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>

<ol>
	<li>Qiao, H., 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=Death+and+proliferation+time+course+of+stem+cells+transplanted+in+the+myocardium.">Death and proliferation time course of stem cells transplanted in the myocardium.</a> Mol Imaging Biol. 11 (6), 408-414 (2009).</li><li>Ye, L., 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=Cardiac+repair+in+a+porcine+model+of+acute+myocardial+infarction+with+human+induced+pluripotent+stem+cell-derived+cardiovascular+cells.">Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells.</a> Cell Stem Cell. 15 (6), 750-761 (2014).</li><li>Zhang, S., Dutton, J. R., Su, L., Zhang, J., Ye, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+a+spatiotemporal+3D+environment+on+endothelial+cell+differentiation+of+human+induced+pluripotent+stem+cells.">The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells.</a> Biomaterials. 35 (12), 3786-3793 (2014).</li><li>Yang, L., 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=Differentiation+of+Human+Induced-Pluripotent+Stem+Cells+into+Smooth-Muscle+Cells:+Two+Novel+Protocols.">Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols.</a> PLoS One. 11 (1), e0147155 (2016).</li><li>Rensen, S. S., Doevendans, P. A., van Eys, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+characteristics+of+vascular+smooth+muscle+cell+phenotypic+diversity.">Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.</a> Neth Heart J. 15 (3), 100-108 (2007).</li><li>Xu, C., Police, S., Hassanipour, M., Gold, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+bodies:+a+novel+culture+method+for+enrichment+of+cardiomyocytes+derived+from+human+embryonic+stem+cells.">Cardiac bodies: a novel culture method for enrichment of cardiomyocytes derived from human embryonic stem cells.</a> Stem Cells Dev. 15 (5), 631-639 (2006).</li><li>Anderson, D., 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=Transgenic+enrichment+of+cardiomyocytes+from+human+embryonic+stem+cells.">Transgenic enrichment of cardiomyocytes from human embryonic stem cells.</a> Mol Ther. 15 (11), 2027-2036 (2007).</li><li>Huber, I., 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=Identification+and+selection+of+cardiomyocytes+during+human+embryonic+stem+cell+differentiation.">Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.</a> FASEB J. 21 (10), 2551-2563 (2007).</li><li>Kita-Matsuo, H., 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=Lentiviral+vectors+and+protocols+for+creation+of+stable+hESC+lines+for+fluorescent+tracking+and+drug+resistance+selection+of+cardiomyocytes.">Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.</a> PLoS One. 4 (4), e5046 (2009).</li><li>Choi, K. D., 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=Hematopoietic+and+endothelial+differentiation+of+human+induced+pluripotent+stem+cells.">Hematopoietic and endothelial differentiation of human induced pluripotent stem cells.</a> Stem Cells. 27 (3), 559-567 (2009).</li><li>Woll, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+promotes+hematoendothelial+cell+development+from+human+embryonic+stem+cells.">Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells.</a> Blood. 111 (1), 122-131 (2008).</li><li>Li, Z., Hu, S., Ghosh, Z., Han, Z., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+and+expression+profiling+of+human+induced+pluripotent+stem+cell-+and+embryonic+stem+cell-derived+endothelial+cells.">Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells.</a> Stem Cells Dev. 20 (10), 1701-1710 (2011).</li><li>Rufaihah, A. J., 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=Endothelial+cells+derived+from+human+iPSCS+increase+capillary+density+and+improve+perfusion+in+a+mouse+model+of+peripheral+arterial+disease.">Endothelial cells derived from human iPSCS increase capillary density and improve perfusion in a mouse model of peripheral arterial disease.</a> Arterioscler Thromb Vasc Biol. 31 (11), e72-e79 (2011).</li><li>Beauchamp, J. R., Morgan, J. E., Pagel, C. N., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+myoblast+transplantation+reveal+a+discrete+minority+of+precursors+with+stem+cell-like+properties+as+the+myogenic+source.">Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.</a> J Cell Biol. 144 (6), 1113-1122 (1999).</li><li>Qu, Z., 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=Development+of+approaches+to+improve+cell+survival+in+myoblast+transfer+therapy.">Development of approaches to improve cell survival in myoblast transfer therapy.</a> J Cell Biol. 142 (5), 1257-1267 (1998).</li><li>Tang, X. L., 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=Intracoronary+administration+of+cardiac+progenitor+cells+alleviates+left+ventricular+dysfunction+in+rats+with+a+30-day-old+infarction.">Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction.</a> Circulation. 121 (2), 293-305 (2010).</li><li>Zeng, L., 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=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Davis, M. 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. W., Wang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiomyocyte+apoptotic+signaling+by+insulin-like+growth+factor+I.">Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I.</a> Circ Res. 83 (5), 516-522 (1998).</li><li>Chong, J. J., 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=Human+embryonic-stem-cell-derived+cardiomyocytes+regenerate+non-human+primate+hearts.">Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.</a> Nature. 510 (7504), 273-277 (2014).</li></ol>

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 ...

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

Research

JoVE Journal

Developmental Biology

8.2K Views.  University of Alabama at Birmingham. 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.

Video: Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

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, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

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 patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

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 direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

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 methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

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

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

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. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human 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 a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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 disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

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 errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

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 cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

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