Name: enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity
Uploaded: 2019-07-10T12:30:00.000+00:00
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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) 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, 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&#8217; 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 &#946;1 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 &#956;L 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.

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>&lt;strong&gt;Reagent&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment and Supplies&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</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&amp;amp;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 &amp;beta;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matlab</td><td>MathWorks</td><td>R2016A</td><td></td></tr><tr><td>ImageJ</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). 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 &#946;1 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 &#946;1 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 &#916;F/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 &#916;F/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 &#956;M, 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59452/59452fig1.jpg" /> 
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 &#60; 0.05 vs. PBS; &#8224;P &#60; 0.05 vs. hiPSC-CMs-RI. (C) Immunostaining of heart sections for hcTnT and HNA. Scale bars = 100 &#181;m (10x images); 20 &#181;m (40x images). n = 5 mice per group. *P &#60; 0.05 vs. hiPSC-CMs-RI. (D) Representative images of heart sections stained with phalloidin and hcTnT. Scale bar = 20 &#181;m. n = 5 mice per group. *P &#60; 0.05 vs. hiPSC-CMs-RI. (E) Representative images of heart sections for TUNEL staining. Scale bar = 20 &#181;m. n = 4-6 mice per group. *P &#60; 0.05 vs. hiPSC-CMs-RI. This figure has been modified from Zhao et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59452/59452fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59452/59452fig2.jpg" /> 
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 &#223;1, 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 &#223;1 (Integ) antibodies. Scale bars = 100 &#181;m. *P &#60; 0.05 vs. hiPSC-CMs-RI; &#8224;P &#60; 0.05 vs. hiPSC-CMs+RI. This figure has been modified from Zhao et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59452/59452fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59452/59452fig3.jpg" /> 
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 &#60; 0.05 vs. hiPSC-CMs-RI; &#8224;P &#60; 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 &#60; 0.05 vs. hiPSC-CMs-RI; &#8224;P &#60; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59452/59452fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59452/59452fig4.jpg" /> 
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 &#60; 0.05 vs. PBS; &#8224;P &#60; 0.05 vs. hiPSC-CMs-VER. (B) Immunostaining of heart sections for hcTnT and HNA. For 10x images, Scale bars = 100 &#181;m (10x images); 20 &#181;m (40x images). n = 5 mice per group. *P &#60; 0.05 vs. hiPSC-CMs-VER. This figure has been modified from Zhao et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59452/59452fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.1K Views.  University of Alabama at Birmingham. Onze gegevens tonen aan dat voorbijgaande remming van Rho-kinase activiteit hechting, overleving en engraftment van hiPSC-afgeleide cardiomyocyten verbetert nadat ze in de infarcted animal hearts zijn getransplanteerd. Wij bieden een eenvoudige, goedkope, terwijl zeer efficiënte methode om de engraftment van getransplanteerde hiPSC-afgeleide cardiomyocyten in de gewonde dierenharten te verhogen. Aangezien deze techniek celengraftment tarieven veilig en efficiënt in acuut hartinfarct en hart...

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

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Developmental Biology

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Bioengineering

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Biology

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Medicine

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

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

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