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
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 purifi...

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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...

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 ...

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