Name: Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization (Video) | JoVE Journal
Uploaded: 2017-02-18T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Learn small-scale propagation of human iPSCs for routine immunocytochemical characterization using serum-free conditions. This cost-effective method enables efficient pluripotency assessment via specific antibody detection.

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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/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Disease-specific+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Successful+function+of+autologous+iPSC-derived+dopamine+neurons+following+transplantation+in+a+non-human+primate+model+of+Parkinson%27s+disease%5BTitle%5D)+AND+%22Cell+stem+cell%22%5BJournal%5D)">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/pubmed?term=((Clinical+Trials+in+a+Dish%3A+The+Potential+of+Pluripotent+Stem+Cells+to+Develop+Therapies+for+Neurodegenerative+Diseases%5BTitle%5D)+AND+%22Annu+rev+pharm+toxicol%22%5BJournal%5D)">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/pubmed?term=((Derivation+and+high+engraftment+of+patient-specific+cardiomyocyte+sheet+using+induced+pluripotent+stem+cells+generated+from+adult+cardiac+fibroblast%5BTitle%5D)+AND+%22Circ+heart+fail%22%5BJournal%5D)">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/pubmed?term=((Enhanced+generation+of+induced+pluripotent+stem+cells+from+a+subpopulation+of+human+fibroblasts%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Generation+and+Characterization+of+Patient-Specific+Induced+Pluripotent+Stem+Cell+for+Disease+Modeling%5BTitle%5D)+AND+%22Methods+mol+bio%22%5BJournal%5D)">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/pubmed?term=((Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Profiling+stem+cells+using+quantitative+PCR+protein+assays%5BTitle%5D)+AND+%22Methods+mol+bio%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+protein+marker+panel+for+characterization+of+human+induced+pluripotent+stem+cells+%28hiPSCs%29+using+global+quantitative+proteome+analysis%5BTitle%5D)+AND+%22Stem+cell+res%22%5BJournal%5D)">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/pubmed?term=((Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus%2C+an+RNA+virus+that+does+not+integrate+into+the+host+genome%5BTitle%5D)+AND+%22Proc+jpn+acad+ser+B+phys+biol+sci%22%5BJournal%5D)">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/pubmed?term=((Adaptation+of+human+embryonic+stem+cells+to+feeder-free+and+matrix-free+culture+conditions+directly+on+plastic+surfaces%5BTitle%5D)+AND+%22J+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging%5BTitle%5D)+AND+%22Methods+mol+bio%22%5BJournal%5D)">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 tec...

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><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&amp;amp;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>0.1 M PO&lt;sub&gt;4&lt;/sub&gt; Buffer</td><td>In-House</td><td>n/a</td><td></td></tr><tr><td>Paraformaldehyde, prill</td><td>Electron Microscopy Sciences</td><td>19202</td><td></td></tr><tr><td>1x Phosphate 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,&lt;br/&gt; Sox2 (D6D9) Rabbit mAb,&lt;br/&gt; SSEA4 (MC813) Mouse mAb,&lt;br/&gt; TRA-1-60(S) (TRA-1-60(S)) Mouse mAb</td><td>Cell Signaling&lt;br/&gt; Cell Signaling&lt;br/&gt; Cell Signaling&lt;br/&gt; Cell Signaling</td><td>2840&lt;br/&gt; 3579&lt;br/&gt; 4755&lt;br/&gt; 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><td></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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>12 545 82</td><td>Available in 12, 15, 18, 22 and 25 mm sizes</td></tr></tbody></table>

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

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 b...

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 ...

The overall goal of this culture method is to propagate human induced pluripotent stem cells, or human iPSCs, at a small scale to specifically enable their routine characterization through immunocytochemistry. This protocol from my laboratory presents a scale down technique to transition human induced pluripotent stem cells from underlying fetal layer to a substrate which is more suitable for antigen detection using specific pluripotency antibodies. The main advantage of this technique is that it provides a time and cost effective means of growing pluripotent cells in small chamber slides or on cover slips, which are ideal for immunocytochemistry.On the day of passaging, remove the matrix-coated plate from four degrees Celsius and allow the plate to acclimate to room temperature for one hour in the tissue culture hood. Next, aspirate the medium from one well of a six well plate of human iPSC culture growing on iMEFs and replace it with 500 microliters of collagenase dissociation reagent. Subsequently, incubate the cells at 37 degrees Celsius for 10 to 30 minutes until the edges of the iPSC colonies appear to lift slightly as observed under the microscope, then carefully aspirate th

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Research

JoVE Journal

Developmental Biology

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

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

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

Here we describe a protocol that facilitates the differentiation of human induced pluripotent stem cells into functional forebrain-specific astrocytes. This enables investigations into the role of glial cells in the pathogenesis of neurodevelopmental disorders, such as Fragile X Syndrome, and modeling of other brain disorders.

Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein

Learn small-scale propagation of human iPSCs for routine immunocytochemical characterization using serum-free conditions. This cost-effective method enables efficient pluripotency assessment via specific antibody detection.

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

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