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>

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	<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. 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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><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

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="iPSC culture process diagram showing transitional passage, immunofluorescence analysis, pluripotency." src="/files/ftp_upload/55260/55260fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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="Cell culture growth phases; electron microscope images; phases A-C; structural differentiation." src="/files/ftp_upload/55260/55260fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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 µm. <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="Immunofluorescence microscopy of stem cell markers: Oct-4, SSE4, Sox2; DAPI staining overlay." src="/files/ftp_upload/55260/55260fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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 µm. <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 µg/mL</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Y-27632 ROCK Inhibitor</td>
			<td></td>
			<td>10 µM</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 µL</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 µL</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 ...

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Nanyang Technological University

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Developmental Biology

7.0K 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

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

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

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.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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 ...

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

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

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 response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

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.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Bioengineering

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

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

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.

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Neuroscience

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Biology

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

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

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.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

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 ...

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

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