Name: maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation
Uploaded: 2017-05-06T13:00:00
Description: Watch this Scientific Journal Video about Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation at JoVE.com

This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Canada (G-14-0006265), operating grants from the Canadian Institutes of Health Research (137352 and 143066), and a J.P. Bickell foundation grant (1013821) to SSN.

1. Master Design and Fabrication
NOTE: Use soft lithography for device fabrication. Make a two-layer SU-8 master for polydimethylsiloxane (PDMS) molding.
<ol>
	<li>Design the device using a design and drafting software (Figure 1A, left). Draw each layer of the master separately. Print the device design on two photomasks at 20,000 dpi, corresponding to the two layers of the master15. Set the device pattern as transparent and the surrounding area as dar...

<ol>
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This manuscript describes the setup and implementation of the engineered platform, biowire, to improve the maturation of hPSC-CMs. The device can be made in standard microfabrication facilities, and biowires can be produced with common cell culture techniques and an electrical stimulator.
To our knowledge, there is no reported method similar to biowires. This strategy reveals that improved maturation properties were dependent on the electrical stimulation regimen, as evidenced by the greater m...

Cell-based therapy is one of the most promising and investigated strategies to achieve cardiac repair/regeneration. It has been aided by cardiac tissue engineering and the co-delivery of biomaterials1,2. Most available cell sources have been studied in animal models for their potentially beneficial effects on damaged, diseased, or aged hearts3. In particular, significant efforts have been made to use human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CM), a potentially unlimited autologous cell source for cardiac tissue engineering. hPSC-CMs can be produced using several established protocols4,5,6. However, the obtained cells display fetal-like phenotypes, with a range of immature characteristics compared to adult ventricular cardiomyocytes7,8. This can be an obstacle to the application of hPSC-CMs as models of adult heart tissue in drug discovery research and in the development of adult cardiac disease models9.
In order to overcome this limitation of phenotypic immaturity, new approaches have been actively investigated to promote cardiomyocyte maturation. Early studies revealed effective pro-maturation properties in neonatal rat cardiomyocytes via cyclic mechanical10 or electrical stimulation11. Gel compaction and cyclic mechanical stimulation were also shown to improve some aspects of hPSC-CM maturation12,13, with minimal enhancement of the electrophysiological and calcium handling properties. Therefore, a platform system called &#34;biological wire&#34; (biowire) was devised by providing both structural cues and electrical field stimulation to enhance the maturation of hPSC-CMs14. This system uses a microfabricated platform to create aligned cardiac tissue that is amenable to electrical field stimulation. This can be used to improve the structural and electrophysiological maturity of hPSC-CMs. Here, we describe the details of making such biowires.

<table border="0" cellpadding="0" cellspacing="0" width="582">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">L-Ascorbic acid</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">A-4544</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AutoCAD</td>
			<td style="width:80px;">Autodesk, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Software to design device</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Carbon rods, &#216; 3 mm</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Electrical stimulator chamber component</td>
		</tr>
		<tr height="206">
			<td height="206" style="height:206px;width:216px;">Collagen, type 1, rat tail</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:119px;">354249</td>
			<td style="width:167px;">Collagen gel: 2.1 mg/ml of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, in 1xM199 media with 10 % of growth factor-reduced Matrigel.</td>
		</tr>
		<tr height="164">
			<td height="164" style="height:164px;width:216px;">Collagenase type I&#160;</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">C0130</td>
			<td style="width:167px;">0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca2+ and Mg2+. Sterilize with 0.22 &#956;m filter and make 12 ml aliquots. Store at -20 &#176;C.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Deoxyribonuclease I (DNase I)</td>
			<td style="width:80px;">EMD Millipore</td>
			<td style="width:119px;">260913-25MU</td>
			<td style="width:167px;">Make 1 mg/ml DNase I stock solution in water. Filter sterile and store 0.5 ml aliquots at &#8722;20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drill &#38; drill bits (&#216; 1mm and 2 mm)</td>
			<td style="width:80px;">Dremel</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Drill holes in polycarbonate frames</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electrical stimulator</td>
			<td style="width:80px;">Grass</td>
			<td style="width:119px;">s88x</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fetal bovine serum (FBS)</td>
			<td style="width:80px;">WISENT Inc.</td>
			<td style="width:119px;">080-450</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">D-(+)-Glucose&#160;</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">G5767</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">L-Glutamine</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030081</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">H2O</td>
			<td style="width:80px;">MilliQ</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">18.2 M&#937;&#183;cm at 25 &#176;C, ultrapure, to make all solutions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">HEPES</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">H4034</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Hot plate</td>
			<td style="width:80px;">Torrey Pines</td>
			<td style="width:119px;">HS40</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Iscove&#39;s Modified Dulbecco&#39;s Medium(IMDM)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12440053</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Mask aligner</td>
			<td style="width:80px;">EVG&#160;</td>
			<td style="width:119px;">EVG 620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Matrigel, growth factor reduced&#160;</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Medium 199 (M199)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Monothioglycerol (MTG)</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">M-6145</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Orbital shaker</td>
			<td style="width:80px;">VWR</td>
			<td style="width:119px;">89032-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Penicillin/Streptomycin (P/S)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15070063</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Phosphate-buffered saline (PBS) with Ca2+ and Mg2+&#160;</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14040133</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plate (6-well)</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">353046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plate (6-well), low attachment</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">3471</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Platinum wires, 0.2 mm</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Electrical stimulator chamber component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:80px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Propylene glycol monomethyl ether acetate (PGMEA)</td>
			<td style="width:80px;">Doe &#38; Ingalls Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To develop the wafer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pouch, peel-open</td>
			<td style="width:80px;">Convertors</td>
			<td style="width:119px;">92308</td>
			<td style="width:167px;">For steam sterilization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Silicon wafer, 4-inch</td>
			<td style="width:80px;">UniversityWafer Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium hydroxide</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">S8045</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprin coater</td>
			<td style="width:80px;">Specialty Coating Systems</td>
			<td style="width:119px;">G3P-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="280">
			<td height="280" style="height:280px;width:216px;">StemPro-34 culture medium</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10639011</td>
			<td style="width:167px;">hPSC-CM culture medium. To make 50 ml, add 1.3 ml supplement, 500 &#956;l of 100&#215; L-Glutamine, 250 &#956;l of 30 mg/ml transferrin, 500 &#956;l of 5 mg/ml ascorbic acid, 150 &#956;l of 26 &#956;l /2 ml monothioglycerol (MTG), and 500 &#956;l (1 %) penicillin/streptomycin.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Stop media</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">&#160;Wash medium:FBS (1:1)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SU-8 50&#160;</td>
			<td style="width:80px;">MicroChem Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">photoresist, master component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SU-8 2050&#160;</td>
			<td style="width:80px;">MicroChem Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">photoresist, master component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Transferrin</td>
			<td style="width:80px;">Roche</td>
			<td style="width:119px;">10-652-202</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Trypsin/EDTA, 0.25%</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25200056</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Wash medium</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">IMDM containing 1% Penicillin/Streptomycin</td>
		</tr>
	</tbody>
</table>

maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The rational for the use of a suture in the biowires is to serve as a template for the formation of 3D constructs that align in one axis and mimic the shape of cardiac fibers. We show that after seven days of culture in the biowire, cells remodeled the gel around the suture (Figure 3A). The cells assembled along the axis of the suture to form aligned cardiac tissue (Figure 3). After 7 days of preculture, the biowires were subjected to 7 days of electrical...

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Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

The overall goal of this procedure is to utilize controlled three dimensional cell cultivation methods in electrical stimulation to promote the maturation of human embryonic and induced pluripotent stem cells derived cardiomyocytes. This method can help answer key questions in the cardiac tissue engineering field including how different electrical stimulation parameters or length of time affect cardiac cell function and maturation state. The end results are engineered human cardiac tissues of improved maturity.The implications of this technique extend toward diagnosis applications such as testing the safety and efficacy of new drugs because cardiomyocyte with mature characteristics such as electrical progress are most likely to serve as more accurate models of human cardiac cells in drug screening studies. Combine the PDMS monomer and curing agent at a 10:1 ratio and mix well. Let it set for five minutes to degas.Then load a Petri dish containing the master for the biowire template with the PDMS mixture. Try to minimize air bubble formation. Next cure the PDMS at 70 degree Celsius for two hours.Then recover the solidified mold by first using a sharp blade around the edges and then peeling it away. Then trim the excess PDMS and collect all the templates in a sterilization pouch. Steam auto clave the templates at 121 degree Celsius for 20 minutes.And then load each template into a sterile Petri dish under the ventilation of a biosafety cabinet. Now for each template use tweezers to mount a piece of sterile 6.0 silk suture into the groove spanning the entire channel. The templates are then ready for cell seeding.The suture will serve as a template for the formation of 3D constructs that align along the suture axis mimicking the shape of cardiac fibers. For each chamber, make a pair of rectangular polycarbonate pieces to serve as the frame for the electrical stimulation chamber. Along the center line of each piece, drill two three millimeter wide holes, two centimeters apart.For each chamber prepare two 1.5 centimeter long carbon rods. About 3 millimeters from the end of each rod drill a one millimeter wide hole. Then thread each hole with a platinum wire and wrap one end of the wire around the rod.Next, for each chamber insert two rods between two polycarbonate pieces making a frame. Then pour about two milliliters of PDMS into a well of a six well plate and place the frame into the well with PDMS, submerging the bottom of the frames. Cure the PDMS at 70 degree Celsius for two hours so the PDMS secures the frame.Then remove the completed chamber from the plate with the PDMS attached. To test the function of an assembled chamber connect the platinum wires to a battery and use a voltmeter to check that electricity is being passed through the rods. Now transfer the functional chambers into a sterilization pouch and use 20 minutes of steam sterilization at 121 degree celsius to prepare them for cell culture.To prepare the gel, precool all the gel components on ice before mixing them in a sterile tube. Make sure to add the collagen and extra cellular matrix last and mix the components using pipetting. For the cells have cardiomyocytes differentiated from human pluripotent stem cells.Collect the cells and for each biowire preparation plant prepare 3.5 microliters of collagen gel containing half a million cells. Now use a P10 pipette tip to load approximately four milliliters of suspension into the 0.5 centimeter long channel template in each dish. Adjust the position of the suture using sterile forceps as needed so it is fully stretched.Once seeding the cells, make sure to always resuspend the gel cell mixture before taking autoclave in order to have consistent results. After loading the channels, cover the bottom of the dish with sterile PBS making sure not to touch the gel. This will ensure that the biowires do not dehydrate during polymerization.After incubating the biowires at 37 degree celsius for 30 minutes, replace the PBS with enough culture medium to cover the cells. 12 milliliters is usually enough. Then culture the dish for a week replacing the medium every other day.In a bio safety cabinet add two milliliters of PBS to each well of a six well plate that will house an electrical stimulator chamber. Then use sterile tweezers to gently place the autoclave devices into the wells. Once in position, discard the PBS and replace it with five milliliters of warm medium which should completely cover the carbon rods.Before culturing the cells for 24 hours, observe the cardiomyocytes contractility in response to electrical stimulation to ensure that the cells are capturing the electrical stimulus and responding with contractions. Next, place the biowires into the electrical stimulator chamber and orient the channels perpendicularly to the carbon rods. Extend the platinum wires away from the plate and then cover the plates.Connect the wires to an electrical stimulator. Set the stimulator to deliver biphasic one millisecond pulses at one hertz and at three volts per centimeter. Then transfer the preparations in the electrical stimulator to an incubator at 37 degree celsius.Increase stimulation rate incrementally every 24 hours from one hertz at day zero to six hertz at day six. During this week of culture, change the medium every other day. And observe the cardiomyocytes contractility on a daily basis.After seven days of preculture in the biowire cells remolded the gel around the suture. The cells assembled along the axis of the suture to form aligned cardiac tissue. The cells expressed cardiac contractile proteins showed sarcomeric banding in the contractile apparatus and their myofibrils were aligned along the axis of the suture.Cells in the biowire exhibited lower proliferation rates than cells in embryoid bodies. Calcium handling was significantly improved in the cardiomyocytes cultivated in biowires. Treatment with caffeine induced an abrupt release of calcium from the sacroplasmic reticulum.Such transients were found only in stimulated cells. There were also significantly higher stimulation frequency depending calcium release amplitudes. In addition, cardiomyocytes cultured in biowires showed improved hERG and inward rectifier current densities compared to cultivation in embryoid bodies which was further enhanced by electrical stimulation.The improved inward rectifier current was consistent with increased expression of the potassium inwardly rectifying channel jean, KCN J2.Automaticity the ability to beat spontaneously was significantly higher in embryoid body derived cardiomyocytes compared to controlled biowires. Control showed automaticity comparable to biowire preparation subjected to the six hertz regimen. Once mastered the setup of the biowires can be done in half a day.The setup of electrical stimulation will take a couple of hours and the final results can be reached in two weeks. While attempting this procedure, it is important to remember that cardiomyocytes differentiated from different cell lines or by different protocols can vary slightly in their maturity and purity. Therefore optimizing the experimental parameters such as the stimulation rate to your cells might be required.Following this procedure, other conditions including variation of the cellular composition and the utilization of mutant cardiomyocytes can be optimized to answer a bevy of additional questions.

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Developmental Biology

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.

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 ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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 ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

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

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

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

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

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

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

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

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

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

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

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