Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00
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 ...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+myocardial+insulin-like+growth+factor+1+(IGF-1)+delivery+with+biotinylated+peptide+nanofibers+improves+cell+therapy+for+myocardial+infarction.">Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction.</a> Proc Natl Acad Sci U S A. 103 (21), 8155-8160 (2006).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+insulin-like+growth+factor-1+in+mice+protects+from+myocyte+death+after+infarction,+attenuating+ventricular+dilation,+wall+stress,+and+cardiac+hypertrophy.">Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy.</a> J Clin Invest. 100 (8), 1991-1999 (1997).</li><li>Wang, L., Ma, W., Markovich, R., Chen, J. 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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 prot...

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 border="0" cellpadding="0" cellspacing="0" width="562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Protocol 1</td>
			<td style="width:180px;"></td>
			<td style="width:155px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td>Stem cell technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth-factor-reduced matrigel</td>
			<td>Corning lifescience</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Stem cell technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement, serum free</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase IV</td>
			<td>Fisher Scientific</td>
			<td>NC0217889</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:163px;">Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)</td>
			<td>Fisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>Corning Lifescience</td>
			<td>356721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm dish</td>
			<td>Corning Lifescience</td>
			<td>354732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene</td>
			<td>Fisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>F8630-5g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T7009-1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMB2 medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VEGF</td>
			<td>ProSpec-Tany</td>
			<td>CYT-241</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EPO</td>
			<td>Life Technologies</td>
			<td>PHC9431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TGF-&#223;</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGM2-MV medium</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB-431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31</td>
			<td>BD Bioscience</td>
			<td>BDB555445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD144</td>
			<td>BD Bioscience</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorff Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR99021</td>
			<td>Stem cell technologies</td>
			<td>720542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDGF-&#223;</td>
			<td>Prospec</td>
			<td>CYT-501-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>O1514</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGF</td>
			<td>R&#38;D</td>
			<td>291-G1-01M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heating plate</td>
			<td>Fisher Scientific</td>
			<td>SP88850200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"><img alt="ezh" src="/files/ftp_upload/55142/ezh.jpg" />-aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0420000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM medium</td>
			<td>Fisher Scientific</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>Fisher Scientific</td>
			<td>1482748</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Datex-Ohmeda</td>
			<td>47810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Ohio Medical</td>
			<td>V5A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defibrillator</td>
			<td>Physiol Control</td>
			<td>LIFEPAK 15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5T MRI</td>
			<td>General Electric</td>
			<td>Signa Horizon LX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7T MRI</td>
			<td>Siemens</td>
			<td>10018532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gadolinium Contrast Medium (Magnevist)</td>
			<td>Berlex</td>
			<td>50419-188-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-0 silk suture</td>
			<td>Ethilon</td>
			<td>685H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 silk suture</td>
			<td>Ethilon</td>
			<td>622H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 e...

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 ...

Research

JoVE Journal

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...