Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00.000+00:00
Duration: 6 min 37 s
Description: Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

All experimental procedures are performed in accordance with the Animal Guidelines of the University of Alabama at Birmingham.
1. Differentiating hiPSCs into hiPSC-CMs
<ol>
	<li>Coat the wells of a 6-well plate with pre-cooled growth-factor-reduced gelatinous protein mixture at 4 &#176;C for overnight. Aspirate the gelatinous protein mixture before use. Seed the hiPSCs onto the pre-coated plates, and culture the cells (1 x 105 cell per well) at 5% CO2 and 37 &#176;C in mTeSR1 medium supplement with 10 &#181;M ROCK inhibitor.</li>
	<li>Refresh the medium daily until the cells reach 90% confluence; then, add growth-factor-reduced gelatinous protein mixture to the medium (0.5 mg gelatinous protein mixture per 6-well plate, 2 ml medium per well), and culture the cells for 5% CO2 and 37 &#176;C two more days.
	<ol>
		<li>To replace the medium, gently suck out the medium from the petri dish via vacuum without touching the cells, and add new medium using a transfer pipette.</li>
	</ol>
	</li>
	<li>Initiate differentiation by replacing the medium with RPMI1640 medium supplemented with growth-factor-reduced gelatinous protein mixture, B27 without insulin, and 100 ng/ml Activin A; culture the cells at 5% CO2 and 37 &#176;C for 24 hr.</li>
	<li>Replace the medium with RPMI1640 medium that has been supplemented with B27 without insulin, 10 ng/ml bone morphogenic protein 4 (BMP-4), and 10 ng/ml basic fibroblast growth factor (bFGF); culture the cells at 5% CO2 and 37 &#176;C for 96 hr.</li>
	<li>Replace the medium with RPMI1640 medium supplemented with B27 and continue culture the cells at 5% CO2 and 37 &#176;C; refresh the medium every 3 days.</li>
	<li>Observe clusters of contracting cells under the microscope ~3 days after initiating differentiation. Collect the clusters ~7 days after first observing contractions and wash them in Hanks Balanced Salt Solution.
	<ol>
		<li>Dissociate the clustered-cells in the plate by incubating them in Hanks buffer containing 100 U/ml collagenase IV for 10 min at 37 &#186;C with gentle shaking; then, add 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) for 5 min.</li>
		<li>Neutralize the enzyme solution with 10% fetal bovine serum in RPMI/B27 medium and then resuspend the cells in RPMI/B27 medium.</li>
		<li>Count the number of cells by hemocytometer. Culture the cells on 10 cm dishes at 5% CO2 and 37 &#176;C for at least 3 hr, which will allow the non-cardiomyocyte cells to adhere to the surface of the culture dish.</li>
	</ol>
	</li>
	<li>Collect the cell suspension, which contains the hiPSC-CMs, and maintain the cells (around 1 x 106 cells per 10 cm dish) by culturing them on a gelatinous protein mixture -coated surface.</li>
</ol>
2. Differentiating hiPSCs into hiPSC-ECs
<ol>
	<li>Dissociate the hiPSC population into single cells by incubating them with chelating agent at 5% CO2 and 37 &#176;C for 5 min. Transfer the cells to a 15 ml centrifuge tube containing fresh mTeSR1 medium.</li>
	<li>Spin down the cells at 200 x g for 5 min. Resuspend the cells in mTeSR1 medium supplemented with 10 &#181;M ROCK inhibitor. Determine the cell density with a hemocytometer.</li>
	<li>Add 250 &#181;l of 20 NIH units/ml thrombin solution to one well of a 24-well plate.</li>
	<li>Add 1 x 106 hiPSCs to 250 &#181;l of a 12.5 mg/ml fibrinogen solution. And add the 250 &#181;l of cell-containing fibrinogen solution to the thrombin-loaded well. Observe the mixture solidify to form a hiPSC-containing fibrin scaffold within min.</li>
	<li>Transfer the solidified, cell-containing scaffolds into the wells of a 6-well plate with the cover glass forceps, and initiate differentiation by culturing the scaffolds in 2 ml EBM2 medium supplemented with 2% B27 without insulin, activin-A (50 ng/ml), and BMP-4 (25 ng/ml) for 24 hr at 5% CO2 and 37 &#176;C.
	<ol>
		<li>Replace the medium by gently sucking out the old medium via vacuum without touching the cells. Add new EBM2 medium supplemented with B27 without insulin, vascular endothelial growth factor (VEGF, 50 ng/ml), erythropoietin (EPO, 100 ng/ml), and transforming growth factor &#946;1 (TGF&#946;1, 10 ng/ml), and culture the scaffolds at 5% CO2 and 37 &#176;C for 48 hr.</li>
		<li>Refresh the medium and culture the scaffolds at 5% CO2 and 37 &#176;C for another 48 hr; then, release the cells from the scaffold by adding 200 U collagenase IV (100 U/ml) to the medium.</li>
		<li>Spin down the cells after they are released. Replace the medium with 2 ml EGM2-MV medium supplemented with 2% of B27, VEGF-A (50 ng/ml), and SB-431542 (10 &#181;M); refresh the medium every two days.</li>
		<li>Approximately 10 days later (i.e., on ~Day 14 after differentiation was initiated), dissociate the cells with 100 U/ml collagenase IV, isolate hiPSC-ECs from the population of differentiated cells via flow-cytometry analyses for the expression of EC-specific marker proteins (e.g., CD31, CD144)3.</li>
		<li>Expand the isolated hiPSC-ECs via an established protocol3.</li>
	</ol>
	</li>
</ol>
3. Differentiating hiPSCs into hiPSC-SMCs
<ol>
	<li>Seed the hiPSCs onto plates that have been coated with gelatinous protein mixture, and culture the cells in mTeSR1 medium at 5% CO2 and 37 &#176;C, with daily medium changes, until confluent (~2 days).</li>
	<li>Initiate differentiation into mesodermal-lineage cells by culturing the cells with CHIR99021 (5 &#181;M) and BMP-4 (10 ng/ml) in RPMI1640 medium and 2% B27 plus insulin for 3 days.</li>
	<li>To produce hiPSC-SMCs with a predominantly synthetic phenotype:
	<ol>
		<li>Culture the cells with VEGF-A (25 ng/ml), fibroblast growth factor &#946; (FGF&#946;, 5 ng/ml) in RPMI1640 medium, and 2% B27 minus insulin at 5% CO2 and 37 &#176;C for 4 days.</li>
		<li>Culture the cells in VEGF-A (25 ng/ml), FGF&#946; (5 ng/ml) in RPMI1640 medium, and 2% B27 plus insulin at 5% CO2 and 37 &#176;C for 2 days.</li>
		<li>Culture the cells in platelet-derived growth factor &#946; (PDGF&#946;, 10 ng/ml), TGF&#946; (3 ng/ml) in RPMI1640, and 2% B27 plus insulin at 5% CO2 and 37 &#176;C for 4 days.</li>
	</ol>
	</li>
	<li>To produce hiPSC-SMCs with a predominantly contractile phenotype:
	<ol>
		<li>Culture the cells for 4 days with VEGF-A (25 ng/ml) and FGF&#946; (5 ng/ml) in RPMI1640 and 2% B27 minus insulin.</li>
		<li>Culture the cells for 7 days with PDGF&#946; (5 ng/ml) and TGF&#946; (2.5 ng/ml) in RPMI1640 and 2% B27 plus insulin.</li>
	</ol>
	</li>
	<li>Purify the final hiPSC-SMC populations by culturing the cells in lactate (4 mM) containing RPMI1640 metabolic medium for ~6 days.</li>
</ol>
4. Creating the IGF-containing Microspheres
<ol>
	<li>Heat olive oil to 45 &#176;C in a water bath.</li>
	<li>Heat 5 ml of 10% gelatin solution to 50 &#176;C.</li>
	<li>Add the gelatin to the olive oil, stir, and then rapidly cool to 5 &#176;C by adding ice to the water bath.</li>
	<li>Twenty-five minutes later, add chilled (4 &#176;C) acetone to the olive oil to induce microsphere formation.</li>
	<li>Maintain the temperature at 5 &#176;C for 1 hr; then, collect the microspheres, wash them 5 times with pre-cooled acetone to remove the olive oil, and allow them to air-dry at 4 &#176;C.</li>
	<li>Resuspend the microspheres in a solution of 70% ethanol and 0.25% glutaraldehyde overnight at 4 &#176;C to induce cross-linking, and then neutralize the mixture with 100 mM glycine.</li>
	<li>Load IGF into the microspheres by mixing 5 mg microspheres with 15 &#181;l distilled H2O containing 5 &#181;g IGF and 0.1% bovine serum albumin for 30 min.</li>
</ol>
5. Creating the Patch over the Site of Injury and Injecting the Cells
<ol>
	<li>Suspend the hiPSC-derived CMs (2 million), SMCs (1 million), and ECs (1 million) together in 1 ml Minimum Essential Medium (MEM).</li>
	<li>Suspend 5 mg of microspheres in 1 ml fibrinogen solution (25 mg/ml).</li>
	<li>Fill one syringe with the cell-containing solution, another syringe with the fibrinogen/microsphere solution, and a third syringe with 1 ml of thrombin solution (80 NIH units/ml, supplemented with 2 &#181;l 400 mM CaCl2 and 200 mM &#949;-aminocaproic acid).</li>
	<li>Surgically induce myocardial infarction in swine heart via ligation of left anterior descending coronary artery as described before2. 
	NOTE: In brief, female Yorkshire swine (~13 kg, 45 days of age) are sedated with intramuscular injection of Telazol/Xylazine (4.4 mg/kg), and&#160;intubated under general anesthesia with isoflurane (0.5-5%). A 4th intercostal thoracotomy is performed and left anterior descending coronary artery is exposed. The coronary artery is occluded by a ligature for 60 min and then the ligature is removed. Immediate electrical defibrillation is performed in the case of ventricular fibrillation.</li>
	<li>Position a sterile plastic ring (~2.5 cm) on the epicardium of the infarcted region of swine hearts, and then simultaneously inject the microsphere/fibrinogen and thrombin solutions into the ring.</li>
	<li>Allow the mixture to solidify (~30 sec), and then insert the needle of the cell-containing syringe through the solidified mixture into the infarcted myocardium and inject the cells. Close the muscle layers, subcutaneous tissue, and the chest wall with 3-0 or 2-0 Monocryl suture depending on animal size.&#160;Buprenorphine 0.01-0.02 mg/kg intramuscular injection every 8 hours will be used to control pain for the first 3 days after surgery.</li>
	<li>Evaluate cardiac function using a cardiac magnetic resonance imaging (MRI) before surgical procedure, one week and four weeks after the surgical procedure2,3,4. After the final MRI studies, sacrifice the animal and process their hearts for histology and molecular analysis2,3,4.</li>
</ol>

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Improved Yield/Purity of hiPSC-CMs
Conventional protocols for differentiating human stem cells into CMs are often limited by low yield and purity; for example, just 35-66% of hESC-CMs obtained via Percoll separation and cardiac body formation expressed slow myosin heavy chain or cTnT6. The purity of differentiated hiPSC-CM populations can be substantially increased by selecting for the expression of a reporter gene that has been linked to the promoter of a CM-specific protein7,8,9, but this technique necessarily requires the use of genetically modified cells, which are less desirable, particularly for clinical investigations. With the protocol presented here, 68% of the hiPSC-CMs expressed cTnT, and the purity was subsequently increased to &#62;90% via micro-dissection and preplating without genetic manipulation or single-cell selection. The total yield was ~3-5 million hiPSC-CMs per well.
Improved Yield/Stability of hiPSC-ECs
The two most commonly used hiPSC-EC differentiation protocols are based on co-culturing with murine stromal cells10,11 and embryonic-body formation12,13. However, the co-culturing method could lead to xenogenic contamination with murine-lineage cells or proteins10, and only ~15% or less of the cells produced via the embryonic-body method assume an EC phenotype10,12,13. The hiPSC-EC differentiation protocol presented here eliminates the risk of xenogenic contamination and can be up to 3-fold more efficient than the methods that use embryonic bodies (depending on which hiPSC line is used). Furthermore, after purification via selection for CD31 expression, ~90% of the hiPSC-ECs maintained the EC phenotype for at least 4 weeks in vitro, compared to just two weeks when hiPSC-ECs are produced via conventional differentiation protocols12.
Phenotypic Specification of hiPSC-SMCs
The phenotype of an SMC (or a population of SMCs) is perhaps best described as a balance between predominantly contractile and synthetic characteristics, which can lead to substantial differences in morphology, marker expression, and activity. Thus, the utility of hiPSC-SMCs for a particular application may depend on which specific phenotype is generated. The hiPSC-SMC differentiation and purification protocols presented here are the first to produce predominantly contractile or synthetic SMC populations that are ~95% pure in just 2-3 weeks, and the risk of xenogenic contamination is minimal, because the procedures do not require the use of feeder cells.
Improved Engraftment Rate of Transplanted Cells
One of the primary obstacles to effective cell therapy is believed to be the extremely low number of administered cells that are engrafted by the myocardium14,15,16,17. Cytokines such as IGF-1 may improve engraftment by enabling the cells to better withstand the toxic microenvironment in ischemic tissues18,19,20, but the high pressures induced during contraction may squeeze the cells out through the needle track and into the peripheral circulation. Thus, the considerably higher rate of engraftment achieved by injecting the cells through an IGF-1-containing fibrin patch, which was more than 2-fold greater than the rate observed when the same cell dose was administered without the patch, can likely be attributed both to the cytoprotective effects of IGF-1 and to the patch itself, which formed a physical barrier that prevented the cells from being ejected into the epicardial space.
Critical Steps
To ensure the pluripotency of the hiPS cells, it is necessary to check the karyotype of the hiPSC line prior to differentiation, determine the expression of pluripotency markers (Nanog, SSEA1, and Oct4, et al.), and confirm their capability of teratoma formation. It is suggested to re-check their pluripotency when hiPSC lines have been passaged for more than 10 generations. The status of undifferentiated hiPSC is also crucial for the correct differentiation. To ensure the status of the hiPSC prior to the initiation of differentiation, it is recommended to: 1) use fresh medium (less than two weeks old), and refresh the medium daily before initiation of hiPSC differentiation; 2) reduce the enzymatic digestion time (less than 5 min), and gently pipette up and down the cells to prevent the unnecessary damage to the cells; 3) replate the cells at a density 1 x 105 cells per well of the 6-well plate; 4) always keep the cells in 37 &#176;C and 5% CO2 incubator, and avoid keeping the cells out of the incubator for longer than 15 min.
Limitations of the Technique
The primary disadvantage of our method for combining injected hiPSC-derived cardiac cells and patch administration is the requirement for open-chest surgery; thus, this approach is unlikely to be directly translatable to clinical applications except as adjunctive therapy for patients who are undergoing concurrent coronary revascularization surgery. More widespread clinical use will require the development of a practical and minimally invasive delivery method, such as an endoscope- and catheter-based approach that could access the heart through the abdomen and diaphragm. Furthermore, one of the most critical safety concerns associated with cardiac cell therapy is the development of arrhythmogenic complications, and when monkeys were treated with intramyocardial injections of 1 billion hESC-derived CMs, all four of the animals developed spontaneous arrhythmias21. However, we observed no evidence of arrhythmogenic complications when 10 million hiPSC-derived CMs were injected into the hearts of swine with IR injury2, perhaps because the cell dose was 100-fold smaller.
Future Applications or Directions
Immune rejection likely plays an important role for the low engraftment rate. The CRISPR/Cas gene editing system may enable researchers to create a line of &#34;universal donor&#34; hiPSCs by knock out (KO) major histocompatibility complex (MHC) class I and class II. The hiPSC MHC KO-derived cells and tissues may have substantially higher rates of engraftment. As techniques improve, engraftment rates are likely to increase. At certain point of the increase the graft size, the large graft is expected to increase the arrhythmogenicity of the recipient heart. The reduction of grafts associated arrhythmogenicity could be achieved by using cells with increase of gap junction proteins expression by the gene editing technology.

Human induced pluripotent stem cells (hiPSCs) are among the most promising agents for regenerative cell therapy because they can be differentiated into a potentially unlimited range and quantity of cells that are not rejected by the patient&#39;s immune system. However, their capacity for self-replication and differentiation can also lead to tumor formation and, consequently, hiPSCs need to be fully differentiated into specific cell types, such as cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs), before administration. One of the simplest and most common methods of cell administration is direct intramyocardial injection, but the number of transplanted cells that are engrafted by the native myocardial tissue is exceptionally low. Much of this attrition can be attributed to the cytotoxic environment of the ischemic tissue; however, when murine embryonic stem cells (ESCs) were injected directly into the myocardium of uninjured hearts, only ~40% of the 5 million cells delivered were retained for 3-5 hr1, which suggests that a substantial proportion of the administered cells exited the administration site, perhaps because they were squeezed out through the needle track by the high pressures produced during myocardial contraction.
Here, we present novel and substantially more efficient methods for generating hiPSC-derived cardiomyocytes (hiPSC-CMs)2, endothelial cells (hiPSC-ECs)3, and smooth muscle cells (SMCs)4. Notably, this hiPSC-SMC protocol is the first to mimic the wide range of morphological and functional characteristics observed in somatic SMCs5 by directing the cells toward a predominantly synthetic or contractile SMC phenotype. We also provide a method of cell delivery that improves the engraftment rate of injected cells by creating a cytokine-containing fibrin patch over the injection site. The patch appears to improve both cell retention, by sealing the needle track to prevent the cells from exiting the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over a period of at least three days.

<table><tbody><tr><td>&lt;strong&gt;Protocol Section 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem cell technologies</td><td>5850</td><td></td></tr><tr><td>Growth-factor-reduced matrigel</td><td>Corning lifescience</td><td>356231</td><td></td></tr><tr><td>Y-27632</td><td>Stem cell technologies</td><td>72304</td><td></td></tr><tr><td>B27 supplement, serum free</td><td>Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>RPMI1640</td><td>Fisher Scientific</td><td>11875-119</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC-010</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>232-FA-025</td><td></td></tr><tr><td>Collagenase IV</td><td>Fisher Scientific</td><td>NC0217889</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (Dextrose, KCl, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, NaHCO&lt;sub&gt;3&lt;/sub&gt;, NaCl, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous)</td><td>Fisher Scientific</td><td>14175079</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10438018</td><td></td></tr><tr><td>6-well plate</td><td>Corning Lifescience</td><td>356721</td><td></td></tr><tr><td>10 cm dish</td><td>Corning Lifescience</td><td>354732</td><td></td></tr><tr><td>Cell incubator</td><td>Panasonic</td><td>MCO-18AC</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Versene</td><td>Fisher Scientific</td><td>15040066</td><td></td></tr><tr><td>Fibrinogen</td><td>Sigma-Aldrich</td><td>F8630-5g</td><td></td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T7009-1KU</td><td></td></tr><tr><td>EMB2 medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>VEGF</td><td>ProSpec-Tany</td><td>CYT-241</td><td></td></tr><tr><td>EPO</td><td>Life Technologies</td><td>PHC9431</td><td></td></tr><tr><td>TGF-&amp;#946;</td><td>Peprotech</td><td>100-21C</td><td></td></tr><tr><td>EGM2-MV medium</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>SB-431542</td><td>Selleckchem</td><td>S1067</td><td></td></tr><tr><td>CD31</td><td>BD Bioscience</td><td>BDB555445</td><td></td></tr><tr><td>CD144</td><td>BD Bioscience</td><td>560411</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>Fisher Scientific</td><td>12565269</td><td></td></tr><tr><td>Eppendorff Centrifuge</td><td>Eppendorf</td><td>5702R</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR99021</td><td>Stem cell technologies</td><td>720542</td><td></td></tr><tr><td>PDGF-&amp;#946;</td><td>Prospec</td><td>CYT-501-10ug</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 4&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Olive oil</td><td>Sigma-Aldrich</td><td>O1514</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td></td></tr><tr><td>IGF</td><td>R&amp;amp;D</td><td>291-G1-01M</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific</td><td>15561020</td><td></td></tr><tr><td>Heating plate</td><td>Fisher Scientific</td><td>SP88850200</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>15-462-10Q</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 5&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>&amp;#949;-aminocaproic acid</td><td>Sigma-Aldrich</td><td>A0420000</td><td></td></tr><tr><td>MEM medium</td><td>Fisher Scientific</td><td>12561-056</td><td></td></tr><tr><td>Syringe</td><td>Fisher Scientific</td><td>1482748</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Datex-Ohmeda</td><td>47810</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Ohio Medical</td><td>V5A</td><td></td></tr><tr><td>Defibrillator</td><td>Physiol Control</td><td>LIFEPAK 15</td><td></td></tr><tr><td>1.5 T MRI</td><td>General Electric</td><td>Signa Horizon LX</td><td></td></tr><tr><td>7 T MRI</td><td>Siemens</td><td>10018532</td><td></td></tr><tr><td>Gadolinium Contrast Medium (Magnevist)</td><td>Berlex</td><td>50419-188-02</td><td></td></tr><tr><td>2-0 silk suture</td><td>Ethilon</td><td>685H</td><td></td></tr><tr><td>3-0 silk suture</td><td>Ethilon</td><td>622H</td><td></td></tr><tr><td>3-0 monofilament suture</td><td>Ethilon</td><td>627H</td><td></td></tr></tbody></table>

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.

Characterization of Differentiated hiPSC-CMs, -ECs, and -SMCs
The differential capacity of hiPSCs were evaluated2,3,4. Flow cytometry analyses of cardiac troponin T (cTnT) expression suggest that the purity of the final hiPSC-CM population can exceed 90% (Figure 1A, 1B, panel B1). Nearly all of the cells expressed slow myosin heavy chain (Figure 1B, panel A1), &#945;-sarcomeric actin (Figure 1B, panel A2), while approximately 25% expressed the 2v isoform of myosin light chain (MLC2v) (Figure 1B, panel B2), which was found only in ventricular CMs4. The efficiency of the hiPSC-EC differentiation protocol (i.e., the percentage of CD31+ cells) was substantially higher when performed with hiPSCs from the GRiPS line (45.6%, Figure 1C, panel C2) than with PCBC16iPS cells (31.3%, Figure 1C, panel D2); populations of &#62;95% purity were achieved via selection for CD31 expression, and &#62;90% of the selected cells continued to express CD31 or CD144 for up to 4 weeks when cultured in EGM2-MV medium (without FBS) supplemented with B27, VEGF, and SB431542 (Figure 1D)3. More than 94% of the purified hiPSC-SMCs expressed smooth muscle actin (SMA), but synthetic hiPSC-SMCs were more likely than contractile hiPSC-SMCs to express collagen 1, connexin 43, or vimentin (Figure 1E). The migration and proliferation capability of hiPSC-SMCs were evaluated4. Synthetic hiPSC-SMCs were also more migratory and proliferative than contractile hiPSC-SMCs (Figure 1F, panels i and ii), while contractile SMCs contracted more strongly in response to carbachol treatment (Figure 1F, panels iii, iv and v).
Growth-factor Release from Gelatin Microspheres
ELISA measurements of IGF-1 levels in the medium from cultured, cell-free patches indicated that the growth factor was released from the microspheres over a period of at least 3 days (Figure 2A)2. The analyses were performed by loading 5 mg of IGF-containing microspheres with 5 &#181;g IGF-1. A patch was generated by mixing the microspheres with 1 ml fibrinogen solution and 1 ml thrombin. The patch was cultured in 2 ml MEM. One ml of the medium was collected and replaced with 1 ml of fresh MEM each day (Figure 2B). Data were presented as Mean &#177; SEM.
Observations from an IR-injury Model
The effectiveness of our patch-mediated cell-transplantation protocol has been investigated in a swine model of ischemia-reperfusion injury2. A total of 6 million hiPSC-CMs, -ECs, and -SMCs (2 million of each cell type) were injected directly into the injured myocardial tissue. For animals in the CELL+Patch group, a patch composed of fibrin and IGF-1-containing microspheres was created over the site of injury before injection, while animals in the CELL group were treated with the same cell dose but without the patch; both treatments were withheld from animals in the MI group. Four weeks after injury and treatment, 9% of the cells delivered to animals in the CELL+Patch group were retained and continued to survive at the site of administration, compared to just 4% of the cells administered to animals in the CELL group (Figure 3A). Treatment with both the cells and the patch, but not with the cells alone, was also associated with significant improvements in measurements of cardiac function and infarct size (Figure 3B).
<img alt="Figure 1" src="/files/ftp_upload/55142/55142fig1.jpg" /> 
Figure 1: Differentiation of hiPSCs into cardiomyocytes, endothelial cells, and smooth muscle cells. Cardiomyocyte differentiation and the purity of hiPSC-CMs were evaluated via flow cytometry (A) and histology with different cardiac markers (B). The endothelial differentiation of hiPSC was determined with flow cytometry (C). The maintenance of the endothelial phenotype of hiPSC-ECs was assessed via analyzing the expression of CD31 and CD144 at 2, 3, and 4 weeks after the purification by flow cytometry (D). The smooth muscle cell differentiation of hiPSC was measured by various markers (E). And the migration and proliferation potential of synthetic smooth muscle cells vs. contractile smooth muscle cells derived from hiPSCs was assessed (F). Nuclei were counterstained with DAPI in the immunofluorescence staining. Scale bar = 100 &#181;m in (B) and 200 &#181;m (E and F). The migration and proliferation capability of synthetic hiPSC-SMCs were evaluated (F, panel i and ii). And contraction of contractile SMCs in response to carbachol treatment was assessed (F, panel iii-v). *p &#60; 0.05, synthetic hiPSC-SMCs vs. contractile hiPSC-SMCs. A and B were modified from Ye L, et al.2. C and D were modified from Zhang S, et al.3. E and F were modified from Yang L, et al.4. Data were presented as mean &#177; standard error of the mean (SEM). Comparison between two groups was evaluated via the Student&#39;s T test, and comparison between multiple groups was assessed via one-way ANOVA. p &#60;0.05 was considered significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55142/55142fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55142/55142fig2.jpg" /> 
Figure 2: Release of growth factors from microspheres. IGF-containing microspheres was synthesized (A). IGF-1 release from microspheres was measured at 1, 3, 5 and 7 days after they were synthesized (B). Scale bar = 200 &#181;m. Panel A was modified from Ye L., et al.2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55142/55142fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55142/55142fig3.jpg" /> 
Figure 3: Assessment of efficiency of cell therapy with hiPSC-derived trilineage cells. The overall engraftment rate for the three cell population was assessed 4 weeks after cell transplantation (A). Cardiac function (as indicated by ejection fraction) and infarction size were evaluated via cardiac MRI at 1 and 4 weeks after cell transplantation (B). *p &#60;0.05 versus MI; #p &#60;0.05 versus Patch. A and B were modified from Ye L., et al.2. Data were presented as mean &#177; SEM. Comparisons among groups were analyzed for significance with one-way ANOVA. p &#60;0.05 was considered significant. Results identified as significant via ANOVA were reanalyzed with the Tukey correction.

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

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 ...

pluripotent-stem-cell-derived-cardiac-cells-for-myocardial-repair

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.2K Views.  University of Alabama at Birmingham.  We presenteren drie nieuwe en efficiëntere protocollen voor differentiatie humane geïnduceerde pluripotente stamcellen in cardiomyocyten, endotheelcellen en gladde spiercellen en bezorg- de engraftment van getransplanteerde cellen verbetert door het combineren celinjectie met patch-gemedieerde cytokine. 

Video: Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Bioengineering

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genetics

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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 ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Medicine

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

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. ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biology

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 ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

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