This work was supported in part by the NIH/NHBI (U01 HL099776-5), the NIH Director&#8217;s New Innovator Award (DP2 OD004411-2), the California Institute of Regenerative Medicine (RB3-05129), the American Heart Association (14GRNT18630016) and the Endowed Faculty Scholar Award from the Lucile Packard Foundation for Children and the Child Health Research Institute at Stanford (to SMW). We also acknowledge funding support from the American Heart Association Predoctoral Fellowship 13PRE15770000, and National Science Foundation Graduate Research Fellowship Program DGE-114747 (AS).

NOTE: Vendor information for all reagents used in this protocol has been listed in Table 1 and Materials List. All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly. Perform all culture incubations in a humidified 37 &#176;C, 5% CO2 incubator unless otherwise specified. In this protocol, all differentiations are performed in 6-well plates, in which the hiPSCs are seeded. Following differentiation and purification, cells can be dissociated and replated for downstream use.
1. Medium Preparation
<ol>
	<li>For the E8 medium, make a stock solution for each medium component (NaHCO3, L-ascorbic acid 2-phosphate, sodium selenite, transferrin, insulin, FGF2, TGFB1) and store the solutions at -20 &#176;C. Add appropriate amount of stock solutions to the bottle of DMEM/F12 and filter to sterilize. The solution concentration and reaction setup for the stock components and E8 medium can be found in Table 1.</li>
	<li>For the extracellular matrix solution, thaw the basement membrane matrix (commonly supplied at 10 mg/ml) at 4 &#176;C O/N. Matrigel is commonly used for pluripotent stem cell culture because human pluripotent stem cells can adhere well to this material using the alpha-6-beta-1 integrin9. Once thawed, make 2.25 mg (250 &#181;l) aliquots on ice using ice cold pipet tips and tubes and store the aliquots at &#8722;80 &#176;C.
	<ol>
		<li>When precoating plates with ECMS, thaw and aliquot the extracellular matrix solution (ECMS) on ice. Mix the ECMS and ice-cold DMEM/F12 medium in a ratio of 1:200 on ice. Keep cool to prevent premature ECMS solidification.</li>
	</ol>
	</li>
	<li>For the RPMI/B27 without insulin medium, add 10 ml of B27 Minus Insulin into 500 ml of RPMI media.</li>
	<li>For the RPMI/B27 (with insulin) medium, add 10 ml of B27 Supplement (with insulin) and 5 ml of Pen-strep antibiotic into 500 ml of RPMI media.</li>
	<li>For the low glucose medium, add 10 ml of B27 Supplement and 5 ml of Pen-strep antibiotic into 500 ml of glucose-free RPMI media.</li>
	<li>For the freezing/cryopreservation medium, mix fetal bovine serum together with dimethylsulfoxide (DMSO) at a 9:1 ratio. The freezing medium can be stored up to 1 month at 4 &#176;C. 
	NOTE: All media should be filtered and stored in 4 &#176;C and used within 2 weeks unless otherwise specified. All reagent/solution volumes used below are intended for a single well in a 6-well plate, unless otherwise specified.</li>
</ol>
2. Pre-coating 6-well Plates with ECMS
<ol>
	<li>Make ECMS by mixing basement membrane matrix (e.g., Matrigel) with ice-cold DMEM/F12 media in a ratio of 1:200, then apply 2 ml of freshly made ECMS to each well for a 6-well plate. Each well in a 6-well plate will receive approximately 90 mg ECMS.</li>
	<li>Incubate the ECMS coated plates for 1 hr at 37 &#176;C. Immediately prior to plating cells, aspirate DMEM/F12 solution from each well. The plates will then be ready to receive cells.</li>
</ol>
3. Thawing the Frozen hiPSCs
NOTE: Closely follow this procedure as it may lead to optimal culturing and downstream differentiation of hiPSCs into hiPSC-CMs following the freeze/thaw cycle.
<ol>
	<li>Thaw one vial of hiPSCs for each well of a 6-well plate. Prepare a 15 ml conical tube with 9 ml of cold E8 medium with ROCK inhibitor (10 &#956;M) for each vial. ROCK inhibitor improves hiPSCs survival following cryopreservation10. Keep the vials in 37 &#176;C water bath to thaw the cells until an approximately 5 mm diameter ice crystal is left.</li>
	<li>Spray the exterior of the vials with 70% ethanol. Wipe the vials with tissue paper before moving them to the sterile laminar hood.</li>
	<li>Transfer the cells into the prepared conical tube. Rinse the vial once with 500 &#956;l of E8 medium and transfer the medium containing the thawed cells to the same conical tube. Centrifuge at RT for 4 min at 200 x g.</li>
	<li>Aspirate the supernatant after centrifugation and gently resuspend the cell pellet with 2 ml of E8 medium with ROCK inhibitor (10 &#956;M). Transfer the resuspended cells to one well of a 6-well plate pre-coated with ECMS.</li>
	<li>Replace the medium with E8 medium without ROCK inhibitor after 24 hr of culturing. Cells should have adhered by this time.</li>
</ol>
4. Passaging of hiPSCs
<ol>
	<li>Typically, passage hiPSCs after reaching 75%-80% confluency in a 6-well plate. Aspirate the culture medium. Quickly wash wells with 1 ml of sterile, RT&#160;PBS. Aspirate PBS.
	<ol>
		<li>Add 500 &#956;l of 0.5 mM EDTA or 1x cell detachment solution (e.g., Accutase) and incubate for 1-7 min at RT. Appropriate time for EDTA incubation varies with the hiPSC line. Expect to see visible curling or thickening of colonies around the edges, as this will indicate that EDTA or cell detachment solution is ready to be removed.</li>
	</ol>
	</li>
	<li>Aspirate the EDTA or the cell detachment solution. Add 1 ml of E8 medium supplemented with 10 &#956;M ROCK inhibitor. Displace hiPSC colonies in the well by repeatedly pipetting up and down and spraying colonies off with a P1000 pipette. Colonies should be broken into 5-10 cell clumps after completion.</li>
	<li>Transfer the colonies that are suspended in 1 ml of E8 medium with ROCK inhibitor into a 15 ml conical tube.</li>
	<li>Disrupt the large cell clumps into small clumps in the E8 medium. Add an appropriate volume of E8 media with inhibitor to the cell suspension to dilute the cells. The media volume to add for dilution depends on the cell density and the number of wells to be seeded. Ideally, a single, 80% confluent well of hiPSCs should be diluted 1:12.
	<ol>
		<li>Add 11 ml E8 medium with ROCK inhibitor to the existing 1 ml of cell solution in aforementioned 15 ml conical tube to obtain a 1:12 dilution.</li>
	</ol>
	</li>
	<li>Further triturate the cell clumps into small cell fragments (around 50-200 cells in each fragment is best). Avoid over-triturating as this reduces cell survival.</li>
	<li>Aspirate ECMS from the pre-coated plates and add 2 ml of resuspended cells into each well. Approximately 100,000 cells per well of a 6-well plate is ideal. Aim to dispense the cells evenly around the well to avoid clustering of cells in the center of the well.</li>
	<li>After 24 hr of culturing, replace the medium with E8 medium without ROCK inhibitor. Change the culture medium every 24 hr until cells become 80% confluent. Then, the cells are ready to passage again. It usually takes 3-6 days between passages to reach 80% confluency.</li>
</ol>
5. Freezing hiPSCs
NOTE: Closely follow this procedure as it may lead to optimal culturing and downstream differentiation of hiPSCs into hiPSC-CMs following the freeze/thaw cycle.
<ol>
	<li>Label cryogenic tubes with the cell line name, cell type, passage number, and freezing date. As a general guideline, use one vial for each well of a 6-well plate. Prepare a 15 ml conical tube filled with 9 ml of E8 medium with ROCK inhibitor (10 &#956;M). Prepare freezing medium with 90% FBS and 10% DMSO, and keep the medium at 4 &#176;C until ready to use.</li>
	<li>Aspirate the culture media, add 500 &#956;l of 0.5 mM EDTA or 1x cell detachment solution and incubate for 2-7 min at RT. The duration of EDTA or cell detachment solution exposure varies from cell line to cell line.</li>
	<li>Aspirate EDTA or cell detachment solution. Add 1 ml of E8 medium.</li>
	<li>Displace hiPSC colonies in the well by pipetting up and down and spraying colonies off with a P1000 pipette. Colonies should not be broken into smaller than 100 cell clumps. Transfer the suspended cells to the prepared conical tube containing E8. Centrifuge at RT for 4 min at 200 x g.</li>
	<li>Aspirate the supernatant and add 500 &#956;l of cold freezing medium to the pellet. Resuspend the pellet by pipetting one to two times. Cell survival is improved by keeping cells in large clumps.</li>
	<li>Transfer the resuspended cells into a labeled cryogenic tube. Quickly move the vials into a freezing container containing isopropanol, which will allow for gradual cooling. Keep the container with cells at -80 &#176;C for 24 hr, then transfer the cells to liquid nitrogen for long-term storage.</li>
</ol>
6. Cardiac Differentiation of Human iPSC
NOTE: All media should be at least at RT when added.
<ol>
	<li>After dissociation with EDTA or 1x cell detachment solution, seed approximately 100,000 human iPSCs on ECMS coated 6-well culture plates for differentiation (same steps as cell passaging procedures). When the cells reach 85% confluency, change the medium to RPMI/B27 without insulin medium with 6 &#181;M GSK3-beta inhibitor CHIR99021 (CHIR) and maintain for 48 hr.</li>
	<li>After 48 hr, replace the CHIR-containing culture medium with RPMI/B27 without insulin medium and leave alone for 24 hr (until day 3).</li>
	<li>At day 3, change the media to RPMI/B27 without insulin with 5 &#181;M Wnt inhibitor IWR1 and maintain for 48 hr (until day 5). 
	NOTE: Wnt inhibition can also be attempted using other small molecule compounds, as described in earlier studies11. IWR1 was selected over other small molecule Wnt inhibitors due to the increased range in which it has been shown to be effective in inhibiting Wnt signaling11.</li>
	<li>At day 5, change the medium back to RPMI/B27 without insulin medium and leave for 48 hr (until day 7).</li>
	<li>At day 7, replace the medium with RPMI/B27 medium (with insulin) and replace medium every 3 days thereafter with the same medium. Spontaneous beating of cardiomyocytes should first be visible at approximately day 8 to day 10.</li>
</ol>
7. Purification of Human Cardiomyocytes through Glucose Starvation
<ol>
	<li>At day 10 post-differentiation, change the medium in each well of the 6-well plate to 2 ml low glucose medium and maintain the cells in this medium for 3 days (until day 13).</li>
	<li>At day 13, return cells to RPMI/B27 medium (with insulin).</li>
	<li>Optionally, replate cardiomyocytes prior to the second round of glucose starvation to help loosen non-cardiomyocytes from the culture plate, allowing for easier dissociation of non-cardiomyocytes during glucose starvation.
	<ol>
		<li>At day 13, aspirate medium, wash once with PBS, and dissociate the cells into single cells using 500 &#956;l of cell disassociation enzyme for 5 min at 37 &#176;C. Specifically, after 5 min of enzyme treatment, use a 1,000 &#181;l pipette to manually dissociate cardiomyocytes from the 6-well plate by repeatedly pulling up the cell disassociation enzyme and spraying it against the cardiomyocyte monolayer. Up to 30 pipetting repetitions may be required to dissociate the cardiomyocytes into single cells.</li>
		<li>After cells are dissociated and are in single-cell form, collect all cells into a 15 ml conical tube filled with 5 ml of RPMI/B27 medium with insulin to dilute out the cell disassociation enzyme and centrifuge for 4 min at 200 x g. Aspirate and discard the supernatant.</li>
		<li>Re-suspend the cells with 2 ml RPMI/B27 medium and plate onto a new ECMS-coated 6-well plate. Typically, higher confluency of cardiomyocytes helps with cell survival during replating. Aim to replate 2 million cells per new 6-well dish for optimal survival during replating.</li>
	</ol>
	</li>
	<li>At day 14, change the medium back to 2 ml of low glucose medium for a second glucose deprivation cycle. Culture the cells in this low glucose state for 3 more days. Most of the non-cardiomyocytes will die in this low-glucose culture condition.</li>
	<li>At day 17, change the medium to 2 ml of RPMI/B27 medium with insulin. The remaining cells will be highly purified cardiomyocytes. These cardiomyocytes can be used for gene expression analysis, drug screening, metabolic analysis, and various other downstream assays.</li>
</ol>

<ol>
	<li>Sharma, A., Wu, J. C., Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-derived+cardiomyocytes+for+cardiovascular+disease+modeling+and+drug+screening.">Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening.</a> Stem Cell Research &amp; Therapy. 4 (6), 150 (2013).</li><li>Kehat, 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=Human+embryonic+stem+cells+can+differentiate+into+myocytes+with+structural+and+functional+properties+of+cardiomyocytes.">Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.</a> The Journal of Clinical Investigation. 108 (3), 407-414 (2001).</li><li>Zhang, 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=Functional+cardiomyocytes+derived+from+human+induced+pluripotent+stem+cells.">Functional cardiomyocytes derived from human induced pluripotent stem cells.</a> Circulation Research. 104 (4), e30-e41 (2009).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell Stem Cell. 8 (2), 228-240 (2011).</li><li>Lian, X., 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=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (27), E1848-E1857 (2012).</li><li>Sharma, A., 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+induced+pluripotent+stem+cell-derived+cardiomyocytes+as+an+in+vitro+model+for+coxsackievirus+B3-induced+myocarditis+and+antiviral+drug+screening+platform.">Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.</a> Circulation Research. 115 (6), 556-566 (2014).</li><li>Lian, X., 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=Directed+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/beta-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions.</a> Nature Protocols. 8 (1), 162-175 (2013).</li><li>Tohyama, 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=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (12), 127-137 (2013).</li><li>Rodin, 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=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nature Biotechnology. 28 (6), 611-615 (2010).</li><li>Li, X., Meng, G., Krawetz, R., Liu, S., Rancourt, D. 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The authors do not declare competing interests.

Obtaining a large amount of highly purified hiPSC-derived cardiomyocytes is critical for basic cardiac research as well as clinical and translational applications. Cardiac differentiation protocols have undergone tremendous improvements in recent years, transitioning from embryoid body-based methods utilizing cardiogenic growth factors2, to matrix sandwich methods12, and finally to small molecule-modulated and monolayer-based methods5. Of the aforementioned protocols, the protocol describ...

Primary human cardiomyocytes are difficult to obtain because of the requirement for invasive cardiac biopsies, difficulty in dissociating to single cells, and because of poor long-term cell survival in culture. Given this lack of primary human cardiomyocytes, patient-specific human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology has been regarded as a powerful alternative cardiomyocyte source for basic research as well as clinical and translational applications such as disease modeling and drug discovery1. Early efforts in differentiating pluripotent stem cells into cardiomyocytes employed differentiation protocols using embryoid bodies (EBs), but this method is inefficient in producing cardiomyocytes because often less than 25% of cells in an EB are beating cardiomyocytes2,3. Comparatively, a monolayer-based differentiation protocol using the cytokines activin A and BMP4 displayed a higher efficiency than EBs, but this protocol is still relatively inefficient, requires expensive growth factors, and only functions in a limited number of human pluripotent stem cell lines4. Recently, a highly efficient, hiPSC monolayer-based cardiomyocyte differentiation protocol was developed by modulating Wnt/&#946;-Catenin signaling5. These hiPSC-CMs express cardiac troponin T and alpha actinin, two sarcomeric proteins that are standard markers of cardiomyocytes6. The protocol describe here is an adaptation of this small molecule-based, feeder cell-free, monolayer differentiation method5,7. We are able to obtain beating cardiomyocytes from hiPSCs after 7-10 days (Figure 1). However, following a cardiomyocyte differentiation resulting in 50% beating cells, immunostaining consistently shows the existence of a population of non-cardiomyocytes that are negative for cardiomyocyte-specific markers such as cardiac-specific troponin T and alpha-actinin. To further purify cardiomyocytes and eliminate non-cardiomyocytes, heterogeneous differentiated cell populations were subjected to glucose starvation by treating them with an extremely low glucose culture medium for multiple days (Figure 2). This treatment selectively eliminates non-cardiomyocytes due to the ability of cardiomyocytes, but not non-cardiomyocytes, to metabolize lactate as the primary energy source in order to survive in a low glucose environment8. After this purification step, a 40% increase in the ratio of cardiomyocytes to non-cardiomyocytes is observed, (Figure 3, Figure 4) and these cells can be used for downstream gene expression analysis, disease modeling, and drug screening assays.

<table border="0" cellpadding="0" cellspacing="0" width="550">
	<tbody>
		<tr height="18">
			<td height="18" style="height:18px;width:207px;">Name</td>
			<td style="width:199px;">Company</td>
			<td style="width:145px;">Catalog number</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Matrigel (9-12 mg/mL)</td>
			<td>BD Biosciences</td>
			<td>354277</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">RPMI media</td>
			<td>Invitrogen</td>
			<td>11835055</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Glucose free RPMI media</td>
			<td>Invitrogen</td>
			<td>11879-020</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">B27 Minus Insulin</td>
			<td>Invitrogen</td>
			<td>A1895601</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">B27 Supplement (w/ insulin)</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Pen-strep antibiotic</td>
			<td>Invitrogen</td>
			<td>15140122</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Fetal bovine serum</td>
			<td>BenchMark</td>
			<td>100-106</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">DMSO</td>
			<td>Sigma</td>
			<td>D-2650</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">ROCK inhibitor Y-27632</td>
			<td>EMD Millipore</td>
			<td>688000</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">CHIR99021</td>
			<td>Thermo Fisher</td>
			<td>508306</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">IWR1</td>
			<td>Sigma</td>
			<td>I0161</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">EDTA</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Accutase</td>
			<td>Millipore</td>
			<td>SCR005</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Cell lifter</td>
			<td>Fisher</td>
			<td>08-100-240</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Cryovial</td>
			<td>Fisher (NUNC tubes)</td>
			<td>375418</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">TrypLE Select Enzyme</td>
			<td>Invitrogen</td>
			<td>12563-011</td>
		</tr>
	</tbody>
</table>

derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation

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

The morphological changes during hiPSC differentiation.
The hiPSC cultured in feeder-free plates grew as flat, two-dimensional colonies. Upon reaching around 85% confluency, hiPSCs were treated with 6 &#181;M CHIR for differentiation (Figure 1A). Substantial amounts of cell death, a normal and common phenomenon, were observed after 24 hr of CHIR treatment. After two days of CHIR treatment, the hiPSCs continued to differentiate towards a mesodermal fate. In com...

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

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

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

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18.6K Aufrufe.  Stanford University School of Medicine. Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening. 

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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 ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

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

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.