Name: derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation
Uploaded: 2015-03-18T15:00:00.000+00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose S

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>

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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 described here displayed the highest and the most reproducible cardiac differentiation efficiency for different hiPSC cell lines by modulating Wnt/&#946;-catenin signaling using the small molecules CHIR and IWR5,7. Specifically, the undifferentiated hiPSCs were induced to undergo mesodermal differentiation with the GSK3-beta inhibitor CHIR and subsequent cardiac differentiation with the Wnt signaling inhibitor IWR1. These sequential steps of Wnt signaling modulation, aided by insulin depletion during the cardiac lineage induction phase, led to highly efficient cardiac differentiation. CHIR is a common small molecule GSK3-beta inhibitor used for mesodermal induction, but other small molecules for GSK3-beta inhibition have been tested in cardiomyocyte differentiation protocols11. Multiple options are also used for the subsequent small molecule Wnt inhibitor. For example, a recent publication focusing on chemically-defined differentiation of pluripotent stem cells to cardiomyocytes uses 2 &#181;M Wnt-C59 for effective Wnt inhibition and cardiac mesoderm induction11.
In addition, the differentiated cardiomyocytes with a glucose starvation method were further purified, which takes advantage of the distinct glucose metabolic flow existing between cardiomyocytes and non-cardiomyocytes8. It is essential to note that the effectiveness of the glucose starvation method is density-dependent (i.e., differentiations that yielded higher percentages of cardiomyocytes are more likely to achieve greater cardiomyocyte survival). However, the transition to the low glucose medium is also a stressful condition for the cardiomyocytes. It is important to change the cells back to the regular RPMI/B27 with insulin medium after 3 days of glucose starvation. In this protocol, a feeder-free growth system was utilized, in which pluripotent stem cells were not grown on feeder mouse embryonic fibroblasts (MEFs). However, prior cardiac differentiation protocols have utilized feeder-based systems to produce cardiomyocytes from pluripotent stem cells5. Likewise, prior protocols have also used mTeSR1 medium for stem cell maintenance, but the E8 media and feeder-free system utilized here is superior due to its simplicity and lack of excess xenogeneic components such as bovine serum albumin and mouse embryonic fibroblasts.
Currently, pluripotent stem cell differentiation towards cardiomyocyte lineages is largely robust, but still suffers from hiPSC line-to-line variability in terms of efficiency. This remains a major issue in the cardiac differentiation field and will require further study. The differentiation efficiency is improved by proper maintenance of hiPSC lines, and in particular, preventing overconfluency during hiPSC maintenance. Additional variability arises from cell seeding during the passaging process, as an evenly seeded monolayer of hiPSCs tends to give rise to the best overall differentiations. Here, a mouse-derived, Matrigel-based ECMS for cell seeding during cell culture was successfully utilized, but an eventual transition to xeno-free substrates is recommended. In regards to cell seeding on ECMS, uneven seeding often results in uneven distribution of cardiomyocytes within a particular well. For example, if hiPSCs are seeded unevenly, the edges of a well in a six-well plate may give rise to higher numbers of cardiomyocytes than the center of the well. These uneven seeding conditions may give rise to low efficiency differentiations and a larger number of non-cardiomyocyte, mesodermal derivatives such as fibroblasts and smooth muscle cells. We have also observed that low efficiency differentiations (below 50%) are much more difficult to purify using the glucose deprivation process mentioned here. Another side effect of long-term glucose starvation is a potential loss in cardiomyocyte viability. Although the cardiomyocytes are able to metabolize lactate in the absence of glucose, we find that this switch to a low-glucose environment is stressful for the cells. Cells may stop spontaneously beating, and some cell loss may be observed if glucose starvation is prolonged beyond the recommended time. This enhanced sensitivity to glucose deprivation may be reflective of the well-established developmental, functional, and electrophysiological immaturity of stem cell-derived cardiomyocytes in comparison to true adult cardiomyocytes13.
In summary, the protocol described here combines the small molecule-based, hiPSC monolayer cardiac differentiation method with a cardiomyocyte-purifying glucose starvation method. This protocol allows for the reproducible generation of highly purified cardiomyocytes and should facilitate various downstream assays relevant to cardiovascular disease modeling and drug screening.

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><tbody><tr><td>Matrigel (9-12 mg/ml)</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>RPMI media</td><td>Invitrogen</td><td>11835055</td><td></td></tr><tr><td>Glucose free RPMI media</td><td>Invitrogen</td><td>11879-020</td><td></td></tr><tr><td>B27 Minus Insulin</td><td>Invitrogen</td><td>A1895601</td><td></td></tr><tr><td>B27 Supplement (w/ insulin)</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Pen-strep antibiotic</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum</td><td>BenchMark</td><td>100-106</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D-2650</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>EMD Millipore</td><td>688000</td><td></td></tr><tr><td>CHIR99021</td><td>Thermo Fisher</td><td>508306</td><td></td></tr><tr><td>IWR1</td><td>Sigma</td><td>I0161</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Cell lifter</td><td>Fisher</td><td>08-100-240</td><td></td></tr><tr><td>Cryovial</td><td>Fisher (NUNC tubes)</td><td>375418</td><td></td></tr><tr><td>TrypLE Select Enzyme</td><td>Invitrogen</td><td>12563-011</td><td></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 comparison to cells in hiPSC colonies, cell size for these day 2 cells increased. Cell number also increased through cell division (Figure 1B). At day 5, the mesodermal cells were directed towards the cardiac lineage (cardiac mesoderm). At this time, cells begin coalescing and forming characteristic, branch-like structures (Figure 1C). At day 10, branch-like structures were highly pronounced, and cardiomyocytes started spontaneously beating (Figure 1D). Terminally-differentiated cardiomyocytes will not proliferate beyond this point. A timeline of the differentiation protocol is shown in Figure 2.
Immunostaining and flow cytometry results showed cardiomyocytes were purified with glucose starvation. 
After 10 days of differentiation, more than 50% of cell areas are beating. However, beating cell sheets sometimes are also intermingled with non-cardiomyocytes. To examine cardiomyocyte purity, cells with immunostaining against cardiomyocyte marker cardiac Troponin-T (cTnT) were characterized to find that most cells were cTnT positive. However, in an unpurified population of differentiated cells, a number of cells also lacked expression of cTnT, suggesting the presence of non-cardiomyocytes in this differentiated population at day 13 (Figure 3A). However, with glucose starvation, almost all remaining cells were cTnT positive at day 13 (Figure 3B), indicating successful purification of the cardiomyocytes following glucose starvation. These data were further corroborated using flow cytometry analysis. A population of differentiated cells at day 13 post-differentiation contained approximately 50% TNNT2+ cells when not glucose starved (Figure 4A). A parallel differentiation was also conducted but with a 3 day glucose deprivation beginning at day 10 after differentiation. In contrast to the unstarved differentiation, glucose deprivation led to a purified population containing 90% TNNT2+ cells at day 13 (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/52628/52628fig1.jpg" /> 
Figure 1. The sequential morphological changes during hiPSC differentiation towards the cardiac lineage. (A) Undifferentiated hiPSCs at day 0 exhibit typical colony morphology and reached around 85% confluency. (B) At day 2, cells after treatment with CHIR for two days reached 100% confluency and entered the mesodermal lineage. (C) At day 5, the cells after treatment with IWR1 for 2 days entered the cardiac mesoderm stage. Some cells began to fuse and form characteristic branch-like morphologies (indicated by arrows). (D) At day 7-10, branch-like structures are highly evident, and cardiomyocytes start spontaneously beating. Bar = 200 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52628/52628fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52628/52628fig2.jpg" /> 
Figure 2. Timeline of hiPSC-CM differentiation protocol and subsequent glucose starvation process. Beating cardiomyocytes are typically first observed at approximately days 7-10. Two rounds of glucose starvation will result in a purified population of cardiomyocytes by day 17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52628/52628fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52628/52628fig3.jpg" /> 
Figure 3. Immunofluorescence reveals purification of cardiomyocytes following glucose starvation. (A) Unpurified cells after 13 days of differentiation were stained with Cardiac Troponin T (cTnT). Most cells have differentiated into cardiomyocytes and were cTnT positive, but a number of non-cardiac, cTnT-negative cells are also present. (B) In contrast, after a 3 day glucose starvation beginning at day 10, almost all of the surviving cells were cTnT positive by day 13. Scale bar = 200 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52628/52628fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52628/52628fig4.jpg" /> 
Figure 4. Flow cytometry reveals purification of cardiomyocytes following glucose starvation. (A) After 13 days of cardiac differentiation without glucose starvation, a population of cells exhibited roughly 50% TNNT2+ cardiomyocytes. Red indicates the differentiated cell population and blue indicates undifferentiated hiPSCs as a negative control. (B) After 10 days of cardiac differentiation and followed by 3 days of glucose starvation, a population of cells from the same batch of differentiations was purified to contain 90% TNNT2+ cardiomyocytes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52628/52628fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">The compositions for E8 medium</td>
		</tr>
		<tr>
			<td>E8</td>
			<td>Volume: 1 L</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>DMEM/F12 with Glutamine and HEPES</td>
			<td>1,000 ml</td>
			<td>Invitrogen</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>NaHCO3 (7.5%, 75 mg/ml)</td>
			<td>7.24 ml</td>
			<td>Invitrogen</td>
			<td>25080-094</td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate (64 mg/ml)</td>
			<td>1 ml</td>
			<td>Sigma</td>
			<td>A8960</td>
		</tr>
		<tr>
			<td>sodium selenite (70 &#181;g/ml)</td>
			<td>200 &#181;l</td>
			<td>Sigma</td>
			<td>S5261</td>
		</tr>
		<tr>
			<td>transferrin (50 mg/ml)</td>
			<td>214 &#181;l</td>
			<td>Sigma</td>
			<td>T3705</td>
		</tr>
		<tr>
			<td>insulin (4 mg/ml)</td>
			<td>5 ml</td>
			<td>Invitrogen</td>
			<td>12585-014</td>
		</tr>
		<tr>
			<td>FGF2 (200 ng/&#181;l)</td>
			<td>500 &#181;l</td>
			<td>Peprotech</td>
			<td>100-18B</td>
		</tr>
		<tr>
			<td>TGFB1 (100 ng/&#181;l)</td>
			<td>20 &#181;l</td>
			<td>Peprotech</td>
			<td>100-21</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="2">The stock solutions for E8 compositions</td>
		</tr>
		<tr>
			<td>L-Ascorbic acid-2-phosphate (64 mg/ml)</td>
			<td>3.2 g in 50 ml Ultrapure water, store 500 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Transferrin (50 mg/ml)</td>
			<td>500 mg in 10 ml of Ultrapure water, store 107 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium selenite (70 &#181;g/ml)</td>
			<td>35 mg sodium selenite into 500 ml ultrapure water, store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>FGF2 (200 ng/&#181;l)</td>
			<td>1 mg in 5 ml cold D-PBS, store 250 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>TGFB1 (100 ng/&#181;l)</td>
			<td>100 &#181;g in 1 ml cold 10 mM citric acid, pH 3, store 10 &#181;l aliquots at -20 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1. Composition of E8 Medium.

Watch this Scientific Journal Video about Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose S

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 ...

derivation-highly-purified-cardiomyocytes-from-human-induced

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

18.6K Views.  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. 

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

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 ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

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

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

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

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

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Bioengineering

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

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

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

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

Biology

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

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

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

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

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

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

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

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