-CMs using the Differential Interference Contrast functionality of the the confocal microscope (Table of Materials 2).&#160;&#160; 
	NOTE: Calcium transient recordings were processed and analyzed using MATLAB R2016A software (Table of Materials 4). Cell contraction change assays were performed using ImageJ software (Table of Materials 4).</li>
</ol>

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The authors have nothing to disclose.

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</...

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

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. ...

enhancing-engraftment-human-induced-pluripotent-stem-cell-derived

Research

JoVE Journal

Biochemistry

6.0K Views.  University of Alabama at Birmingham. 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.

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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...