Funding Sources: The University of Arizona, The Jim Himelic Foundation, and the Arizona Center for the Biology of Complex Diseases.

hiPSCs were derived from human dermal fibroblasts isolated from 4 mm skin punch biopsies and reprogrammed in house via Sendai virus-mediated reprogramming11. The University of Arizona Institutional Review Board approved all procedures for subject recruitment and biopsy collection.
1. Preparation of Extracellular Matrix Coated Surface for iPSC Culture
<ol>
	<li>One day prior to the confluence of hiPSC cultures growing on iMEFs, prepare extracellular matrix (Matrigel) coated plates.</li>
	<li>Slow thaw an aliquot of the extracellular matrix (lot dependent, follow instructions on the specification sheet) on ice in 4 &#176;C for 2 h.</li>
	<li>In a biosafety hood, using cold pipet tips (stored at -20 &#176;C), add 1 aliquot of extracellular matrix to cold Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12/HEPES) according to the lot specific dilution factor.</li>
	<li>Dispense ~ 600 &#181;L of extracellular matrix per well of each new cell culture treated 12-well plate needed.</li>
	<li>Incubate the plate at room temperature for several hours. Here, use a 3 h incubation time. 
	NOTE: Plates can be used immediately or stored at 4 &#176;C for up to two weeks.</li>
</ol>
2. Transfer of hiPSCs Grown on iMEF Feeder Cells onto Extracellular Matrix for Propagation
<ol>
	<li>On the day of passaging, remove the matrix coated plate from 4 &#176;C and allow the plate to acclimate to room temperature for 1 h in the tissue culture hood.</li>
	<li>Aspirate the medium from 1 well of a 6-well plate of hiPSC culture growing on iMEFs and replace with 500 &#181;L collagenase dissociation reagent (1 mg/mL in basal culture medium).</li>
	<li>Incubate at 37 &#176;C for ~ 10 - 30 min until the edges of the iPSC colonies appear to lift slightly. 
	NOTE: Incubation time is line dependent and will vary. Monitor dissociation under a microscope.</li>
	<li>Carefully aspirate the dissociation reagent and wash gently 2 times with phosphate buffered saline (PBS, pH 7.4).</li>
	<li>Add 1 mL of room temperature mTeSR1 complete medium with 10 ng/mL Rho-associated protein kinase inhibitor (Y-27632 ROCK inhibitor) to the well. 
	NOTE: Using a 50:50 mixture of iMEF-conditioned medium and mTeSR1 during this initial passage is suggested and can be beneficial for the survival of the hiPSCs during this media transition.</li>
	<li>Using a small phase contrast microscope placed partially inside the hood, manually score and pick 1 - 2 hiPSC colonies (~ 700 - 1,000 &#181;m in diameter) using a syringe and needle (25 G, 1&#189; in). Push off the scored pieces of the colony into the medium with a P200 pipet tip.</li>
	<li>Aspirate and dispose of the matrix from the new plate, being careful not to disturb the coating.</li>
	<li>Transfer 1 mL of the cell suspension from the old well containing the scored iPSCs into the new matrix coated well.</li>
	<li>Place the plate in the incubator at 37 &#176;C, 5% CO2. Rock the plate in several quick, back-and-forth, and side-to-side movements to evenly spread out the cells. Do not disturb the plate for 24 h.</li>
	<li>Perform daily full media changes with mTeSR1 complete medium (addition of Y-27632 ROCK Inhibitor is not needed after the initial plating in step 2.5).</li>
</ol>
3. Small-scale Passaging of hiPSCs on Matrix-coated Plates
NOTE: Generally, after the initial transfer of the hiPSCs to matrix coated plates, the cells should be passaged once more in a similar manner to ensure all iMEFs have been eliminated and cells have adapted to feeder-free conditions.
<ol>
	<li>One day prior to iPSC confluency prepare freshly coated matrix plates as discussed in section 1.</li>
	<li>On the day of passage, remove the matrix coated plate from 4 &#176;C incubation and allow the plate to acclimate to room temperature for 1 h in the tissue culture hood.</li>
	<li>Aspirate the medium from 1 well of a 6-well plate of hiPSCs (originally transitioned from feeder to matrix) and replace with 500 &#181;L cell dissociation buffer. 
	NOTE: The commercial cell dissociation buffer used here (see Materials Table) is more suitable for use with mTeSR1 medium compared to collagenase.</li>
	<li>Incubate at room temperature for 3 - 7 min until the edges of the iPSC colonies appear to lift slightly. As mentioned before, the incubation time is line dependent and variable. Therefore, monitor the cellular dissociation under a microscope to determine the optimal time.</li>
	<li>Carefully aspirate the dissociation buffer and wash gently 2 times with PBS.</li>
	<li>Add 500 &#181;L of room temperature mTeSR1 complete medium with 10 ng/mL Y-27632 ROCK inhibitor to the well.</li>
	<li>Instead of scoring colonies as in Step 2.6, push the desired number of colonies into the media using a P200 tip and gently triturate the cell suspension 2 times against one corner of the well to break the hiPSC colonies into clumps of ~ 50 - 200 &#181;m in size. For example, for a 12-well plate, pick 1 - 2 colonies to transfer into each 1 new well. Transfer the cell suspension from the old well into a single new well of a 12-well plate.</li>
	<li>Rock the plate in several quick, back-and-forth, and side-to-side movements to evenly spread out the cells, and let the plate rest for 24 h in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Perform daily full media changes with mTeSR1 complete medium. As mentioned before, it is not necessary to add Y-27632 ROCK Inhibitor after the initial plating. 
	NOTE: Unused iPSC colonies from Step 3.7 may be easily cryopreserved at this point.</li>
</ol>
4. Preparation of Chamber Slides and Coverslips for Seeding of hiPSCs for Immunocytochemistry
NOTE: The following protocol utilizes 4-well plastic chamber slides and 12 mm glass coverslips with appropriate volumes of media in the multi-wells to support cost-effective immunocytochemistry. However, media volumes can be increased if the use of larger well and coverslip sizes is desired.
<ol>
	<li>One day prior to hiPSC confluence, prepare chamber slides freshly coated with extracellular matrix as discussed in section 1. Add enough matrix solution (~ 300 - 500 &#181;L per well of a 4-well chamber slide) to fully coat each well without any concern of evaporation. Alternatively, after placing 1 cleaned and sterilized coverslip into each well of a 24-well plate, coat with 500 &#181;L extracellular matrix and make sure the coverslip is not floating on top of the solution.</li>
	<li>On the day of passage, remove the coated chamber slides/coverslips from 4 &#176;C and allow acclimation to room temperature for 1 h in the tissue culture hood.</li>
	<li>Aspirate the medium from 1 well of a 12-well plate of hiPSCs and replace with 500 &#181;L dissociation buffer.</li>
	<li>Incubate at room temperature for ~ 3 - 7 min until the edges of the iPSC colonies appear to lift slightly.</li>
	<li>Carefully aspirate the dissociation buffer and add 1 mL of room temperature mTeSR1 complete medium with 10 ng/mL Y-27632 ROCK Inhibitor to the well.</li>
	<li>Using a small phase contrast microscope placed inside the tissue culture hood, manually pick 1 colony/new well of a 4-well chamber slide or 1 new coverslip to be plated (~ 1,000 - 1,500 &#181;m in diameter) using a P200 pipet tip by pushing it off the plate surface into the medium.</li>
	<li>Aspirate the matrix from the new chamber slide/coverslip, being careful not to disturb the coating.</li>
	<li>Gently triturate the cell suspension 2 times against one corner of the well to break the hiPSC colonies into clumps of ~ 50 - 200 &#181;m in size. Transfer 250 &#181;L of the cell suspension from the old well into each new well of a 4-well chamber slide/coverslip.</li>
	<li>Bring the volume of each new well up to 500 &#181;L with mTeSR1 containing 10 ng/mL Y-27632 ROCK Inhibitor.</li>
	<li>Place the chamber slide/coverslips in the incubator at 37 &#176;C, 5% CO2.</li>
	<li>After 24 h, perform daily full media changes with mTeSR1 complete medium. Again as mentioned previously, the addition of Y-27632 ROCK Inhibitor is not necessary after the initial plating.</li>
</ol>
5. Preparation of iPSCs for Immunocytochemistry
<ol>
	<li>Once confluent, preserve the hiPSC colonies via fixation by removing the media from the wells and adding pre-warmed CAUTION 4% paraformaldehyde (~ 300 &#181;L/well of a 4-well chamber slide/coverslip). Incubate at room temperature for 20 min.</li>
	<li>Discard the paraformaldehyde appropriately according to institutional biohazard and chemical safety guidelines.</li>
	<li>Wash the cells 3 times for 5 min each on a bench-top shaker with PBS. 
	NOTE: Cells on chamber slides/coverslips can be stored with ample PBS at 4 &#176;C indefinitely at this stage. Alternatively, they can be immediately used for ICC.</li>
</ol>
6. Immunostaining of hiPSCs with Pluripotency Markers
NOTE: Immunostain hiPSCs with standard pluripotency antibodies. As an example, here cells are double-stained with antibodies targeting one 1 surface and 1 intracellular antigen in a sequential manner as follows: Anti-Stage-Specific Embryonic Antigen-4 (SSEA4) with anti-Octamer-binding protein 4 (OCT-4) and anti-Tra-1-60 with anti-SRY related HMG BOX gene 2 (SOX2) (Please see the Materials Table for antibody details).
<ol>
	<li>Aspirate the PBS from the wells and add in 500 &#181;L/well Blocking Solution without Triton-X-100. Incubate for 1 h at room temperature.</li>
	<li>Prepare a pre-determined concentration (here, use 1:200) of the surface antibodies, either SSEA4 or Tra-1-60, by diluting in Blocking Solution without Triton X-100.
	<ol>
		<li>For chamber slides, prepare 300 &#181;L/well of a 4-well chamber slide by mixing 298.5 &#181;L of Blocking Solution above and 1.5 &#181;L of anti-SSEA4 or Tra-1-60 primary antibody.</li>
		<li>For coverslips, prepare 40 &#181;L/coverslip by mixing 199 &#181;L of Blocking Solution and 1 &#181;L of anti-SSEA4 or Tra-1-60 primary antibody.</li>
	</ol>
	</li>
	<li>Aspirate the Blocking Solution from the chamber slide and dispense the primary antibody into appropriate wells. For coverslips, place a piece of parafilm firmly inside a Petri dish and dispense 40 &#181;L of the primary antibody on top as a droplet. Using a bent syringe needle and forceps, carefully lift the coverslip out of the culture well and place it cell side down onto the drop of antibody on the parafilm. Incubate overnight at 4 &#176;C.</li>
	<li>The next morning, wash the cells 3 times, 5 - 10 min each, by removing the antibody from the chamber slide and adding in PBS. For coverslips, lift each one off the parafilm and place it back in a well of a 24-well plate, cell side up. Carefully add PBS.</li>
	<li>Prepare appropriate secondary antibodies at 1:500 in the Blocking Solution.
	<ol>
		<li>For chamber slides, prepare 300 &#181;L/well of a 4-well chamber slide by mixing 499 &#181;L of Blocking Solution and 1 &#181;L of goat anti-mouse IgG3 Alexa Fluor 488 (for SSEA4) or 1 &#181;L of goat anti-mouse IgM Alexa Fluor 488 (for Tra-1-60).</li>
		<li>For coverslips, prepare 40 &#181;L/coverslip by mixing 499 &#181;L of Blocking Solution and 1 &#181;L of goat anti-mouse IgG3 Alexa Fluor 488 (for SSEA4) or 1 &#181;L of goat anti-mouse IgM Alexa Fluor 488 (for Tra-1-60).</li>
	</ol>
	</li>
	<li>Aspirate the PBS from the chamber slide and dispense the secondary antibody into appropriate wells. For coverslips, following Step 6.2, invert the coverslips onto the antibody solution placed on parafilm at room temperature for 2 h. 
	NOTE: If using fluorescent tagged secondary antibodies, the cells must be protected from light. Place the chamber slides and the coverslips in a dark environment during incubations and washes.</li>
	<li>Wash the cells 3 times for 5 - 10 min each by removing the antibody from the chamber slide and adding in PBS. For coverslips, lift each one carefully from the parafilm and place back in a well of a 24-well plate, cell side up. Gently add PBS during washes.</li>
	<li>Prior to probing for the second antigen, intracellular OCT-4 or SOX2 in this case, incubate again in Blocking Solution made with permeabilization reagent (Triton-X-100) for 1 h at room temperature.</li>
	<li>To immunostain for OCT-4 or SOX2, simply follow the methodology described in Steps 6.1-6.6 for applying the primary and secondary antibodies. For the purposes of this protocol, use OCT-4 and SOX2 at a concentration of 1:200 and the secondary antibody goat anti-rabbit IgG Alexa Fluor 594 at 1:500.</li>
	<li>After the abovementioned primary and secondary antibody treatments, wash the cells 3 times for 5 - 10 min each with PBS and counterstain with 4&#39;,6&#39;-diamidino-2-phenylindole, dihydrochloride (DAPI) following standard protocols if nuclear visualization is desired.</li>
</ol>
7. Preparation for Viewing
<ol>
	<li>For chamber slides, carefully remove the upper chamber from the slide, following the manufacturer&#39;s instructions. Then, rinse in distilled water, and add mounting medium and a coverglass to enable microscopy.</li>
	<li>For coverslips, invert the coverslips on to a drop of mounting medium placed on a glass microscope slide.</li>
	<li>Image under a standard or confocal fluorescence microscope.</li>
</ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Park, I. H., 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=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</li><li>Hallett, P. J., 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=Successful+function+of+autologous+iPSC-derived+dopamine+neurons+following+transplantation+in+a+non-human+primate+model+of+Parkinson's+disease.">Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease.</a> Cell stem cell. 16, 269-274 (2015).</li><li>Haston, K. M., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Trials+in+a+Dish:+The+Potential+of+Pluripotent+Stem+Cells+to+Develop+Therapies+for+Neurodegenerative+Diseases.">Clinical Trials in a Dish: The Potential of Pluripotent Stem Cells to Develop Therapies for Neurodegenerative Diseases.</a> Annu rev pharm toxicol. 56, 489-510 (2016).</li><li>Zhang, L., 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=Derivation+and+high+engraftment+of+patient-specific+cardiomyocyte+sheet+using+induced+pluripotent+stem+cells+generated+from+adult+cardiac+fibroblast.">Derivation and high engraftment of patient-specific cardiomyocyte sheet using induced pluripotent stem cells generated from adult cardiac fibroblast.</a> Circ heart fail. 8, 156-166 (2015).</li><li>Byrne, J. A., Nguyen, H. N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+generation+of+induced+pluripotent+stem+cells+from+a+subpopulation+of+human+fibroblasts.">Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.</a> PloS one. 4, 7118 (2009).</li><li>Sivapatham, R., Zeng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+Characterization+of+Patient-Specific+Induced+Pluripotent+Stem+Cell+for+Disease+Modeling.">Generation and Characterization of Patient-Specific Induced Pluripotent Stem Cell for Disease Modeling.</a> Methods mol bio. 1353, 25-44 (2016).</li><li>Park, I. H., 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=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Ruff, D., Lieu, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+stem+cells+using+quantitative+PCR+protein+assays.">Profiling stem cells using quantitative PCR protein assays.</a> Methods mol bio. 997, 225-236 (2013).</li><li>Pripuzova, N. S., 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=Development+of+a+protein+marker+panel+for+characterization+of+human+induced+pluripotent+stem+cells+(hiPSCs)+using+global+quantitative+proteome+analysis.">Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis.</a> Stem cell res. 14, 323-338 (2015).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proc jpn acad ser B phys biol sci. 85, 348-362 (2009).</li><li>Bigdeli, N., 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=Adaptation+of+human+embryonic+stem+cells+to+feeder-free+and+matrix-free+culture+conditions+directly+on+plastic+surfaces.">Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces.</a> J Biotechnol. 133, 146-153 (2008).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods mol bio. 767, 137-146 (2011).</li></ol>

The authors have nothing to disclose.

The systematic protocol presented here offers a timesaving and cost-effective method, in the form of a scaled-down culture technique, specifically designed to support effective pluripotency analysis via immunocytochemistry.
The main advantages of the described methodology are as follows. Traditionally more than 3 to 4 passages are needed to transition iPSCs from feeder layers to feeder-free culture conditions in order to eliminate residual iMEFs remaining after the use of bulk dissociation techniques and for typical monolayer morphology to appear12,13. In contrast, the method presented here involves a shortened timeline, which allows for the relatively quick transfer of iPSCs to serum-free conditions via the manual picking of iPSC colonies. The iPSCs are grown in limited amounts in small chamber slides or coverslips conducive for immunocytochemistry. In fact, the volume of culture medium needed per well is just 0.5 mL and the volume of diluted antibody solution required for a single coverslip is as nominal as 40 &#181;L or 300 &#181;L per chamber well, when using this protocol.
We find that the addition of equal parts iMEF conditioned media to the mTeSR1 during the initial passage (Step 2.5) is an important step that aids in the survival and maintenance of the iPSC&#39;s pluripotent morphology. In addition, if cell adherence is not optimal after passaging (Steps 2.2, 3.3 and 4.3), troubleshooting can be performed by further optimizing the dissociation times used.
Finally, a limitation of the presented small-scale method, when compared to other techniques such as flow cytometry also used to verify pluripotency, is that it does not allow for the continued propagation of analyzed cells for downstream applications. The usefulness of our method arises from its specific design which supports the cost-effective and efficient culture of the hiPSCs for confirming pluripotency via routine immunocytochemistry.

Reprogramming human adult somatic cells into induced pluripotent stem cells (iPSCs) provides a way to obtain a potentially unlimited supply of patient-specific cells to study disease1,2. Recapitulating a disease phenotype in vitro would make it plausible to examine cellular and molecular mechanisms associated with disease, and enhance drug discovery and personalized medicine3. In addition, human iPSCs (hiPSCs) offer the possibility of deriving specific cell types which can be used as a unique resource to replace dead or dysfunctional cells and restore function in the context of several disorders4,5.
An important prerequisite to using iPSCs in the above applications is ensuring that their pluripotent and undifferentiated state is maintained during expansion in culture. Typically, techniques such as flow cytometry, western blotting, polymerase chain reaction and functional assays, which require large quantities of cells and specialized equipment, are used for the detailed analysis of iPSC pluripotency6,7,8,9,10. However, routine assessment of the iPSCs&#39; undifferentiated state might effectively be achieved through the limited propagation of these cells specifically for immunocytochemistry (ICC), thus involving reduced time and resources.
Recent advances allow for the growth of iPSCs in defined serum-free conditions, which is a significant improvement over conventional culture systems that require murine fibroblast feeder layers and serum containing media. However, the current literature does not include clear stepwise protocols that describe how to transition iPSCs from feeder layer to feeder-free systems.
In this context, the present protocol systematically details how hiPSCs grown on irradiated mouse embryonic fibroblast (iMEF) feeder layers can be (1) adapted to propagate in serum-free medium, and (2) cultured on a small-scale to specifically support robust immunocytochemical analysis. Overall, this methodology represents a timely and cost-effective procedure for propagating human iPSCs in serum-free conditions for confirming their pluripotency on a routine basis using immunocytochemistry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM-F12/HEPES</td>
			<td>Life Technologies</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-Basic</td>
			<td>Cell Sciences</td>
			<td>CRF001B</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 ROCK Inhibitor</td>
			<td>R&#38;D</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Life Technologies</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 Basal Medium</td>
			<td>StemCell Technologies</td>
			<td>05850</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 5X Supplement</td>
			<td>StemCell Technologies</td>
			<td>05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Buffer</td>
			<td>StemCell Technologies</td>
			<td>07174</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M PO4 Buffer</td>
			<td>In-House</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldeyde, prill</td>
			<td>Electron Microscopy Sciences</td>
			<td>19202</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phoshate Buffered Saline</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Life Technologies</td>
			<td>16210072</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-4A (C30A3) Rabbit mAb, 
			Sox2 (D6D9) Rabbit mAb, 
			SSEA4 (MC813) Mouse mAb, 
			TRA-1-60(S) (TRA-1-60(S)) Mouse mAb</td>
			<td>Cell Signaling 
			Cell Signaling 
			Cell Signaling 
			Cell Signaling</td>
			<td>2840 
			3579 
			4755 
			4746</td>
			<td>Alternatively, a combination of 6 pluripotency primary antibodies can be purchased together as a kit in Catalog #9656</td>
		</tr>
		<tr>
			<td>Goat anti-Ms IgM Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21042</td>
		</tr>
		<tr>
			<td>Goat anti-Ms IgG3 Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21151</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rb IgG Alexa Fluor 594</td>
			<td>Life Technologies</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Cell Culture Plates</td>
			<td>Fisher Scientific&#160;</td>
			<td>0720080/0720081</td>
			<td>Available in 6, 12, 24, 48, 96 well sizes</td>
		</tr>
		<tr>
			<td>Chamber Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12 565 21</td>
			<td>Available in Glass or Permanox Plastic in 1, 2, 4, 8, 16 well sizes</td>
		</tr>
		<tr>
			<td>Coverglass for growth</td>
			<td>Fisher Scientific&#160;</td>
			<td>12 545 82</td>
			<td>Available in 12, 15, 18, 22 and 25mm sizes</td>
		</tr>
	</tbody>
</table>

small-scale propagation of human ipscs in serum-free conditions for routine immunocytochemical characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

This protocol provides a step-wise description of how human iPSCs can be transferred from feeder layer to feeder-free conditions, and subsequently propagated in a limited manner to specifically enable cost-effective immunocytochemistry for confirming pluripotency maintenance. Figure 1 shows a schematic representation of this protocol. Figure 2A shows hiPSC colonies growing on iMEFs in 6-well plates. These colonies exhibit typical morphology with defined borders and dense phase bright centers. As shown in Figure 2B, after the transfer of iPSCs to feeder-free conditions in 12-well plates, colony morphology appears somewhat chaotic with less distinct edges. Also, some iMEFs may remain in the culture at this stage. Figure 2C shows that after an additional passage in the feeder-free system, iPSC colonies display a classic monolayer morphology with a high nucleus to cytoplasm ratio. At this stage, it is seen that iMEFs have been virtually eliminated from the culture.
Colonies growing under feeder-free conditions are mechanically picked and plated onto small multi-well chamber slides or glass coverslips, and probed for specific pluripotency antigens via immunocytochemistry. Figure 3 shows representative colonies that are immunopositive for SSEA4 or Tra-1-60 (3B, 3F, surface pluripotency antigens) and Oct-4 or Sox2 (3 A, 3E, intracellular pluripotency antigens).
<img alt="Figure 1" src="/files/ftp_upload/55260/55260fig1.jpg" /> 
Figure 1: Schematic Representation of the Protocol. Overall schema of the described method used to transition human iPSCs from iMEF feeders to feeder-free culture and subsequent probing of cells with pluripotency markers. iMEF: irradiated Mouse Embryonic Fibroblast; iPSC: Induced Pluripotent Stem Cell. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55260/55260fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55260/55260fig2.jpg" /> 
Figure 2. Representative Images of iPSC Colonies Transitioning from iMEF Feeders to Feeder-free Culture Conditions. A) Dense iPSC colony (white arrow) grown on iMEF feeder cells (black arrow). B) After the initial passage on to a matrix-coated 12-well plate in serum-free medium, a few iMEFs may still be observed in culture (black arrow). C) Typical morphology of a monolayer iPSC colony grown feeder-free. No iMEFs remain in culture at this stage. Scale Bar (A-C) = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55260/55260fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55260/55260fig3.jpg" /> 
Figure 3. Immunocytochemical Characterization of Human iPSCs Plated in Small Well Plates. Representative immunofluorescence images of iPSCs, obtained via a confocal microscope, showing the positive expression of pluripotency markers A) Oct-4 (red) and B) SSEA4 (green) with C) the nuclear stain DAPI (blue), and E) Sox2 (red) and F) Tra-1-60 (green) with G) DAPI (blue). D and H show the overlay images relating to A-C and E-G. Scale bar (A-H) = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55260/55260fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">hiPSC Media for maintenance on Feeders</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock Concentration</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td>DMEM-F12/HEPES</td>
			<td>100%</td>
			<td>80%</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>100%</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>200 mM</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>10 mM</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>55 mM</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-Basic</td>
			<td>10 &#181;g/mL</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Y-27632 ROCK Inhibitor</td>
			<td></td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td colspan="3">Matrigel coating for Feeder-Free cultures</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Amount</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified Matrix</td>
			<td>Dilution Factor 269 &#181;L</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12/HEPES</td>
			<td>25 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Note: Dilution Factor is lot dependent and must be ascertained from certificate of analysis</td>
		</tr>
		<tr>
			<td colspan="3">mTeSR1 complete media for Feeder-Free cultures</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Amount</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 Basal Medium</td>
			<td>400 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 5x Supplement</td>
			<td>100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Note: Once mixed, mTeSR1 complete media may be frozen in aliquots and used until component expiration date</td>
		</tr>
		<tr>
			<td colspan="3">Note: mTeSR1 complete media must be warmed at room temperature only. Do not place in water bath</td>
		</tr>
	</tbody>
</table>
Table 1: iPSC Culture Media Recipes and Plate Coatings.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">4% Paraformaldehyde fixative</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>0.1 M PO4 Buffer</td>
			<td>2 L</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, prill</td>
			<td>80 g</td>
		</tr>
		<tr>
			<td colspan="2">ICC Blocking Solution without Triton-X-100</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline</td>
			<td>49 mL</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>0.5 g</td>
		</tr>
		<tr>
			<td colspan="2">ICC Blocking Solution with Triton-X-100</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline</td>
			<td>49 mL</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>0.5 g</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>200 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: Fixative and Immunocytochemistry Recipes.

Watch this Scientific Journal Video about Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization at JoVE.com

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

small-scale-propagation-human-ipscs-serum-free-conditions-for-routine

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.9K Views.  The University of Arizona. Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

Video: Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...