We thank Lars Moen for assistance during preliminary experiments and the Norwegian Core Facility for Human Pluripotent Stem Cells at the Norwegian Center for Stem Cell Research, Oslo University Hospital, for the use of&#160;facilities. The H9 hESC line was obtained from WiCell, and the HS429 hESC line was obtained from Outi Hovatta at the Karolinska Institute. Both were used in accordance with Material Transfer Agreements. The NCS001 and NCS002 hiPSC lines were generated by the Norwegian Core Facility for Human Pluripotent Stem Cells. That reprogramming and all the work reported here were performed with the approval of the Southeastern Norway Regional Ethics Committee (approval REK 2017/110).

See the Table of Materials for details about all the materials, reagents, and instruments used in this protocol.
1. Cultivation of human fibroblast cells and preparation of the feeder cell layer
<ol>
	<li>Seed 0.5 &#215; 106 human foreskin fibroblasts (hereafter called &#34;feeder cells&#34;) per each T-75 culture flask (number of flasks as needed) with 20 mL of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) with (w/) 10% fetal bovine serum (FBS), hereafter called &#34;feeder cell medium.&#34;</li>
	<li>When the feeder cells reach 90% confluence, remove the medium, and wash 3x with 10 mL of Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) per flask to avoid the inhibition of trypsin by factors in the medium. Add 2 mL of trypsin-EDTA to each flask, and place the flask(s) in a 37 &#176;C/5% CO2 incubator for 5 min or until the feeder cells have detached from the flask(s). Observe the detachment of the cells under a microscope as floating aggregates of cells or single cells.</li>
	<li>Add 5 mL of fresh pre-warmed feeder cell medium to each flask to inactivate the trypsin-EDTA, and gently suspend the feeder cells by pipetting.</li>
	<li>Transfer the feeder cells to a 15 mL centrifuge tube. Cap the tube, and pellet the feeder cells by centrifugation at 200 &#215; g for 5 min.</li>
	<li>Carefully remove the supernatant without disturbing the feeder cell pellet. Then, carefully resuspend the pellet in 4 mL of fresh feeder cell medium. Ensure that the feeder cells are thoroughly resuspended before counting using a cell counting chamber or other cell counting apparatus.</li>
	<li>Add 0.5 &#215; 106 feeder cells to the required number of new T-75 culture flasks for expansion, and add 20 mL of fresh feeder cell medium to each flask. Incubate the culture flask(s) in a 37 &#176;C/5% CO2 incubator until the feeder cells have reached 90% confluence. 
	NOTE: Feeder cells can be used up to at least passage 25.</li>
	<li>Calculate the number of feeder cells required for the number of 35 mm tissue culture dishes that will be used for culturing the hESCs/hiPSCs. 
	NOTE: Usually, 3.0 &#215; 105 feeder cells per tissue culture dish are sufficient to generate a confluent layer of feeder cells.</li>
	<li>To avoid the proliferation of the feeder cells, ensure that they are mitotically arrested in either of two ways. 
	NOTE: For both methods, a large batch of mitotically arrested feeder cells can be generated and frozen down in aliquots for later use.
	<ol>
		<li>Perform mitotic arrest by gamma-irradiation by transferring all the feeder cells needed to a 50 mL centrifuge tube and topping up with feeder cell medium to a total volume of 5 mL. Transport immediately at room temperature to a gamma-irradiation machine, and irradiate to mitotically arrest the feeder cells (300 kV and 10 mA for 20 min). 
		NOTE: A delay in transport can lead to undesired attachment of the feeder cells to the wall of the 50 mL centrifuge tube. If the transport requires more than a few minutes, ensure that the feeder cells remain suspended during transport by continuously agitating the tube.</li>
		<li>Perform mitotic arrest using mitomycin C by transferring all the feeder cells needed in 5 mL of feeder cell medium to a 50 mL centrifuge tube, and then add 15 mL of feeder cell medium containing 20 &#956;g/mL mitomycin C, and incubate in&#160; a 37 &#176;C/5% CO2 incubator for 3 h. Add 20 mL of 37 &#176;C PBS, pellet the cells by centrifugation at 200 &#215; g for 5 min, repeat the PBS wash two additional times, and resuspend in feeder cell medium.</li>
	</ol>
	</li>
	<li>After the feeder cells have been mitotically arrested, return to the tissue culture hood, and plate out the feeder cells at 3.0 &#215; 105 cells per 35 mm tissue culture dish, as follows. Ensure that the feeder cells are fully resuspended, add feeder cell medium to reach a feeder cell concentration of 1.5 &#215; 105 per mL, and add 2 mL of this feeder cell suspension to each 35 mm tissue culture dish.</li>
	<li>Transfer the culture dishes to a 37 &#176;C/5% CO2 incubator. To ensure an even distribution of the feeder cells, move the culture dishes slowly but firmly on the incubator shelf forward and backward 3x, followed by a pause, and then perform the same action from left to right 3x. Do not move the dishes again, and gently close the incubator door.</li>
	<li>After 24 h, switch from feeder cell medium to IMDM w/ 10% serum replacement (SR). Replace this medium thereafter every third day. The feeder cells are ready for use after the first 3 days.</li>
</ol>
2. Mechanical harvesting of the hESC or hiPSC colonies
<ol>
	<li>Prewarm hESC medium consisting of 80% Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), 20% SR, 1 mM glutamine substitute 100x, 1 mM non-essential amino acids (NEAA), 1 mM penicillin/streptomycin (P/S), 0.1 mM 2-mercaptoethanol, and 10 ng/mL basic fibroblast growth factor (bFGF). The hESC medium is used for the culture of either the hESCs or hiPSCs on the feeder cells.</li>
	<li>Take fresh 35 mm tissue culture dishes containing mitotically arrested feeder cells, and replace the feeder cell medium with 1.2 mL of hESC medium containing bFGF at least 30 min before the transfer of the hESC/hiPSC colonies.</li>
	<li>Place a culture dish containing hESC/hiPSC colonies on mitotically arrested feeder cells under a microscope with 10x magnification placed within a laminar flow hood. Use a sterile scalpel to cut carefully around the circumference of each colony and then cut each colony into 5-6 roughly equal pieces. Carefully lift up the colony pieces with the tip of the scalpel blade so that they detach from the feeder cell layer and float freely in the medium.
	<ol>
		<li>Try to avoid regions of the colonies that contain differentiating cells, which appear as islands of smaller cells with less distinct nuclei compared to hESCs/hiPSCs within a colony.</li>
	</ol>
	</li>
	<li>Transfer the freely floating colonies with a 1 mL pipette to the new culture dishes containing the feeder cells. Try to keep the colonies separate so that they do not grow into each other later. Move the culture dishes carefully to a cell incubator, and avoid disturbing the dishes until the next day.</li>
	<li>On the following day, carefully add 600 &#181;L of hESC medium containing bFGF to a final volume of 1.8 mL. Replace the hESC + bFGF medium each day thereafter until the next passage (generally after 1 week).</li>
</ol>
3. Harvesting of the hESC or hiPSC colonies using EDTA-mediated dis-adhesion
<ol>
	<li>Take fresh culture dishes with mitotically arrested feeder cells, and switch from IMDM w/ 10% SR to 1.2 mL of prewarmed hESC + bFGF medium at least 30 min before the transfer of the colonies.</li>
	<li>Handle one culture dish containing hESC or hiPSC colonies at a time. Remove the hESC + bFGF medium, and wash the colonies with 1 mL of room-temperature DPBS to eliminate any possible unattached cells and cell debris. Add 1 mL of 0.5 mM EDTA, and incubate for 1 min at 37 &#176;C. If the laminar flow hood has a warming plate, perform this step and the steps in section 4 on the warming plate for better dis-adhesion.</li>
	<li>After the 1 min incubation, remove the EDTA solution, and carefully add 1 mL of hESC + bFGF medium using a 1 mL pipette. Gently triturate with the same pipette to release the colonies from the feeder cell layer. Continue to triturate carefully until the feeder cell layer loosens and folds on itself in a separate clump. Pull away the feeder cell layer with the pipette tip.</li>
	<li>Transfer the suspended hESC/hiPSC colonies with a new 1 mL pipette to new culture dishes containing the feeder cells and hESC + bFGF medium, splitting at a ratio of 1:5. The colonies tend to distribute evenly within each new culture dish, but facilitate this by moving the dish gently from side to side. Replace the hESC + bFGF medium each day thereafter until the next passage (generally after 1 week).</li>
</ol>

<ol>
	<li>Skottman, H., Hovet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+human+embryonic+stem+cells.">Culture conditions for human embryonic stem cells.</a> Reproduction. 132 (5), 691-698 (2006).</li><li>Hovatta, O., 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=A+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Human Reproduction. 18 (7), 1404-1409 (2003).</li><li>Desai, N., Rambhia, P., Gishto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell+cultivation:+historical+perspective+and+evolution+of+xeno-free+culture+systems.">Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems.</a> Reproductive Biology and Endocrinology. 13, 9 (2015).</li><li>Villa-Diaz, L. G., 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=Synthetic+polymer+coatings+for+long-term+growth+of+human+embryonic+stem+cells.">Synthetic polymer coatings for long-term growth of human embryonic stem cells.</a> Nature Biotechnology. 28 (6), 581-583 (2010).</li><li>Watanabe, M., 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=TGFb+superfamily+signaling+regulates+the+state+of+human+stem+cell+pluripotency+and+capacity+to+create+well-structured+telencephalic+organoids.">TGFb superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.</a> Stem Cell Reports. 17 (10), 2220-2238 (2022).</li><li>Garitaonandia, I., 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=Increased+risk+of+genetic+and+epigenetic+instability+in+human+embryonic+stem+cells+associated+with+specific+culture+conditions.">Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.</a> PLoS One. 10 (2), e0118307 (2015).</li><li>Inzunza, 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=Derivation+of+human+embryonic+stem+cell+lines+in+serum+replacement+medium+using+postnatal+human+fibroblasts+as+feeder+cells.">Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells.</a> Stem Cells. 23, 544-549 (2005).</li><li>Watanabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>Rivera, T., Zhao, Y., Ni, Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cell+culture+methods+under+cGMP+conditions.">Human-induced pluripotent stem cell culture methods under cGMP conditions.</a> Current Protocols in Stem Cell Biology. 54 (1), 117 (2020).</li><li>Castro-Vi&#241;uelas, R., 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=Tips+and+tricks+for+successfully+culturing+and+adapting+human+induced+pluripotent+stem+cells.">Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells.</a> Molecular Therapy. Methods &amp; Clinical Development. 23, 569-581 (2021).</li><li>Meng, G., Rancourt, D. E. <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+maintenance+of+undifferentiated+human+embryonic+stem+cells.">Derivation and maintenance of undifferentiated human embryonic stem cells.</a> Methods in Molecular Biology. 873, 69-90 (2012).</li><li>Schindelin, 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li></ol>

Joel C. Glover is the director and Hege Brincker Fjerdingstad is the daily manager of the Norwegian Core Facility for Human Pluripotent Stem Cells. The authors have no competing financial interests or other conflicts of interest to disclose.

We have described a rapid and cost-efficient method for harvesting hESCs and hiPSCs&#160;cultured on feeder cells using EDTA-mediated dis-adhesion and compared this primarily to the conventional method of mechanical harvesting using a scalpel. We also compared EDTA-based harvesting to enzymatic harvesting with respect to the speed of the method but not aspects of the resultant colony quality. The reason for this is that enzymatic harvesting is inherently more variable and has been linked to a higher prevalence of genomic aberrations5, which could obscure the inter-method differences.
We demonstrate that&#160;EDTA-based harvesting is faster and more efficient than either of the other methods and generates smaller and morphologically more homogeneous colonies than mechanical harvesting. This latter feature is beneficial with respect to cell survival, since the larger clumps obtained with mechanical harvesting are prone to central necrosis, while enzymatic digestion tends to generate isolated hESCs and hiPSCs, which are more prone to apoptosis and require extra treatment, for example with ROCK inhibitors, to survive. EDTA-based harvesting can be used for at least 20 passages. The EDTA-based and mechanical harvesting methods are comparable when it comes to colony cell density, mRNA and protein expression of stemness genes, differentiation of the three germ layers in embryoid bodies, and genomic abnormalities. If the goal is efficiency, higher yield, less variability, and gentler handling of the hESCs and hiPSCs, EDTA-based harvesting is preferable.
We also note that EDTA-based harvesting of hESCs and hiPSCs cultured on feeder cells is an inexpensive way to maintain a more naive state and provides a smooth transition from feeder-based to feeder-free culture where this is desirable.
Critical steps within the protocol 
The most critical steps of EDTA-mediated dis-adhesion are protocol section 3 (incubation in the EDTA solution) and section 4 (trituration). If the exposure to the EDTA solution is longer than 1 min, the risk of complete dissociation to single cells increases. This can also occur if the trituration is too protracted or too harsh. The latter is affected by the pipette tip size. Using 1 mL cell culture pipettes as described here is ideal. Using a different type of pipette with a smaller tip diameter is risky.
Troubleshooting 
If the feeder cells continue to proliferate, mitotic arrest has not been effective, and a fresh batch must be taken and the procedure restarted. If the colonies do not loosen from the feeder cell layer, one must make sure there is no Ca2+ in the EDTA and that the culture dish containing the colonies is rinsed well with PBS to remove any remaining cell culture medium before adding the EDTA. Too much dissociation, which generates isolated cells or cell clumps that are too small, can arise due to excessive trituration and compromises the establishment of new colonies. The degree of trituration should be determined empirically in trial runs of the protocol to confirm&#160;that the resultant cell clumps are ~60 &#181;m in diameter. If the feeder layer detaches spontaneously from the culture dish, especially before the hESCs/hiPSCs are ready for harvesting, it could be because the feeder cells have not been used within ~7 days after being prepared. Therefore, the time frame of use of the feeder cells should be monitored carefully. If the feeder layer dissociates during EDTA exposure (something we have never observed with the feeder cells used here), either the type of feeder cell or their culture method must be changed.
Limitations of the technique 
The main limitation of the technique is that it requires visual inspection of the dis-adhesion process to achieve a successful result. This means that users must learn how to identify when the colonies release from the feeder cell layer and the feeder cell layer loosens from the substrate. However, this is not difficult, and in our experience, new users of the technique can master it within a couple of trials.
There is also an inherent possibility that the harvested hESCs or hiPSCs may be contaminated by a few feeder cells. If the intention is to transfer to non-feeder conditions or to isolate the hESCs or hiPSCs for assays, such contamination would compromise&#160;purity. We note that with the feeder cells used here (human foreskin fibroblasts), it is extremely difficult to dissociate the feeder cell layer, even with enzymatic digestion (not shown). Since the undissociated feeder cell layer is removed in toto,&#160;contamination of the harvested hESCs or hiPSCs is likely to be negligible. As the feeder cells are, moreover, mitotically arrested, any contamination would eventually diminish to nil with further passaging of the hESCs or hiPSCs.
Significance with respect to existing methods 
The current norm for culturing hESCs and hiPSCs is to do so under feeder-free conditions, for which the use of EDTA for passaging is widespread. Feeder-free culture depends on the use of specially formulated media and culture substrates that ensure adherence. These reagents entail an additional expense that may exceed some laboratory budgets. In addition, culture under feeder-free conditions has been associated with a perturbed differentiation potential due to the lack of specific factors in the feeder-free culture media and a resultant transition from the naive state to the primed state. Growth on mitotically arrested feeder cells avoids this transition and can bring overall costs down to a manageable level, thus facilitating the broader use of pluripotent stem cells in laboratory research.

The proper maintenance of hESCs and hiPSCs in vitro is a basic and convenient methodology for several avenues of research in human cell and developmental biology. Due to the inherent drive of hESCs and hiPSCs to differentiate, maintaining the undifferentiated state in vitro demands particular care and attention. Thus, developing cost-efficient protocols for the maintenance and passaging of hESCs and hiPSCs with as little methodological variability as possible is of great general utility.
Originally, hESCs and hiPSCs were cultured on different types of feeder cells to assist in the long-term culture and maintenance of the undifferentiated state1,2,3. More recently, culture under feeder-free conditions has become the norm, as it avoids dealing with feeder cells altogether4. However, some laboratories and core facilities still culture hESCs or hiPSCs on feeder cells. Feeder-free culture is more expensive because it requires the use of culture media of special compositions and some form of coating of the culture surface to ensure colony adherence (major extracellular matrix [ECM] components or a commercial ECM compound, or using commercially available coated plates). The expense is not trivial and presents a potential financial hindrance for some laboratories interested in pursuing hESC- or hiPSC-based research and development. Moreover, culture under feeder-free conditions tends to drive the hESCs and hiPSCs to a less naive state than is maintained on feeder cells5, and this can compromise&#160;subsequent differentiation and lead to genetic variations6.
Historically, the passaging of hESCs and hiPSCs cultured on feeder cells involved mechanical harvesting - using a scalpel to excise colonies under a microscope7&#160;- but this was later largely supplanted by enzymatic digestion with or without gentle scraping to isolate colonies or dissociated cells. Mechanical harvesting is tedious and requires precision microsurgery. Enzymatic harvesting can vary in efficiency due to batch-to-batch enzyme differences and tends to favor complete dissociation, which promotes cell death unless counteracted by ROCK inhibitors8,9 and increases the incidence of abnormal karyotypes9.
To take advantage of the lower expense and greater differentiation potential of culturing hESCs and hiPSCs on feeder cells while avoiding the disadvantages of mechanical and enzymatic harvesting, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC and hiPSC colonies maintained on a feeder layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion. We have compared the yield, variability, and stem cell quality to that obtained with mechanical harvesting (we did not compare to enzymatic digestion because of the additional variability this approach entails). We note that EDTA-mediated dis-adhesion also works well for transferring colonies from feeder-based culture to feeder-free conditions, if desired for downstream use and analyses. This method provides a transition with a consistent passaging method, since EDTA-induced dis-adhesion is a popular approach employed for feeder-free cultures.

<table><tbody><tr><td>0.5 M EDTA pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>31350-010</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>PeproTech</td><td>AF-100-18B-250UG</td><td></td></tr><tr><td>Brand B&amp;uuml;rker Chamber</td><td>Fisher Scientific</td><td>10628431</td><td></td></tr><tr><td>Disposable scalpels no.15</td><td>Susann-Morton</td><td>505</td><td></td></tr><tr><td>DPBS (1x) without Ca/Mg</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Easy Grip tissue culture dish, 35 x 10 mm</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Eppendorf pipette 1 mL</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Eppendorf pipette 200 &amp;mu;L</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Filter tip 1,000 &amp;mu;L</td><td>Sarstedt</td><td>70.1186.210</td><td></td></tr><tr><td>Filter tip 200 &amp;mu;L</td><td>Sarstedt</td><td>70.760.211</td><td></td></tr><tr><td>Gamma Cell 3000 ELAN irradiation machine (alternatively, use Mitomycin C to arrest proliferation)</td><td>Best Theratronics</td><td>BT/MTS 8007 GC3000E</td><td></td></tr><tr><td>Glutamax 100x</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Growth Factor Reduced Matrixgel</td><td>Corning</td><td>734-0269</td><td></td></tr><tr><td>H9 hESC line</td><td>WiCell</td><td>WAe009-A</td><td></td></tr><tr><td>hPSC Genetic Analysis Kit</td><td>Stem Cell Technologies</td><td>#07550</td><td></td></tr><tr><td>HS429 hESC line&amp;nbsp;</td><td>ECACC</td><td>KIe024-A</td><td></td></tr><tr><td>Human Foreskin Fibroblasts -CRL2429 line</td><td>ATTC</td><td>CRL2429</td><td></td></tr><tr><td>IMDM (1x)</td><td>Gibco</td><td>21980-032</td><td></td></tr><tr><td>iPSC lines</td><td>Norwegian Core Facility for Human Pluripotent Stem Cells</td><td>NCS001 &amp;amp; NCS002</td><td></td></tr><tr><td>Knockout DMEM</td><td>Gibco</td><td>10829-018</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope or equivalent (we use the LSM 700 from Zeiss)</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Microscope</td><td>CETI</td><td></td><td></td></tr><tr><td>Mitomycin C</td><td>Sigma Aldrich</td><td>M4287</td><td></td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Gibco</td><td>11140.035</td><td></td></tr><tr><td>Pipettes, plastic 10 mL</td><td>Sarstedt</td><td>86.1254.001</td><td></td></tr><tr><td>Pipettes, plastic, 5 mL</td><td>Sarstedt</td><td>86.1253.001</td><td></td></tr><tr><td>Serum Replacement (SR)</td><td>Gibco</td><td>10828-028</td><td></td></tr><tr><td>Sterile filters 0.22 um</td><td>Sarstedt</td><td>83.1826.102</td><td></td></tr><tr><td>T-75 culture flask</td><td>ThermoScientific</td><td>156499</td><td></td></tr><tr><td>Trypan Blue Stain (0.4 %)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Trypsin-EDTA, 500 mL</td><td>Gibco</td><td>25300062</td><td></td></tr></tbody></table>

rapid, cost-efficient, enzyme-free passaging of human pluripotent stem cells on feeder cells by ethylenediaminetetraacetic acid-mediated dis-adhesion

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells&#39; differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures.
To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting).
In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for&#160;colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

In the assays and comparisons documented below, we used two hESC lines (H9 and HS429, from WiCell and the Karolinska Institute, respectively) and two hiPSC lines (NCS001 and NCS002, both generated by the Norwegian Core Facility for Human Pluripotent Stem Cells). The data presented in the figures and tables are from the hESC lines, but entirely similar results were obtained from the hiPSC lines.
In our hands, mechanical harvesting resulted in the colonies being split into approximately five to six clumps of ~200-250 &#181;m in diameter, whereas with EDTA-induced dis-adhesion followed by trituration, each colony was split into ~10-20 clumps of ~60 &#181;m in diameter. We estimate that the number of cells in each EDTA-harvested clump is ~20. As it is impractical to split a colony into clumps of this size with a scalpel, in this respect, EDTA-induced dis-adhesion is superior, as it generates clumps of a size that is more favorable for colony cell survival10,11.
The hESC/hiPSC colonies harvested using EDTA were also more homogeneous in size and shape compared to the colonies harvested mechanically (Figure 1A-F). This is because the cutting required for mechanical harvesting generates uneven edges and varying clump sizes. To evaluate this quantitatively, we assessed the colony circularity (as a measure of how rounded the colony edges were; a value of 1 indicates a perfect circle) 5 days after passaging using the ImageJ-win64 protocol12. The colony circularity was significantly lower in the colonies harvested mechanically (mechanical harvesting: 0.61 &#177; 0.10; EDTA-based harvesting: 0.84 &#177; 0.01; n = 10, p &#60; 0.001, Mann-Whitney U-test, U = 10).
The cell density in the harvested and replated colonies, which is a measure of post-harvesting cell-cell interactions during colony formation, was similar with EDTA-based harvesting and mechanical harvesting (Table 1 and Figure 1G,H). The mechanically harvested colonies had a greater tendency to develop necrosis in their central regions (Figure 1J). This was likely due to variability in the shape and, particularly, the size of the mechanically isolated cell clumps, as when these clumps are too large, they can easily fold upon themselves when being transferred to new culture dishes. This was not the case with the colonies harvested using EDTA, which uniformly exhibited a translucent appearance with distinct edges (Figure 1I).
Using EDTA-based harvesting, we were able to collect essentially all the colonies that had been established in a well within 2-3 min. Using mechanical harvesting, collecting all the colonies in a well would be tedious and time-consuming. We typically managed to collect only ~30%, or ~20-25 colonies, using mechanical harvesting, and this took ~20 min. Likewise, using collagenase digestion followed by gentle scraping, it was typically difficult to harvest all the colonies, although the total procedure only took a few minutes. Thus, EDTA-based harvesting is as fast or faster than enzymatic harvesting and more efficient than either mechanical or enzymatic harvesting.
To assess the effect of the different harvesting methods on stemness and pluripotency, we first subjected the colonies obtained after 20 passages using EDTA-based or mechanical harvesting to qPCR analysis (Figure 2) and immunocytochemical staining (Figure 3 and Figure 4) for stemness markers. The colonies obtained using either method exhibited a stable expression of stemness markers both at the mRNA and protein levels. We then assessed pluripotency by differentiation to the three germ layers in embryoid bodies (Figure 5 and Figure 6). The embryoid bodies generated from the hESCs or hiPSCs obtained after 20 passages using either method contained a mixture of cells expressing commonly assessed markers for ectoderm, mesoderm, and endoderm.
Finally, we assessed the incidence of genomic aberrations in hESCs and hiPSCs passaged by each method using qPCR-based genetic analysis (see the Table of Materials). The colonies obtained after 20 passages using either harvesting method exhibited some examples of modest deviation from a reference diploid chromosomal pattern (the abnormalities assessed were those commonly associated with the reprogramming of hiPSCs but can also be obtained in hESCs) (Figure 7). However, the pattern of these deviations was essentially the same in the colonies obtained after either harvesting method, indicating that they were not linked to the harvesting method.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63788/63788fig01.jpg" /> 
Figure 1: Colony morphology and cell density following EDTA-based or mechanical harvesting. (A-F) Representative brightfield images of H9 hESC colonies established in feeder-free culture for 5 days after 20 passages using (A-C) EDTA-based or (D-F) mechanical harvesting. (G,H) Representative fluorescence images of the cell density in H9 hESC colonies established after 20 passages using (G) EDTA-based or (H) mechanical harvesting. The cell nuclei are stained with DAPI. (I,J) Representative brightfield images of H9 hESC colonies established after 20 passages using (I) EDTA-based or (J) mechanical harvesting. Note the necrotic central region in the colony harvested mechanically (arrow in J). All the images were acquired 5 days following the 20th passage. Scale bars = 100 &#181;m. Abbreviations: hESC = human embryonic stem cell; EDTA = ethylenediaminetetraacetic acid; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63788/63788fig02.jpg" /> 
Figure 2: Expression of stemness marker mRNA in two hESC lines (H9 and HS429) generated after EDTA-based or mechanical harvesting.&#160;Quantitative real-time polymerase chain reaction of the indicated markers in H9 (upper panel) and HS429 (lower panel) hESCs after a single passage using mechanical harvesting, after 20 passages using mechanical harvesting, and after 20 passages using EDTA-based harvesting (1:5 dilution). The expression level is relative to that of the housekeeping gene ACTB (beta-actin). The error bars indicate the standard deviation. Abbreviations: hESC = human embryonic stem cell; EDTA = ethylenediaminetetraacetic acid. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63788/63788fig03.jpg" /> 
Figure 3: Expression of stemness marker proteins in the H9 hESC line after different harvesting conditions.&#160;Representative immunofluorescence staining of H9 hESC colonies harvested mechanically (A-E) before further passaging, (F-J) after 20 passages using mechanical harvesting, and (K-O) after 20 passages using EDTA-based harvesting. Scale bars = 100 &#181;m. Abbreviations: hESC = human embryonic stem cell; EDTA = ethylenediaminetetraacetic acid; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63788/63788fig04.jpg" /> 
Figure 4: Expression of stemness marker proteins in the HS429 hESC line after different harvesting conditions.&#160;Representative immunofluorescence staining of HS429 hESC colonies harvested mechanically (A-E) before further passaging, (F-J) after 20 passages using mechanical harvesting, and (K-O) after 20 passages using EDTA-based harvesting. Scale bars = 100 &#181;m. Abbreviations: hESC = human embryonic stem cell; EDTA = ethylenediaminetetraacetic acid; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63788/63788fig05.jpg" /> 
Figure 5: Expression of markers for the three germ layers in embryoid bodies generated from the H9 hESC line following mechanical or EDTA-based harvesting.&#160;Representative immunofluorescence staining of markers for (A and D rows) ectoderm (ECTO, TUJI), (B and E rows) endoderm (ENDO, AFP), and (C and F rows) mesoderm (MESO, SMA). EBs generated (A-C) after 20 passages of mechanical harvesting or (D-F) after 20 passages of EDTA-based harvesting. Scale bars = 40 &#181;m. Abbreviations: hESC = human embryonic stem cell; EDTA = ethylenediaminetetraacetic acid; EBs = embryoid bodies. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63788/63788fig06.jpg" /> 
Figure 6: Expression of markers for the three germ layers in embryoid bodies generated from the HS429 hESC line following mechanical or EDTA-based harvesting.&#160;Representative immunofluorescence staining of markers for (A and D rows) ectoderm (ECTO, TUJI), (B and E rows) endoderm (ENDO, AFP), and (C and F rows) mesoderm (MESO, SMA). EBs generated (A-C) after 20 passages of mechanical harvesting (A-C) or (D-F) after 20 passages of EDTA-based harvesting. Scale bars = 40 &#181;m. Abbreviations: hESC = human embryonic stem cell; EDTA = ethylenediaminetetraacetic acid; EBs = embryoid bodies. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63788/63788fig07.jpg" /> 
Figure 7: qPCR-based genetic analysis of common genomic aberrations in the HS9 and HS429 ESC lines and the NCS002 iPSC line following 20 passages using mechanical or EDTA-based harvesting. The baseline at value 2 represents normal diploidy at all the chromosomal markers. A value of 1 or 3 would represent a loss or gain, respectively, of the indicated chromosomal marker in all the cells. Intermediate values between 1 and 2 or between 2 and 3 indicate the presence of a loss or gain of the indicated marker in a fraction of the cells. Note that the pattern of aberrations is similar in the two harvesting conditions. Abbreviations: ESC = embryonic stem cell; EDTA = ethylenediaminetetraacetic acid; iPSC = induced pluripotent stem cell. <a href="https://www.jove.com/files/ftp_upload/63788/63788fig07large.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>Cell density (cells/mm2)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H9</td>
			<td>mean</td>
			<td>stdev</td>
		</tr>
		<tr>
			<td>Mechanical harvest before further passaging</td>
			<td>3918</td>
			<td>263.3</td>
		</tr>
		<tr>
			<td>Mechanical harvest 20 times</td>
			<td>3868</td>
			<td>197.7</td>
		</tr>
		<tr>
			<td>EDTA harvest 20 times</td>
			<td>4080</td>
			<td>127.8</td>
		</tr>
		<tr>
			<td>HS429</td>
			<td>mean</td>
			<td>stdev</td>
		</tr>
		<tr>
			<td>Mechanical harvest before further passaging</td>
			<td>5249</td>
			<td>565.4</td>
		</tr>
		<tr>
			<td>Mechanical harvest 20 times</td>
			<td>5247</td>
			<td>726.3</td>
		</tr>
		<tr>
			<td>EDTA harvest 20 times</td>
			<td>4963</td>
			<td>448.8</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of the cell densities in the colonies from the two hESC lines (H9 and HS429) generated after EDTA-based or mechanical harvesting. The cell densities were assessed either after a single passage using mechanical harvesting, after 20 passages using mechanical harvesting, or after 20 passages using EDTA-based harvesting (at 1:5 dilution). In all cases, n = 5 colonies.

Watch this Scientific Journal Video about Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion at JoVE.

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

To avoid the limitations associated with the enzymatic or mechanical passaging of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) cultured on feeder cells, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC or hiPSC colonies maintained on a feeder cell layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different ...

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Research

JoVE Journal

Biology

555 Views.  Oslo University Hospital. Dit protocol biedt een snelle, kostenefficiënte methode voor enzymvrije passaging van menselijke pluripotente stamcellen die op voedingscellen zijn gekweekt. Het voordeel van dit protocol is dat er geen dure voedingsvrije kweekmedia en groeisubstraten nodig zijn, het risico van volledige dissociatie door enzymen wordt vermeden en het risico van stamcellen die overgaan naar een geprimed toestand wordt verminderd. De procedure zal worden gedemonstreerd door Hege Brincker Fjerdingstad, dagelijk...

Video: Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Developmental Biology

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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 ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent epiblast cells by orchestrated exposures to cytokines, including bone morphogenetic protein 4 (Bmp4). In human embryos, the earliest PGCs have been identified on the endodermal wall of yolk sac around the end of the 3rd week of gestation, but little is known about the process of human PGC specification and their early development. To circumvent the technical and ethical barriers of studying human embryonic PGCs, surrogate cell culture models have been recently generated from pluripotent stem cells. Here, we describe a 13-day protocol for robust production of human PGC-Like Cells (hPGCLCs). Human induced pluripotent stem cells (hiPSCs) maintained in the primed pluripotency state are incubated in the 4i na&#239;ve reprogramming medium for 48 hours, dissociated to single cells, and packed into microwells. Prolonged maintenance of hiPSCs in the na&#239;ve pluripotency state causes significant chromosomal aberrations and should be avoided. hiPSCs in the microwells are maintained for an additional 24 hours in the 4i medium to form embryoid bodies (EBs), which are then cultured in low-adherence plasticware under a rocking condition in the hPGCLC induction medium containing a high concentration of recombinant human BMP4. EBs are further cultured for up to 8 days in the rocking, non-adherent condition to obtain maximum yields of hPGCLCs. By immunohistochemistry, hPGCLCs are readily detected as cells strongly expressing OCT4 in almost all EBs exclusively on their surface. When EBs are enzymatically dissociated and subjected to FACS enrichment, hPGCLCs can be collected as CD38+ cells with up to 40-45% yield.

Primordial germ cells (PGCs) are common precursors of both sperm and eggs. Human embryonic PGCs are specified from pluripotent epiblast cells through interactions of cytokines. Here, we describe a 13-day protocol of inducing human cells transcriptomally resembling PGCs at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells.

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Medicine

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

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