We thank Dr. Kati Andreasson (Department of Neurology and Neurological Science, Stanford University) for use of the qRT-PCR machine. The work was supported by National Institutes of Health Grants K08NS098876 (to P.M.G.) and Stanford School of Medicine Dean's Postdoctoral Fellowship (to B.O.).

All stem cell and animal procedures were approved by Stanford&#39;s Stem Cell Research Oversight committee and by Stanford University&#39;s Administrative Panel on Laboratory Animal Care (SCRO-616 and APLAC-31909).
1. Etching of ITO Glass
<ol>
	<li>Prepare the indium tin oxide (ITO)-covered glass slide by placing a masking agent over the conductive side of the glass. 
	NOTE: Most commercial transparent tapes can be used as a masking agent.
	<ol>
		<li>Remove the excess mask using a blade, leaving only the desired shape of the final ITO design covered on the glass.</li>
	</ol>
	</li>
	<li>Combine 5% (v/v) of nitric acid and 45% (v/v) of HCl in a glass beaker inside a fume hood to prepare the etching solution.
	<ol>
		<li>Use a hot plate and a thermometer to ensure that the temperature of etching solution is maintained at 70 &#176;C.</li>
	</ol>
	</li>
	<li>Once the etching solution reaches 70 &#176;C, place the masked-ITO glass slide into the solution for 2 minutes to remove the excess ITO layer. 
	NOTE: Tape masking protects the ITO layer from the exposure to acid solution.</li>
	<li>Remove the slide from the solution and rinse it with deionized (DI) H2O.</li>
	<li>Carefully remove the mask and measure resistance using a multimeter to ensure that the remaining ITO is intact. Readouts of the desired etched area should be approximately 0 &#937;.</li>
</ol>
2. Preparation of Pyrrole Solution
<ol>
	<li>In a 1000 mL glass bottle, dissolve 70 g of sodium dodecylbenzenesulfonate (NaDBS) in 600 mL of DI H2O.</li>
	<li>Once the NaDBS is dissolved, add 14 mL of 0.2 M pyrrole and 386 mL of DI H2O to the solution.</li>
	<li>Cover the solution with aluminum foil and store at 4 &#176;C.</li>
</ol>
3. Electroplating of Polypyrrole on ITO glass
<ol>
	<li>Warm the pyrrole solution to room temperature until NaDBS is fully redissolved. 
	NOTE: This takes about 15 to 20 minutes.</li>
	<li>Pour 25 mL of the pyrrole solution into a beaker.</li>
	<li>Connect the etched ITO glass to a multimeter and submerge the slide into the pyrrole solution.
	<ol>
		<li>Ensure that the side with 0 &#937; resistance is facing towards the platinum mesh reference electrode.</li>
	</ol>
	</li>
	<li>Apply an electrical current to initiate electroplating of PPy on the ITO surface using a multimeter (current controlled at 3 mA/cm2 for 4 hours).</li>
	<li>Remove the ITO glass from the multimeter and wash electroplated-PPy in DI H2O to remove residual surfactant.</li>
	<li>Gently detach the electroplated-PPy plate from the ITO glass using a razor blade.
	<ol>
		<li>Store the PPy plate at room temperature.</li>
	</ol>
	</li>
</ol>
4. Preparation of Polydimethylsiloxane (PDMS)
<ol>
	<li>Using a spatula and a weigh dish, mix 18 g of base agent with 2 g of curing agent and pour the mixture into two 10 cm x 8 cm glass molds.</li>
	<li>Remove the bubbles from the molds using a vacuum chamber for 15 - 20 minutes.</li>
	<li>Place the molds in a 70 &#176;C oven for 3 h.</li>
	<li>Once cooled, use a flat spatula to remove the PDMS from the molds.</li>
</ol>
5. Fabrication of the In Vitro Electrical Stimulation Chamber
<ol>
	<li>Autoclave the metal plates (WxL, 2.5 cm x 12.5 cm) and flow valves at 120 &#176;C for 1 hour.</li>
	<li>Using a chamber slide as a template, cut two pieces of PDMS. 
	NOTE: One piece of PDMS serves as the perimeter of the cell chamber with cutouts of two adjoining squares from within the cell chamber. The other PDMS piece is an outline of the chamber&#39;s perimeter.
	<ol>
		<li>Cut out the inside cell chamber so that it is square shaped and matches the shape of the cell chamber. Ensure that the overall shape of both pieces of PDMS is rectangular and matches that of the chamber slide.</li>
	</ol>
	</li>
	<li>Layer the materials as following from bottom to top: (1) Metal plate, (2) PDMS (without cutouts), (3) PPy plate (perpendicular to PDMS),&#160;(4) PDMS (with cutouts), and (5) cell chamber.</li>
	<li>Clamp the apparatus together once it is fully aligned.</li>
	<li>Cut two 60 cm pieces of a metal wire and use silver paste to attach one wire to each end of the PPy plate that protrudes from the outside of the chamber. Ensure that the wires are long enough to extend from the apparatus to the outside of an incubator.</li>
	<li>Once the silver paste is cured, reinforce and seal the wire connection with epoxy.
	<ol>
		<li>Record the resistance using a multimeter to make sure that the applied field is the same in each chamber.</li>
		<li>For implantation, remove the wires; unclamp the cell chambers and PDMS and then them separate from the conductive scaffold. The dimension of the implanted scaffolds is approximately 1 mm x 3 mm x 0.25 mm.</li>
	</ol>
	</li>
</ol>
6. Plating human Neural Progenitor Cells (hNPCs) on PPy
<ol>
	<li>Sterilize the assembled chambers under UV light for 2 hours.
	<ol>
		<li>After 1 hour, rotate the assembled chambers 90&#176;.</li>
	</ol>
	</li>
	<li>After sterilization, coat the surface of PPy at the bottom of the chamber with 800 &#181;L of a coating substrate and allow the substrate to solidify on the PPy plate in an incubator at 37 &#176;C at 5% CO2 for 1 h.</li>
	<li>After 1 hour, remove the supernatant from chambers and gently rinse with 1 mL of DPBS +Ca +Mg per well. 
	NOTE: Avoid washing the surface of PPy forcefully as this will cause the detachment of the coating substrate.</li>
	<li>Using fresh cell media, mechanically dissociate cultured cells for use with the chambers from a tissue culture plate with gentle pipetting. 
	NOTE: Cells should be 90 - 100% confluent upon dissociation.</li>
	<li>Collect hNPC pellets using centrifugation at 8000 x g for 5 minutes.</li>
	<li>Using a hemocytometer, count and resuspend the cells at a volume of 100,000 cells/cm2.</li>
	<li>Plate 100,000 cells into each chamber well (100,000 cells/cm2 in 1 mL of media).</li>
	<li>Return chambers to incubator at 37 &#176;C at 5% CO2.</li>
</ol>
7. Electrical Stimulation of hNPCs
<ol>
	<li>One day after seeding the cells on the chambers, use a waveform generator to apply electrical stimulation to the cells.</li>
	<li>Prior to stimulation, remove old media from each chamber well and replenish with 800 &#181;L of fresh media.</li>
	<li>After the media is changed, place the chambers back into the incubator, extend the wires out of the incubator, and attach them to a waveform generator</li>
	<li>Apply the stimulation to the cells with a square wave signal at &#177;400 mV with 100 Hz for 1 h.</li>
	<li>After the stimulation, remove the wires and incubate the chambers for another day in an incubator set at 37 &#176;C with 5% CO2.</li>
	<li>Perform subsequent biological analysis including cell viability and quantitative real-time polymerase chain reaction (qRT-PCR) following the manufacturer&#39;s protocol.</li>
</ol>
8. In Vivo PPy Implantation
<ol>
	<li>Perform distal middle cerebral artery (dMCA) occlusion stroke models on T cell-deficient adult male nude rats (NIH-RNU 230 &#177; 30 g) as described previously2. Perform implantation one week post-dMCA occlusion.</li>
	<li>One day prior to surgery, give ampicillin (1 mg/mL) to the rats in their cage water.</li>
	<li>Anesthetize the rat in an induction chamber using 0.5 - 3% mg/kg of isoflurane administered via inhalation.</li>
	<li>Confirm anesthetization of the rat by a lack of toe pinch reflex response.
	<ol>
		<li>While the animal is under anesthesia, continue to monitor its membrane color, breathing pattern, and rectal temperature every 15 minutes.</li>
	</ol>
	</li>
	<li>Once anesthetization is confirmed, apply artificial tears on eyes of rat to prevent dryness.</li>
	<li>Ensure that aseptic technique is maintained during this procedure by using sterile gloves, and a sterile surgical drape13. Keep sterile surgical instruments within a sterile field.</li>
	<li>While the rat is under anesthesia, drill a craniectomy above the left cortex. Open the dura.
	<ol>
		<li>Remove the dura mater from the brain using a micro-thin surgical needle.</li>
	</ol>
	</li>
	<li>Implant the conductive scaffolds from in vitro system onto the rat cortex. 
	NOTE: For our experiments, we implanted scaffolds from in vitro day 3 after hNPC plating.
	<ol>
		<li>Place the implant primarily on the penumbral cortex medial to the stroke lesion.</li>
	</ol>
	</li>
	<li>After the scaffold is placed, place surgicel over the implant to prevent movement with skin closure.</li>
	<li>Suture the wound shut and subcutaneously inject the rats with buprenorphine SR at a dosage of 1 mg/kg to manage the pain associated with stereotaxic surgery and dMCA occlusion.</li>
	<li>Place the rat in a recovery cage as it regains consciousness.
	<ol>
		<li>Never leave the animal unattended while it is unconscious.</li>
		<li>Do not place the animal with others until it has fully gained consciousness.</li>
	</ol>
	</li>
	<li>Monitor animals daily for signs of pain including body weight loss, decreased food/water intake, reduced grooming/unkempt coat, decreased/increased spontaneous activity, abnormal posture, dehydration/skin tenting, sunken eyes, hiding, self-mutilation, rapid breathing, open mouthed breathing, twitching, trembling, tremor, and vocalization.
	<ol>
		<li>Monitor the animals daily until it appears that they are eating, drinking, grooming, and gaining weight; and then weekly after weight gain.</li>
	</ol>
	</li>
	<li>Early euthanization via CO2 inhalation and a secondary confirmation of euthanization (as to ensure the animal will not revive due to normal oxygen supply post CO2 inhalation) will occur to&#160; &#160;any animal that appears to be not eating, drinking and gaining weight after the initial surgical procedure; appears in pain; or appears unable to complete the behavioral tests due to a larger than expected motor cortex deficit.&#160;</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Outcomes+of+Transplanted+Modified+Bone+Marrow-Derived+Mesenchymal+Stem+Cells+in+Stroke:+A+Phase+1/2a+Study.">Clinical Outcomes of Transplanted Modified Bone Marrow-Derived Mesenchymal Stem Cells in Stroke: A Phase 1/2a Study.</a> Stroke. 47 (7), 1817-1824 (2016).</li><li>George, P. M., Steinberg, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Stroke+Therapeutics:+Unraveling+Stroke+Pathophysiology+and+Its+Impact+on+Clinical+Treatments.">Novel Stroke Therapeutics: Unraveling Stroke Pathophysiology and Its Impact on Clinical Treatments.</a> Neuron. 87 (2), 297-309 (2015).</li><li>Schabitz, W. 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The authors have no conflicts of interest to declare with this work.

Growing evidence has demonstrated the promise of stem cells as a novel stroke therapy. This promise has resulted in a major effort to advance stem cell therapeutics to the bedside with at least 40 ongoing or completed clinical trials. Stroke pathology offers a unique neurological disorder that lends itself to stem cell therapy because after the acute insult, there is no neurodegenerative process preventing recovery. The exact mechanism of stem cell-enhanced stroke recovery remains unclear. Angiogenesis, synaptogenesis, and synaptic remodeling have all been shown to be important. When important molecules for synapse formation such as BDNF and THBS-1 are removed, stroke recovery is impaired6,19. hNPCs have improved functional recovery after stroke in numerous animal models20,21. The mechanisms of the hNPCs effect remains not fully understood, and the ideal delivery method for these cells is unknown. By developing a system that allows one to manipulate the important molecules released from hNPCs, one can help differentiate the integral recovery mechanisms and optimize stem cell therapy. 
Successful cell delivery to the brain remains a challenge. Currently, a number of technologies have been developed using different biomaterial scaffolding systems to deliver stem cells to improve the survival and integration of stem cells9,10. However, due to the inert characteristics of these materials, it is difficult to modulate stem cells after their transplantation. 
This article describes a method using a PPy scaffold to electrically manipulate the transcriptome of hNPCs as shown by the upregulation of BDNF and THBS-1 in our qRT-PCR data. Our previous study revealed that an increase in VEGFA expression on hNPCs after exposure to electrical stimulation improved functional recovery after stroke2. Here we demonstrate that additional neurotrophic factors such as BDNF and THBS-1 are also modulated after exposure to electrical stimulation in a different hNPC cell line, showing that our system can be used with numerous cell lines. 
During the fabrication of the in vitro electrical stimulation chamber, it is critical that the metal wire is completely connected to PPy plate using silver paint and epoxy. If this connection is not sound, it causes varying electric fields even with the same parameters. Moreover, at times media may leak after the assembly of the scaffolding system. To resolve this issue, vacuum grease can be applied to seal the space between the PDMS and the PPy plate with care not to apply to the cell-seeding surface. One limitation due to the non-transparent nature of the PPy plate is that checking cells' confluency with standard light microscopy is limited. 
The advantage of the method described in this protocol allows for modulation of hNPCs to help determine the mechanistic changes of the cells. Currently, electrical modulation has not been approached using conventional hydrogel or inert scaffolding systems. After electrical stimulation using the PPy scaffold, the hNPCs can be delivered without removal from the polymer surface to an in vivo model. Developing methods to further study disease models of stem cell applications allows us to better design translational applications. The electrical preconditioning approach, made possible by the conductive polymer system, can be applied to different cell types and allows for better understanding of the impact of electrical stimulation on a cell. The ability to optimize the cells in vitro and then without further manipulation (such as passaging or removal) allows for testing of these in vitro manipulations in in vivo disease states such as stroke. The ultimate aim is to utilize new techniques, such as the described conductive polymer scaffold, to advance stroke therapeutics and improve understanding of stroke recovery mechanisms.

Stroke is the second leading cause of death in the world and the fifth leading cause of death in the United States. Despite these high death rates, treatments for stroke recovery currently remain a challenge with no viable medical options currently available1. There are currently about 300 clinical trials dealing with ischemic strokes, of which only 40 utilize stem cells. Previous studies have shown that stem cell therapies have a beneficial effect on stroke repair2,3. Paracrine factors such as brain-derived neurotrophic factor (BDNF) and thrombospondin-1 (THBS-1) released from transplanted human neural progenitor cells (hNPCs) have shown improved functional recovery through mechanisms associated with an increase in synapse formation, angiogenesis, dendritic branching and new axonal projections, as well as modulating the immune system4,5,6. However, the optimal delivery methods of the stem cells remain elusive.
Successful stem cell delivery to the brain remains a challenge. Currently, injectable hydrogel and polymeric scaffolding systems have been introduced to deliver stem cells. These delivery methods protect stem cells during transplantation as well as offer protection from the harsh post-stroke environment including the host&#39;s inflammatory response and hypoxic conditions7,8,9,10. However, the most commonly used materials are inert, which limits the use of continuous modulation (i.e., electrical stimulation) of the cells11. Electrical stimulation is a cue that influences differentiation, ion channel density, and neurite outgrowth of stem cells12. As compared to inert polymers, conductive polymers can carry a current allowing for electrical stimulation and manipulation of stem cells2. However, the precise mechanism by which electrical stimulation modulates neurotrophic factor release (i.e., BDNF and THBS-1) is still not fully explored.
In this protocol, we describe the steps to construct a cell culture system consisting of a conductive polymer scaffold, polypyrrole (PPy), that allows for in vitro electrical stimulation. Because of the manner in which the cell culture system is fabricated, subsequent implantation of the stem cell-seeded scaffold onto the peri-infarct cortex is possible. For this system, we electrically precondition stem cells on the scaffold for a short time period prior to implantation. Following electrical stimulation, the conductive polymer scaffold carrying the cells is successfully implanted intracranially using a minimally invasive method.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">FGF-Basic</td>
			<td style="width:103px;">Invitrogen</td>
			<td style="width:119px;">CTP0261</td>
			<td style="width:188px;">20 ng/mL for working media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Matrigel</td>
			<td style="width:103px;">Corning</td>
			<td style="width:119px;">cb40234a</td>
			<td style="width:188px;">1:200 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">LIF Protein, Recom. Hum. (10 &#181;g/mL)</td>
			<td style="width:103px;">EMD Millipore</td>
			<td style="width:119px;">LIF1010</td>
			<td style="width:188px;">10 ng/mL for working media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sylgard 184 silicone 3.9 kg</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">NC0162601</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">SA56-4</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">SA95</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">ITO Glass</td>
			<td style="width:103px;">Delta Technologies</td>
			<td style="width:119px;">CG-40IN-0115</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Sodium dodecylbenzenesulfonate</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">289957-1KG</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Pyrrole</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">131709-500ML</td>
			<td style="width:188px;">Protect pyrrole solution from light and room temperature</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">8 well glass slide chambers</td>
			<td style="width:103px;">Thermo Sci Nuc&#160;</td>
			<td style="width:119px;">125658</td>
			<td style="width:188px;">Detach the cell chamber and keep it under sterilized conditions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Flat-Surface Bracket, 3&#34;x1&#34;</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">1030A4</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">TWO PART SILVER PAINT 14G</td>
			<td style="width:103px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:119px;">1264214</td>
			<td style="width:188px;">Mix two parts (1:1) in plastic plate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">DPBS, 1x, with Ca and Mg, No Phenol Red</td>
			<td style="width:103px;">Genesse&#160;</td>
			<td style="width:119px;">25-508C</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">AB2 ArunA Neural Cell Culture Media Kit</td>
			<td style="width:103px;">Aruna Biomedical&#160;</td>
			<td style="width:119px;">ABNS7013.2</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">hNP1 Human Neural Progenitor Expansion Kit</td>
			<td style="width:103px;">Aruna Biomedical&#160;</td>
			<td style="width:119px;">&#160;hNP7013.1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&#34; to 5/8&#34; Tube OD</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">5330K22</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Multimeter</td>
			<td style="width:103px;">Keysight&#160;</td>
			<td style="width:119px;">E3641A</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Wavefoam generator</td>
			<td style="width:103px;">Keysight</td>
			<td style="width:119px;">33210A-10MHz</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Pt meshes</td>
			<td style="width:103px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">298107-425MG</td>
			<td style="width:188px;">Reference electrode with dimensions, 1x1 cm</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">L3224</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">BDNF</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02718934_s1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">THBS1</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs00962908_m1</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">GAPDH</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02758991_g1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">RNeasy Mini Kit (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">QIAshredder (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">79656</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">RNase-Free DNase Set (50)</td>
			<td style="width:103px;">QIAGEN</td>
			<td style="width:119px;">79254</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">iScript cDNA Synthesis Kit, 100 x 20 &#181;L rxns</td>
			<td style="width:103px;">BIORAD</td>
			<td style="width:119px;">1708891</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">4369510</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">7-8 Week Old, male, RNU Rats</td>
			<td style="width:103px;">NCI-Frederick</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">4-0 Ethicon Silk Suture</td>
			<td style="width:103px;">eSutures.com</td>
			<td style="width:119px;">683G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Isoflurane</td>
			<td style="width:103px;">Henry Schein</td>
			<td style="width:119px;">29405</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">V-1 Tabletop Laboratory Animal Anesthesia System</td>
			<td style="width:103px;">VetEquip</td>
			<td style="width:119px;">901806</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Surgicel Original Absorbable Hemostat</td>
			<td style="width:103px;">Ethicon</td>
			<td style="width:119px;">1952</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Lab Standard Stereotaxic Instrument, Rat</td>
			<td style="width:103px;">Stoelting</td>
			<td style="width:119px;">51600</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:213px;">Kimberly-Clark Professional&#160;Safeskin Purple Nitrile Sterile Exam Gloves</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">19-063-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sterile Drape</td>
			<td style="width:103px;">Medline</td>
			<td style="width:119px;">DYNJSD1092</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:213px;">Thermo Scientific&#160;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">53-34</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Walter Stern Scalpel Blade Series 300</td>
			<td style="width:103px;">Fisherbrand</td>
			<td>17-654-456</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4484642</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Frazier Micro Dissecting Hook</td>
			<td style="width:103px;">Harvard Apparatus</td>
			<td>52-2706</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrically conductive scaffold to modulate and deliver stem cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

The schematic shown in Figure 1 represents the overall workflow of the electrical stimulation of hNPCs and potential downstream applications. A current limitation in stem cell therapy is that stem cells are exposed to a harsh post-transplantation environment including inflammation and ischemic conditions. These difficult conditions likely limit their therapeutic efficacy14,15. The use of a conductive scaffold to protect hNPCs from this environment may augment hNPCs therapeutic benefits through electrical preconditioning. The first step in this stem cell delivery technique is the development of a conductive scaffold using an electroplating approach2,16. We characterized the scaffolds biocompatibility and optimized electrical preconditioning characteristics with hNPCs. Controls were defined as unstimulated stem cells grown on a tissue culture plate.
Various voltages were evaluated to determine the safety of electrical stimulation and to maximize the preconditioning efficacy. To ensure that the applied field is same in each chamber, the resistivity of assembled chamber was measured using a multimeter (Resistances (Ohm, &#937;) of chambers were approximately 10 k&#937;, and resistivity&#8776;3 &#937;m). According to the commercial vendor, the hNPCs used have shown no significant cytotoxicity in normal culture systems. hNPCs with or without exposure to electrical stimulation were stained with cell viability assays (Live: green; Dead: red) (Figure 2). These results indicate that hNPCs were viable after the electrical stimulation (&#177;400 mV, 100 Hz for 1 h). To validate the cell viability assay, we performed cell viability testing using a resazurin assay. The results also demonstrate no significant cytotoxicity of electrical stimulation on hNPCs.
Our previous in vivo data demonstrated that paracrine factors released from hNPCs (SD56, NPCs derived from embryonic stem cells) with an exposure to electrical stimulation improve the recovery after stroke2. To explore further candidate factors known to be important in stroke recovery that are released from hNPC (Aruna Biomedical, NPCs derived from embryonic stem cells), BDNF and THBS-1 were evaluated. These factors have been extensively studied due to their role in neuronal outgrowth and increase in cell-to-cell interactions17,18. To investigate the efficacy of electrical stimulation on transcriptome changes for BDNF and THBS-1, qRT-PCR was performed including BDNF and THBS-1 genes with GAPDH as a housekeeping gene. Approximately 1 &#181;g of total RNA was reverse-transcribed into cDNA according to the manufacturer&#39;s protocol. Normalized fold-change ratios were calculated by &#916;&#916;Ct method comparing gene expression in hNPCs on PPy and hNPCs on PPy with an exposure to electrical stimulation. Statistical analysis showed significant upregulations in gene expression of BDNF and THBS-1 between groups that were electrically stimulation dependent (p &#8804;0.01) (Figure 3). This data suggests that optimization is possible to maximize hNPC efficacy without significant cell death.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57367/57367fig1.jpg" /> 
Figure 1. In vitro conductive polymer scaffold system for electrical stimulation. (A) A slide chamber is placed on top of PPy plate. (B) The schematic shows the fabrication of the in vitro electrical stimulation chamber with hNPCs plated on the surface of PPy. Flow valves are used to hold the slide chamber, PDMS, PPy, and metal plate together. (C) Image of the chamber. <a href="/files/ftp_upload/57367/57367fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57367/57367fig2.jpg" /> 
Figure 2. Cell viability assay in hNPCs with or without electrical stimulation. (A) Bar graph demonstrating live cell stained with Calcein-am. Data presented are mean &#177; S.D.; n = 4. (B) Cell viability assay using resazurin. Bar graph shows there was no cytotoxicity of electrical stimulation on hNPCs; n = 4. (C) Images of cell viability assay before and after electrical stimulation treatment. Green signal indicates live cells (Calcein-am), whereas red signal indicates dead cells (EtHD-1). ES indicates electrical stimulation. ES + or - indicates the presence or absence of electrical stimulation on cells. <a href="/files/ftp_upload/57367/57367fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57367/57367fig3.jpg" /> 
Figure 3. Gene expression changes with electrical stimulation. Bar graph demonstrating fold changes in gene expressions of BDNF (A) and THBS1 (B) in hNPCs (** indicates statistically significant between electrically preconditioned and all other groups, p &#60;0.01, error bars show S.D.; n = 4, one-way ANOVA). Negative indicates the control where cells were cultured on a regular tissue culture plate. ES + or - indicates the presence or absence of electrical stimulation on cells. <a href="/files/ftp_upload/57367/57367fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

electrically-conductive-scaffold-to-modulate-and-deliver-stem-cells

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.3K Views.  Stanford University School of Medicine. This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique. 

Video: Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

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 ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

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 ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...