Name: high-throughput cardiotoxicity screening using mature human induced pluripotent stem cell-derived cardiomyocyte monolayers
Uploaded: 2023-03-24T14:30:00
Description: Watch this Scientific Journal Video about High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers at JoVE.com

This work has been supported by NIH grants HL148068-04 and R44ES027703-02 (TJH).

hiPSC usage in this protocol was approved by the University of Michigan HPSCRO Committee (Human Pluripotent Stem Cell Oversight Committee). See the Table of Materials for a list of materials and equipment. See Table 1 for media and their compositions.
1. Thawing and plating commercially available cryopreserved hiPSC-CMs for maturation on a maturation-inducing extracellular matrix (MECM)
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	<li>Warm all the reagents to room temperature and...

<ol>
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E., Anzai, T., Chanthra, N., Uosaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+current+maturation+methods+for+human+induced+pluripotent+stem+cells-derived+cardiomyocytes.">A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes.</a> Frontiers in Cell and Developmental Biology. 8, 178 (2020).</li><li>Guo, Y., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+maturation:+new+phase+in+development.">Cardiomyocyte maturation: new phase in development.</a> Circulation Research. 126 (8), 1086-1106 (2020).</li><li>Yang, X., Pabon, L., Murry, C. 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There are several different approaches to in vitro cardiotoxicity screening using hiPSC-CMs. A recent &#34;Best Practices&#34; paper on the use of hiPSC-CMs presented the various in vitro assays, their primary readouts, and importantly, each assay&#39;s granularity to quantify human cardiac electrophysiological function20. In addition to using membrane-piercing electrodes, the most direct measure of human cardiac electrophysiological function is provided by VSDs. VSD assay readou...

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been validated on an international scale, and are available for in vitro cardiotoxicity screening1. Highly pure hiPSC-CMs can be generated in virtually unlimited numbers, cryopreserved, and thawed. Upon replating, they also reanimate and begin contracting with a rhythm reminiscent of the human heart2,3. Remarkably, individual hiPSC-CMs couple to each other and form functional syncytia that beat as a single tissue. Nowadays, hiPSCs are routinely derived from patient blood samples, so any person can be represented using in vitro hiPSC-CM cardiotoxicity screening assays4,5. This creates the opportunity to perform &#34;Clinical Trials in a Dish&#34;, with significant representation from diverse populations6.
One critical advantage over existing animal and animal cell cardiotoxicity screening approaches is that hiPSC-CMs utilize the full human genome and offer an in vitro system with genetic similarities to the human heart. This is especially attractive for pharmacogenomics and personalized medicine - the use of hiPSC-CMs for medication and other therapy development is predicted to provide more accurate, precise, and safe medication prescriptions. Indeed, two-dimensional (2D) hiPSC-CM monolayer assays have proven to be predictive of medication cardiotoxicity, using a panel of clinically used medications with a known risk of causing arrhythmias1,7,8,9. Despite the vast potential of hiPSC-CMs and the promise to streamline and make drug development cheaper, there has been a reluctance to use these novel assays10,11,12.
Until now, one major limitation of widespread adoption and acceptance of hiPSC-CM screening assays is their immature, fetal-like appearance, as well as their function. The critical issue of hiPSC-CM maturation has been reviewed and debated in the scientific literature ad nauseum13,14,15,16. Likewise, many approaches have been employed to promote hiPSC-CM maturation, including extracellular matrix (ECM) manipulations in 2D monolayers and the development of 3D engineered heart tissues (EHTs)17,18. At the moment, there is a widely held belief that the use of 3D EHTs will provide superior maturation relative to 2D monolayer-based approaches. However, 2D monolayers provide a higher efficiency of cell utilization and increased success in plating compared to 3D EHTs; 3D EHTs utilize greater numbers of cells, and often require the inclusion of other cell types that can confound results. Therefore, in this article, the focus is on using a simple method to mature hiPSC-CMs cultured as 2D monolayers of electrically and mechanically coupled cells.
Advanced hiPSC-CM maturation can be achieved in 2D monolayers using an ECM. The 2D monolayers of hiPSC-CMs can be matured using a soft, flexible polydimethylsiloxane coverslip, coated with basement membrane matrix secreted by an Engelbreth-Holm-Swarm mouse sarcoma cell (mouse ECM). In 2016, reports showed that hiPSC-CMs cultured on this soft ECM condition matured functionally, displaying action potential conduction velocities near adult heart values (~50 cm/s)18. Further, these mature hiPSC-CMs displayed many other electrophysiological characteristics reminiscent of the adult heart, including hyperpolarized resting membrane potential and expression of Kir2.1. More recently, reports identified a human perinatal stem cell-derived ECM coating that promotes the structural maturation of 2D hiPSC-CMs19. Here, easy-to-use methods are presented to structurally mature 2D hiPSC-CM monolayers for use in high-throughput electrophysiological screens. Further, we provide validation of an optical mapping instrument for the automated acquisition and analysis of 2D hiPSC-CM monolayer electrophysiological function, using voltage-sensitive dyes (VSDs) and calcium-sensitive probes and proteins.

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			<td>0.25% Trypsin EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
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			<td>0.5 mg/mL BSA (7.5 &#181;mol/L)</td>
			<td>Millipore Sigma</td>
			<td>A3294</td>
			<td></td>
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			<td>2.9788 g/500 mL HEPES (25 mmol/L)</td>
			<td>Millipore Sigma</td>
			<td>H4034</td>
			<td></td>
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			<td>AdGCaMP6m</td>
			<td>Vector biolabs</td>
			<td>1909</td>
			<td></td>
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			<td>Albumin human</td>
			<td>Sigma</td>
			<td>A9731-1G</td>
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			<td>alpha actinin antibody</td>
			<td>ThermoFisher</td>
			<td>MA1-22863</td>
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			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
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			<td>Blebbistatin</td>
			<td>Sigma</td>
			<td>B0560</td>
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			<td>CalBryte 520AM</td>
			<td>AAT Bioquest</td>
			<td>20650</td>
			<td></td>
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			<td>CELLvo MatrixPlus 96wp</td>
			<td>StemBiosys</td>
			<td>N/A</td>
			<td>https://www.stembiosys.com/products/cellvo-matrix-plus</td>
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		<tr>
			<td>CHIR99021</td>
			<td>LC Laboratories</td>
			<td>c-6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Assay medium (fluorobrite)</td>
			<td>ThermoFisher</td>
			<td>A1896701</td>
			<td>For adenovirus transduction</td>
		</tr>
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			<td>DAPI</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt</td>
			<td>ThermoFisher</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell CM maintenance media</td>
			<td>FUJIFILM/Cellular Dynamics</td>
			<td>M1003</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell2 CMs</td>
			<td>FUJIFILM</td>
			<td>1434</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Zoom&#160;</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iPS DF19-9-11T.H</td>
			<td>WiCell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>MilliporeSigma</td>
			<td>CAS-51-30-9</td>
			<td></td>
		</tr>
		<tr>
			<td>IWP4</td>
			<td>Tocris</td>
			<td>5214</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td>
			<td>Sigma</td>
			<td>A8960-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyii Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Buffer (autoMACS Running Buffer)</td>
			<td>Miltenyii Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red</td>
			<td>ThermoFisher</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Nautilus HTS Optical Mapping&#160;</td>
			<td>CuriBio</td>
			<td></td>
			<td>https://www.curibio.com/products-overview</td>
		</tr>
		<tr>
			<td>Nikon A1R Confocal Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nonessential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>pre-separation filter</td>
			<td>Miltenyii Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr>
			<td>PSC-Derived Cardiomyocyte Isolation Kit, human</td>
			<td>Miltenyii Biotec</td>
			<td>130-110-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse</td>
			<td>CuriBio</td>
			<td></td>
			<td>https://www.curibio.com/products-overview</td>
		</tr>
		<tr>
			<td>Quadro MACS separator (Magnet)</td>
			<td>Miltenyii Biotec</td>
			<td>130-091-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 (+HEPES, +L-Glutamine)</td>
			<td>Gibco</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS iPS-Brew XF</td>
			<td>Miltenyii Biotec</td>
			<td>130-107-086</td>
			<td></td>
		</tr>
		<tr>
			<td>TnI antibody (pan TnI)</td>
			<td>Millipore Sigma</td>
			<td>MAB1691&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene (ethylenediaminetetraacetic acid - EDTA solution)</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
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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.

hiPSC-CM maturation characterized by phase contrast and immunofluorescent confocal imaging 
The timeline for ECM-mediated maturation of commercially available hiPSC-CMs using MECM coated 96-well plates is presented in Figure 1A. These data are collected using commercially available cardiomyocytes that arrive in the laboratory as cryopreserved vials of cells. Each vial contains &#62;5 &#215; 106 viable cardiomyocytes. The cells are ~98% pure and rigorously tes...

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

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 ...

This protocol is significant because it enables researchers to mature commercial or in-house-made hiPSC-CMs to an adult-like phenotype that significantly improves the predictive value of hiPSC-CMs in cardiotoxicity screening. The main advantage of this protocol is that it delivers mature hiPSC-CMs in a high throughput format for optical mapping of calcium or voltage change. This protocol may be used to investigate disease mechanisms or to screen drugs for cardiotoxicity.Demonstrating the procedure will be Jeffrey Creech, a research associate from my laboratory. Begin by washing the maturation-inducing extracellular matrix, or MECM, plates two times with 200 microliters of Hank's balanced salt solution, or HBSS, 1 hour prior to cardiomyocyte plating. Keep the wells hydrated.Transfer the cardiomyocyte tubes from the liquid nitrogen tank to dry ice, and release the pressure by slightly opening the tube caps. Next, reseal the tube caps and thaw them in the water bath for 4 minutes. After the thawing of the cells, spray the tubes with 70%ethanol before opening them.Transfer the cells into 15-milliliter conical tubes with a 1-milliliter pipette. Add 1 milliliter of plating medium to the cryovial and transfer the wash into the 15-milliliter conical tube. Then, slowly drip 1 milliliter of plating medium and agitate the tube.Repeat this until the transfer of 8 milliliters of plating medium. Centrifuge the 15-milliliter tube at 300 x g for 5 minutes and resuspend the pellet in 1 milliliter of the plating medium. Remove an aliquot, and perform live cell counting with a hemocytometer before adding a plating medium to obtain 7.5 x 10 to the 5th cells per milliliter.Dispense 100 microliters of the cell suspension per well of an MECM-coated 96-well plate using a multichannel pipette, and incubate the cells for 2 days. Next, replace the medium with 200 microliters of maintenance medium, and maintain the plates for 7 days, with a change of medium on day 5. On day 7, perform EP assay, as described in the text.Ensure to change the medium every other day when opting for extending the cell culture. To purify hiPSC-CM via magnetic-activated cell sorting, or MACS, aspirate the cell culture medium and wash the cells with 1 milliliter of HBSS-Then, dissociate the cells by adding 1 milliliter of 0.25%trypsin-ethylenediaminetetraacetic, or EDTA, and incubating the cells for 10 minutes. Then, inactivate the trypsin/EDTA by resending and singularizing the cells with 2 milliliters of plating medium.From the six wells, collect the cells into a 50-milliliter conical tube using a 70-micrometer strainer. After washing the strainer with 3 milliliters of plating medium, count the cells. After counting, centrifuge and wash the cell pellet with 2 milliliters of ice-cold MACS separation buffer before pelleting and resending the cells in 80 microliters of cold MACS separation buffer per 5 x 10 to the 6th cells.Add 20 microliters of cold non-cardiomyocyte depletion cocktail and mix the suspension before incubating on ice for 10 minutes. After washing the cells with 4 milliliters of cold MACS separation buffer at 300 x g for 5 minutes, resuspend the pellet in 80 microliters of cold MACS separation buffer. Add 20 microliters of cold anti-biotin microbeads per 5 x 10 to the 6th cells, and gently mix the cell suspension before incubating on ice for 10 minutes.While the samples are incubating, place the positive depletion columns fitted with the 30 micrometer pre-separation filters on the MACS separator, and place the labeled 15-milliliter collection tubes below the columns. Next, prime each column with 3 milliliters of cold MACS separation buffer. Mix the antibody-treated cell suspension with 2 milliliters of MACS separation buffer and add it to the column.Add 2 milliliters of MACS separation buffer to each column and collect 12 milliliters of flowthrough cardiomyocyte suspension. Centrifuge the cardiomyocytes and discard the supernatant before resuspending the cardiomyocytes in a 1-milliliter plating medium. To determine the concentration, count the cells and adjust the volume to the desired seeding density before plating the purified cardiomyocyte cells on the MECM 96-well plates.Aspirate and replace the cardiomyocyte maintenance medium with 100 microliters of voltage-sensitive dyes, or VSD, or calcium-sensitive fluorophores, or CSF, per well of a 96-well plate. After 30 minutes of incubation, replace the dyes with an assay medium or HBSS. Equilibrate the cells at 37 degrees Celsius before acquiring baseline data optical mapping using a high-throughput optical mapping device.To treat the cells with drugs for acute exposure testing or map the cells chronically exposed to drugs of interest, dilute the drugs in dimethyl sulfoxide, or DMSO, and store them as stock solutions at 20 degrees Celsius before diluting them in HBSS to the desired concentrations. For cardiotoxicity testing in 96-well plates, add four doses of a compound with at least eight wells per dose. Use doses ranging from below to above the clinically-effective therapeutic plasma concentration, including the effective dose.Similar to the baseline electrophysiology measurements before drug application, capture electrophysiology recordings at least 30 minutes after drug treatment for chronic studies. After baseline recordings, apply at least four doses of each medication to mature hiPSC-CMs monolayers expressing GCaMP6m, with at least eight wells per dose. Equilibrate the medications on the cells for 30 minutes, and warm the cells to 37 degrees Celsius before and during the data acquisition.Following baseline recordings of an entire plate, add 500 nanomolar isoproterenol to every well to enable robust drug response data and quantify the effects of isoproterenol on monolayer beat rate, contraction amplitude, and calcium transient duration. To acquire optical mapping data, open the front drawer and position the plate on the plate heater. After opening the acquisition software and determining the file saving location in the software, select 10 to 30 seconds duration and frame rate of acquisition for higher temporal resolution, and click Start Acquisition.Open the analysis software, and in the Import Filter tab, select either Browse for a single file or Tile Multiple to reconstruct a plate. Select the files and enter a number of rows and columns and select Automatic. Next, select Update Pixel Size, enter distance per pixel, and use the Well Wizard to determine the wells'location in the image before clicking Process Save and moving to the next tab.Open the ROIs, or Regions of Interest tab, and choose to draw the ROIs manually, automatically, or not use ROIs at all, which will be considered for the analysis of the entire plate. After selecting the region in the well, click the Process Save button to move to the next step. Check the Hide Wells and Only Show Filtered boxes to visualize the ROIs.Open the Analysis tab, select each well or ROI to confirm the accuracy of automatic beat detection. Add or remove beats from the traces by selecting an individual beat and pressing Delete on the keyboard. Once done, click on Save.Next, click the Time-Space Plot button in the Analysis tab and add the line to determine the time-space plot to visualize data for each well or the ROI. After selecting a horizontal or vertical line, click on Generate Plot. After ensuring the accurate beat detection, proceed to the Export tab and select File Format, enter Beat Statistic option before clicking Export.Once exported, open data files and run the chosen statistical analysis routine. Phase contrast imaging showed that the hiPSC-CMs plated on the MECM matured and became structurally distinct from the same batch of hiPSC-CMs replated on the mouse ECM. Mature cells became rod-shaped, while immature cells retained a circular shape.Cardiomyocytes stained with alpha-actinin antibodies showed the typical shape of the cells cultured on each ECM condition. Consistent with the TnI staining, the alpha-actinin staining indicated that the hiPSC-CMs matured on the MECM promoted a rod-shaped mature phenotype and induced greater sarcomere organization. Mitochondrial content and activity were distinct between cells cultured on the mouse ECM and the MECM.The electrophysiological data for action potentials recorded using VSDs and the optical mapping system showed that the atrial-specific cells have a significantly faster spontaneous beat rate and shorter action potential duration, or APD80, than the ventricular-specific cells. Whole-plate heat maps for parameters such as APD80 revealed the reproducibility of a given parameter within a plate. Typical action potentials of mature hiPSC-CM monolayers indicated the action potential morphology of isolated and tested adult cardiomyocytes.A typical action potential spontaneous rhythm was also displayed. In response to isoproterenol, activation of the beta-1-adrenergic receptors caused positive chronotropy, positive inotropy, and positive lusitropy. HiPSC-CM response to human Ether-a-go-go-related gene, or hERG, channel blocker, including E-4031, domperidone, vandetanib, and sotalol, was studied using GCaMP6m calcium fluorescence to monitor rhythm.The study showed the detection of early after-depolarizations caused by the E-4031 hERG channel blockade. Work fast with the cells in suspension and avoid shear stress by pipetting cells gently. Additionally, perform media change carefully to avoid damaging the hiPS syncytia.Following this procedure, cells are amenable for protein, RNA, DNA, and lipids collection for analysis with investigators'favorite technique. The technique empowers researchers to investigate diseases and test for cardiotoxicity of new drugs using adult-like cardiomyocytes.

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Bioengineering

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...