Name: electrically conductive scaffold to modulate and deliver stem cells
Uploaded: 2018-04-13T13:30:00
Description: Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

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 blad...

<ol>
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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, a...

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>
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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 ...

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 ...

The overall goal of this procedure is to create a platform for in vitro electrical stimulation of stem cells on a conductive polymer scaffold, with subsequent in vivo implantation. This method can help answer key questions in stroke research and stem cell biology by providing a means to modulate stem cells. The main advantage is that the stem cells can be optimized in vitro, and subsequently implanted.Although this method can provide an insight into stroke recovery, it can also be applied to other systems as a new cell delivery method. Generally, users new to this technique will struggle due to the precision required for cell chamber assembly, and the technique involved with the craniectomy. In preparation, autoclave the 2.5 by 12.5 centimeter metal plates and the flow valves.Next, using a chamber slide as a template, cut two pieces of PDMS. One piece serves to outline of the chamber's perimeter. On the second piece, cut out two adjacent pieces from the inside of the chamber slide.Both pieces should be rectangular and match the chamber slide. Now, layer the materials. Start with the metal plate, then the uncut PDMS, then the polypyrrole plate, oriented perpendicularly, then the PDMS with cut-outs, and finally, the cell chamber.Align the layers carefully, and clamp the apparatus together. Next, cut two lengths of a metal wire to extend from the apparatus to the outside of the incubator that will contain it. 60 centimeter lengths are usually long enough.Next, use silver paste to attach a wire to each end of the polypyrrole plate protruding from the outside of the chamber. Once the silver paste is cured, reinforce and seal the connections using an epoxy. Now, record the resistance across the assembly using a multimeter.The applied field must be the same in each chamber. Next, plate the cells onto the assembly, and electrically stimulate them as described in the text protocol. Prior to implanting the cells, remove the wires from the assembly.Then, unclamp the cell chambers and separate the PDMS from the conductive scaffold. The scaffold is now ready for implanting. One day prior to the surgery, provide the subject animal ampicillin in its drinking water.On the day of the surgery, anesthetize the rat with isoflurane, and confirm the anesthesia with a toe pinch. Now, apply artificial tears to the rat's eyes to keep them moist. After shaving the skull, drape the animal, and set up for a sterile surgery.Wear protective garments, unpack the sterilized tools, and sterilize the scalp. For the craniectomy, drill a hole above the left cortex down to the dura mater. Make the opening slightly larger than 1 by 3 millimeters, the size of the implant.Then, open the dura and remove the dura mater from the brain using a micro-thin surgical needle. Remove the rig from the TC-culture incubator and disable the rigs. Next, carefully implant the conductive scaffold from the in vitro system onto the rat cortex.Then cover the implant with Surgicel to secure it, or it may move during the skin closure. Now, suture the wound shut using 4-0 silk suture. After completing the sutures, subcutaneously inject the rats with buprenorphine SR.Then, place the rat in a recovery cage until it regains consciousness.The post-operative procedure is detailed in the text protocol. Human neural progenitor cells were exposed to electrical stimulation, and then stained with cell viability assays. The green color is indicative of a living cell.The cells were clearly able to survive the electrical stimulation. Factors released from the neuro-progenitor cells that are important in stroke recovery were evaluated from one microgram of total RNA using quantitative RTPCR. After normalizing the results using the delta delta cycle threshold method, they reveal then the exposure to electrical stimulation up-regulated BDNF and THBS1 expression.After watching this procedure, you should have a good understanding of how to create a cell culture system for electrically-conducting stem cells, and subsequent implantation for those stem cells into a rat model. Once it is mastered, the procedure can be done in 45 minutes if it is performed properly. While you're attempting this procedure, it is important to remember to always monitor the animals for proper breathing pattern and membrane color, to ensure proper anesthesia.Don't forget that working with rats and stem cells can be hazardous, and precautions, such as wearing proper PPE, should always be taken.

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

Research

JoVE Journal

Bioengineering

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 ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...