This study was supported by a grant from the New Business Promotion Center, Panasonic Production Engineering Co., Ltd., Osaka, Japan.

The Ethics Committee of the Kansai Medical University approved the generation and use of the healthy volunteer-derived iPSCs named KMUR001 (approval No. 2020197). The donor, who was openly recruited, provided formal informed consent and agreed with the scientific usage of the cells.
NOTE: The current interface (the special software named &#34;ccssHMI&#34; running under the Windows XP operating system) is the fundamental operation screen. Under the aforementioned interface, a series of tabs are arranged, allowing users to initiate various operations.
1. Loading operations
<ol>
	<li>Click the Loading button on the software&#39;s top screen. Click the Loading Preparation Start button.</li>
	<li>Place the dish(es) or plate(s) to be entered into the apparatus in position in the apparatus. 
	NOTE: Necessary information for dish identification should be written on each lid.</li>
	<li>Manually close the front sliding window and push the mechanical safety-confirmation button.</li>
	<li>Select the type and quantity of dish(es) or plate(s) in the software.</li>
	<li>Click the Loading Preparation Completed button. Click the Loading Start button.</li>
	<li>After a dish is uploaded into the system, select the information on the dish, such as with iPS cells or without iPS cells, on the software. Register notes about each dish in the software.</li>
	<li>Click the Registration button at the end to complete the loading operation.</li>
</ol>
2. Unloading operation
<ol>
	<li>Click the Unload button on the software&#39;s top screen. Select the dish(es) to be removed in the software.</li>
	<li>After selecting the dish(es), click the Unloading Preparation Start button. Click the Unloading Start button.</li>
	<li>After the dish(es) have been transferred from the incubator to the workbench in the system, press the Dish Removal button.</li>
	<li>Manually open the front sliding window and take out the dish(es). Manually close the front sliding window and push the mechanical safety-confirmation button.</li>
</ol>
3. Supplement of consumables: pipettes, tubes, and medium
<ol>
	<li>To replenish consumables such as pipettes, tubes, and medium, click the Consumables button on the software&#39;s top screen, then select the item to be replenished.
	<ol>
		<li>Pipettes
		<ol>
			<li>Click the Pipette button. Select the Replenish button.</li>
			<li>Select a rack that the user wants to replenish on the software. Click the Replenish Start button.</li>
			<li>After confirming that the lid of the pipette storage area on the workbench is opened, manually open the front sliding window and replenish the pipettes as needed.</li>
			<li>Manually close the front sliding window and push the mechanical safety-confirmation button. Click the Replenish Completed button.</li>
			<li>Click the Replenishment Setup button and enter the information for the replenishment, then click the Registration button.</li>
			<li>Click the Replenish Completed button.</li>
		</ol>
		</li>
		<li>Tubes
		<ol>
			<li>Click the Tube button. Select the Replenish button.</li>
			<li>Select a rack that the user wants to replenish on the software. Click the Replenish Start button.</li>
			<li>After confirming that the rack has moved to the top, click the Replenish Tube button.</li>
			<li>Manually open the front sliding window and replenish the tubes as needed.</li>
			<li>Manually close the front sliding window and push the mechanical safety-confirmation button.</li>
			<li>Click the Replenish Completed button.</li>
			<li>Click the Replenishment Setup button and enter the information for the replenishment, then click the Registration button. Click the Close button.</li>
		</ol>
		</li>
		<li>Medium
		<ol>
			<li>Click the Medium button. Select the Replenish button in the software.</li>
			<li>Select one rack from three that users want to replenish. Click the Replenish Start button.</li>
			<li>After confirming that the lid of the medium storage area has been opened, manually open the front sliding window and replenish the medium.</li>
			<li>Manually close the front sliding window and push the mechanical safety-confirmation button.</li>
			<li>Click the Replenish Completed button.</li>
			<li>Click the Replenish button and enter the information for the medium, including the name and the amount of the medium. Enter additional comments if necessary.</li>
			<li>Click the Registration button.</li>
			<li>Click the Close button.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Task selection
<ol>
	<li>Click the Task button on the software&#39;s top screen.</li>
	<li>Select the Task Setting button. Select the desired task from the task list and click the Next Step button.</li>
	<li>Specify the date and time to perform the task, and then click the Registration button. Select a dish or plate to perform the task, and then click the Registration button.</li>
	<li>After reconfirming the selected task, click the Registration button. Confirm that the task has been registered on the next screen.</li>
	<li>If necessary, set the next task in the same way.</li>
	<li>Click the Start button at the end. Then, the task will start automatically at the specified date and time. 
	NOTE: Immediately after finishing every task, UV lights (located on two sides of the hood) automatically turn on, and after 5-30 min, in accordance with the auxiliary setting, they are turned off to keep the hood in aseptic condition. To stop, users can click the Start button.</li>
	<li>If users want to cancel a scheduled task in advance, click the Stop button.</li>
	<li>After selecting the task to be aborted, click the Edit Task button.</li>
	<li>Click the Task Cancel button. Confirm that the task has been deleted on the next screen.</li>
</ol>
5. Check cell pictures
<ol>
	<li>Every task includes microscopic observations(taking photos) before and after the task work. Observe the progress of cell culture photographically by incorporating a microscopic photography task before or after each task.</li>
	<li>Select pre-set task programs, including selecting multiple specific locations of the dish or the well plate in advance for fixed-point observation to monitor the same location over time.</li>
</ol>
6. Passaging and differentiation
<ol>
	<li>Follow the steps in sections 1-5 and set the automated cell culture system to perform passaging and differentiation. The instrument settings for passaging, cardiomyocyte differentiation, hepatocyte differentiation, neural precursor cell differentiation, and keratinocyte differentiation are shown in Table 1, Table 2, Table 3, Table 4, and Table 5, respectively.</li>
</ol>

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

<ol>
	<li>Okita, 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+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8 (5), 409-412 (2011).</li><li>Tanaka, T., 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=In+vitro+pharmacologic+testing+using+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes.</a> Biochemical and Biophysical Research Communications. 385 (4), 497-502 (2009).</li><li>Egashira, T., 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+characterization+using+LQTS-specific+induced+pluripotent+stem+cells.">Disease characterization using LQTS-specific induced pluripotent stem cells.</a> Cardiovascular Research. 95 (4), 419-429 (2012).</li><li>Sasamata, 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=Establishment+of+a+robust+platform+for+induced+pluripotent+stem+cell+research+using+Maholo+LabDroid.">Establishment of a robust platform for induced pluripotent stem cell research using Maholo LabDroid.</a> SLAS technology. 26 (5), 441-453 (2021).</li><li>Tristan, C. A., 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=Robotic+high-throughput+biomanufacturing+and+functional+differentiation+of+human+pluripotent+stem+cells.">Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells.</a> Stem Cell Reports. 16 (12), 3076-3092 (2021).</li><li>Konagaya, S., Ando, T., Yamauchi, T., Suemori, H., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+human+induced+pluripotent+stem+cells+by+automated+cell+culture+system.">Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.</a> Scientific Reports. 5, 16647 (2015).</li><li>McClymont, D. W., Freemont, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=With+all+due+respect+to+Maholo,+lab+automation+isn't+anthropomorphic.">With all due respect to Maholo, lab automation isn't anthropomorphic.</a> Nature Biotechnology. 35 (4), 312-314 (2017).</li><li>Gonzalez, F., Zalewski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+joint-level+robot+programming+with+a+new+robotics+software+tool.">Teaching joint-level robot programming with a new robotics software tool.</a> Robotics. 6 (4), 41 (2017).</li><li>Bando, K., Yamashita, H., Tsumori, M., Minoura, H., Okumura, K., Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+automated+culture+system+for+human+induced+pluripotent+stem+cell+maintenance+and+differentiation.">Compact automated culture system for human induced pluripotent stem cell maintenance and differentiation.</a> Frontiers in Bioengineering and Biotechnology. 10, 1074990 (2022).</li><li>Tohyama, 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=Glutamine+oxidation+is+indispensable+for+survival+of+human+pluripotent+stem+cells.">Glutamine oxidation is indispensable for survival of human pluripotent stem cells.</a> Cell Metabolism. 23 (4), 663-674 (2016).</li><li>Yamashita, H., Fukuda, K., Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte-like+cells+derived+from+human+pluripotent+stem+cells+can+be+enriched+by+a+combination+of+mitochondrial+content+and+activated+leukocyte+cell+adhesion+molecule.">Hepatocyte-like cells derived from human pluripotent stem cells can be enriched by a combination of mitochondrial content and activated leukocyte cell adhesion molecule.</a> JMA journal. 2 (2), 174-183 (2019).</li><li>Shimojo, D., 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=Rapid,+efficient+and+simple+motor+neuron+differentiation+from+human+pluripotent+stem+cells.">Rapid, efficient and simple motor neuron differentiation from human pluripotent stem cells.</a> Molecular Brain. 8 (1), 79 (2015).</li><li>Nishimoto, R., Kodama, C., Yamashita, H., Hattori, F. <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-derived+keratinocyte-like+cells+for+research+on+Protease-Activated+Receptor+2+in+nonhistaminergic+cascades+of+atopic+dermatitis.">Human induced pluripotent stem cell-derived keratinocyte-like cells for research on Protease-Activated Receptor 2 in nonhistaminergic cascades of atopic dermatitis.</a> The Journal of Pharmacology and Experimental Therapeutics. 384 (2), 248-253 (2023).</li><li>International Stem Cell Initiative. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+ethnically+diverse+human+embryonic+stem+cells+identifies+a+chromosome+20+minimal+amplicon+conferring+growth+advantage.">Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.</a> Nature Biotechnology. 29 (12), 1132-1144 (2011).</li><li>Keller, A., 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=Genetic+and+epigenetic+factors+which+modulate+differentiation+propensity+in+human+pluripotent+stem+cells.">Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells.</a> Human Reproduction Update. 24 (2), 162-175 (2018).</li></ol>

The author has nothing to disclose.

A critical step in the protocol is that if a user finds any faults, click the cancel, stop, or reset button anytime and start over from the first step. The software can avoid human mistakes, including double booking, opening doors while the system tasks are active, and a lack of replenishment. Another critical point for successful and efficient differentiation to the desired somatic cell is the proper selection of pluripotent stem cell lines because each pluripotent stem cell has an uncontrollable bias in its differentiation properties14,15.
This automated culture system for induced pluripotent stem cells can maintain and differentiate them into various somatic cell types. The size of this system is 200 cm (width), 110 cm (depth), and 233 cm (height), with a total weight of 1.2 tons. The incubator built into the device can simultaneously hold thirty-six 10-cm dishes and nine multiwell plates.
Improvements over previous models are that first, as a handling arm, the X-Y-Z-axis rail system was newly converted from a 6-axis articulated arm, which works by changing the tip tool of the arm to three different parts for dishes, tubes, and pipettes. The arm is suspended from the ceiling of the system and moves along the X-Y-Z axis. Second, a cell-counting system was introduced for feeder-free maintenance cultures and differentiation induction protocols that are performed continuously from the passage. After mixing the single-cell suspension solution with the trypan blue solution, it was transferred to a disposable hemocytometer for microscopic observation and imaging. The software automatically analyzes the obtained image to count the number of live cells and calculate the volume required to seed the required number of cells. The accuracy of cell counts obtained by this image analysis was consistent with that obtained manually.
The X-Y-Z-axes rail system was applied to handle each task quicker than the previous version (multi-axes articulated arm). With this automated culture system, five continuous passages were performed for 16 days, and iPSCs were expanded 625 &plusmn; 93 fold. Although the proof of endurance for the time of cell maintenance without feeder cells was shorter than our previous demonstration with feeder cells, the cell expansion was more efficient compared with the previous demonstration6. The shortened task time seemed to contribute to preventing cell damage from drying during the tasks. 
Furthermore, differentiation tasks from maintenance culture to differentiation into cardiomyocytes, hepatocytes, neuronal precursor cells, and keratinocytes were performed without human intervention. After a series of these courses, it was confirmed by fluorescence immunostaining that the cells obtained in the automated culture system alone had differentiated into the desired cells. Therefore, by sharing a task program and using this system, researchers who are not familiar with iPS cell management can obtain iPSC-derived somatic cells for their own research. Moreover, differences in reproducibility between researchers or facilities could be eliminated as much as possible, and it could assure that cells of homogeneous quality are obtained every time.
By adopting a new rail system, the equipment could be downsized and reduced in weight to 1.2 tons, ~200 kg lighter than before. Furthermore, parts and program setting costs could be reduced by approximately $10,000 US dollars. The maintenance and programming costs were estimated to decline by 13%. In order to motivate more researchers to enter iPS cell-related research easily, it is also important to keep the equipment costs low6. We hope that current and next-generation automatic culture systems will be one of the contributions to helping provide iPSCs and iPSC-derived differentiated cells of more uniform quality. 
This system's major limitations are the refrigerator's limited capacity for tentative storage of cultures and its weakness in handling small amounts (&lt;100 &#181;L) of liquid. Furthermore, the lack of sophisticated artificial intelligence disables automatic optimization according to the on-time cell condition. The minor limitation is the requirement of special pipette tips provided by the system manufacturers4,5. We are developing the next generation of culture systems that will be able to handle small amounts of liquid for medium preparations and include a feedback optimization system based on high-performance artificial intelligence (system learning). Our collaborating manufacturer9 has enough know-how to build custom-made systems according to the users' special needs. In addition to this, next-generation systems equipped with more general and wider functions will be on the market soon. We are also considering a subscription-based rental system to supply up-to-date systems to all users.

This article aims to provide actual and detailed handling procedures for an automated culture system for human induced pluripotent stem cells (iPSC), which we produced by collaborating with a company, and to show representative results.
Since the publication of the article in 2007, iPSC has been attracting attention all over the world1. Due to its greatest feature of being able to differentiate into any type of somatic cell, it is expected to be applied in various fields such as regenerative medicine, elucidating the causes of intractable diseases, and developing new therapeutic drugs2,3. In addition, using human iPSC-derived somatic cells could reduce animal experiments, which are subject to significant ethical restrictions. Although numerous homogeneous iPSCs are constantly required to research new methods with iPSCs, it is too laborious to manage them. Moreover, handling iPSC is difficult because of its high sensitivity, even to subtle cultural and environmental changes.
To solve this problem, automated culture systems are expected to perform tasks instead of humans. Some groups have developed a few automated human pluripotent stem cell culture systems for cell maintenance and differentiation and published their achievements4,5,6. These systems equip multiarticulated robotic arm(s). Robotic arms have not only merit in that they highly mimic human arm movements but also demerit in that they require higher cost(s) for the arm(s), larger and heavier system packaging, and time-consuming education efforts by the engineers to obtain the aimed movements7,8. In order to make it easier to introduce the apparatus to more research facilities at the points of economic, space, and human resource consumption, we have developed a novel automated culture system for the maintenance and differentiation of iPSC into various cell types9.
Our rationale for the new system was to adopt an X-Y-Z axis rail system instead of multiarticulated robotic arms9. To replace the complex hand-like functions of robotic arms, we applied a new idea to this system, which can automatically change three types of specific functional arm tips. Here, we also indicate how users can easily make task schedules with simple orders on software because of the lack of requirements for engineers&#39; contributions throughout the process.
One of the robotic culture systems has demonstrated the making of embryoid bodies using 96-well plates as 3D cell aggregates for differentiation4. The system reported here cannot handle 96-well plates. One achieved the current good manufacturing practice (cGMP) grade using a cell line, although it was not a human pluripotent stem cell5. The automated culture system detailed here has now been developed with the specific aim of helping laboratory experiments (Figure 1). However, it has enough systems to keep clean levels equivalent to a level IV safety cabinet.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.15% bovine serum albumin fraction V</td>
			<td>Fuji Film Wako Chemical Inc., Miyazaki, Japan</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>1% GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm plastic plates&#160;</td>
			<td>Corning Inc., NY, United States</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>253G1</td>
			<td>RKEN Bioresource Research Center</td>
			<td>HPS0002</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinin&#160; mouse</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A&#160;</td>
			<td>Nacali Tesque</td>
			<td>18585-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Thermo Fisher Scientific</td>
			<td>A14906.30</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin&#160; rabbit</td>
			<td>Dako</td>
			<td>A0001</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid</td>
			<td>Fuji Film Wako Chemical Inc.&#160;</td>
			<td>186-01114</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated culture system</td>
			<td>Panasonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Fuji Film Wako Chemical Inc.&#160;</td>
			<td>062-06661</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHC9531</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Merck</td>
			<td>810037</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021&#160;</td>
			<td>MCE, NJ, United States #HY-10182</td>
			<td>252917-06-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Defined Keratinocyte-SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10744019</td>
			<td>Human keratinocyte medium</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Merck</td>
			<td>266785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihexa&#160;</td>
			<td>TRC, Ontario, Canada</td>
			<td>13071-60-8</td>
			<td>rac-1,2-Dihexadecylglycerol</td>
		</tr>
		<tr>
			<td>Disposable hemocytometer</td>
			<td>CountessTM Cell Counting Chamber Slides, Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Thermo Fisher Scientific</td>
			<td>1219168-18-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium/F12&#160;</td>
			<td>Fuji Film Wako Chemical Inc.</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Fuji Film Wako Chemical Inc.&#160;</td>
			<td>053-07751</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1517001</td>
			<td>Human pluripotent stem cell medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Biowest, FL, United States</td>
			<td>S140T</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-basic&#160;</td>
			<td>Nacalai Tesque Inc.</td>
			<td>19155-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Thermo Fisher Scientific</td>
			<td>J63292.MF</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>Glutamine supplement</td>
		</tr>
		<tr>
			<td>Goat IgG(H+L) AlexaFluo546</td>
			<td>Thermo Scientific</td>
			<td>A11056</td>
			<td></td>
		</tr>
		<tr>
			<td>HNF-4A&#160; goat</td>
			<td>Santacruz</td>
			<td>6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Thermo Fisher Scientific</td>
			<td>A16292.06</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone 21-hemisuccinate</td>
			<td>Merck</td>
			<td>H2882</td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix511 Silk&#160;</td>
			<td>Nippi Inc., Tokyo, Japan</td>
			<td>892 021</td>
			<td>Cell culture matrix</td>
		</tr>
		<tr>
			<td>Insulin-transferrin-selenium</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 1&#160; mouse</td>
			<td>Santacruz</td>
			<td>376224</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 10&#160; rabbit</td>
			<td>BioLegend</td>
			<td>19054</td>
			<td></td>
		</tr>
		<tr>
			<td>KMUR001</td>
			<td>Kansai Medical University&#160;</td>
			<td></td>
			<td>Patient-derived iPSCs&#160;</td>
		</tr>
		<tr>
			<td>Knockout serum replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate&#160;</td>
			<td>A8960, Merck</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 medium&#160;</td>
			<td>Fuji Film Wako Chemical Inc.</td>
			<td>128-06075</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning Inc.</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG(H+L) AlexaFluo488</td>
			<td>Thermo Scientific</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin mouse</td>
			<td>Santacruz</td>
			<td>23927</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofilament&#160; rabbit</td>
			<td>Chemicon</td>
			<td>AB1987</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutristem</td>
			<td>Sartrius AG, G&#246;ttingen, Germany</td>
			<td>05-100-1A</td>
			<td>cell culture medium&#160;</td>
		</tr>
		<tr>
			<td>Oct 3/4&#160; mouse</td>
			<td>BD</td>
			<td>611202</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS(-)</td>
			<td>Nacalai Tesque Inc., Kyoto, Japan</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG(H+L) AlexaFluo488</td>
			<td>Thermo Scientific</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG(H+L) AlexaFluo546</td>
			<td>Thermo Scientific</td>
			<td>A10040</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human albumin&#160;</td>
			<td>A0237, Merck, Darmstadt, Germany</td>
			<td>A9731</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho kinase inhibitor, Y-27632&#160;</td>
			<td>Sellec Inc., Tokyo, Japan</td>
			<td>129830-38-2</td>
			<td></td>
		</tr>
		<tr>
			<td>RIKEN 2F</td>
			<td>RKEN Bioresource Research Center</td>
			<td>HPS0014</td>
			<td>undifferentiated hiPSCs&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Thermo Fisher Scientific #11875</td>
			<td>12633020</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Thermo Fisher Scientific</td>
			<td>301836-41-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-ascorbate</td>
			<td>Merck</td>
			<td>A4034-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>SSEA-4&#160; mouse</td>
			<td>Millipore</td>
			<td>MAB4304</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFit AK02N&#160;</td>
			<td>Ajinomoto, Tokyo, Japan</td>
			<td>AK02</td>
			<td>cell culture medium&#160;</td>
		</tr>
		<tr>
			<td>TnT rabbit</td>
			<td>Abcam</td>
			<td>ab92546</td>
			<td></td>
		</tr>
		<tr>
			<td>TRA 1-81 mouse</td>
			<td>Millipore</td>
			<td>MAB4381</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Thermo Fisher Scientific</td>
			<td>H34068.06</td>
			<td></td>
		</tr>
		<tr>
			<td>TripLETM express enzyme&#160;</td>
			<td>Thermo Fisher Scientific, Waltham, MA, United States</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution&#160;</td>
			<td>Nacalai Tesque, Kyoto, Japan</td>
			<td>20577-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose phosphate broth</td>
			<td>Merck</td>
			<td>T8782-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt-C59&#160;</td>
			<td>Bio-techne, NB, United Kingdom</td>
			<td>5148</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;&#160;<img alt="Equation 1" src="/files/ftp_upload/65672/65672eq01v2.jpg" style="margin: auto;" /> Tublin&#160; mouse</td>
			<td>Promega</td>
			<td>G712A</td>
			<td></td>
		</tr>
	</tbody>
</table>

an automated culture system for maintaining and differentiating human-induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Maintenance of human-induced pluripotent stem cells 
We used three hPSC lines (RIKEN-2F, 253G1, and KMUR001). We have optimized the maintenance protocol through daily manually performed experiments and further optimized the detailed programs through the seven preliminary experiments performed by the system. For example, shear stresses caused by the liquid speeds of the spit flow from different pipets handled by humans and the system are quite different; therefore, we optimized the time length of the enzymatic digestion and the number of pipetting for cell dispersion by the system.
Human induced pluripotent stem cells were maintained on a 10 cm dish coated with 0.5 &#181;g/cm2 cell culture matrix with hiPSC culture medium (e.g., Neutristem or StemFit AK02N). For passage, the system was programmed to wash hiPSC three times with 5 mL of phosphate buffer saline without calcium (PBS[-]), then treat with 3 mL of TrypLE express enzyme supplemented with 10 &#181;M of the Rho kinase inhibitor (Y-27632) for 15 min at 37 &#176;C. After that, the system added 9 mL of PBS(-) with 0.15% bovine serum albumin fraction V (BSA) to the plate and performed two sets of motion that aspirated 10 mL of cell-containing liquid and dispensed the liquid from the pipette tip in a zig-zag motion across the top of the plate, which was tilted 20&#176; closer to the plate surface, and repeated the above sequence of operations with the plate tilted at 20&#176; on the opposite side. Then, the system collected the cell-containing medium in a 15-mL tube and capped it, after which it was centrifuged at 115 x g for 5 min to deposit the cells. Thereafter, the system aspirated the supernatant and dispersed the cell pellet into single cells using 10 mL of culture medium with 10 &#181;M Y-27632 by pipetting 10 times. The system transferred 1 mL of trypan blue solution into a new 15 mL tube, then added 1 mL of the cell suspension and mixed them by pipetting five times. After that, 100 &#181;L of the Trypan blue-stained cell solution was aspirated and dispensed into the inlet window of the disposable hemocytometer in the holder. The system moved the hemocytometer holder to the microscopic observation area, and captured a photo, which was immediately analyzed by software, and the cell concentration obtained was applied to the next step. For iPSC maintenance, the system seeded the cells at a cell density of 15,000 cells/cm2 in new 10-cm diameter plastic plates coated with 0.5 &#181;g/cm2 cell culture matrix. For differentiation experiments, the system seeded the cells at the required cell density in the medium prepared for each somatic cell type in 6-well plates coated with a cell culture matrix diluted to 1/100. Finally, the system performed cross-locking five times before transferring the plates to the incubator. Three new 10-cm plates containing iPSCs and three cell culture matrix-pre-coated plates were loaded into the system, and other necessary items were also prepared according to the respective procedures mentioned above. One cycle of maintenance culture consists of a passaging task on day 1 followed by a culture medium change once a day for 3 days. All through the experiment, this cycle was set up for five cycles. In addition, consumables were replenished in turn.
According to the ordered schedule, five cycles of maintenance culture were successfully performed as planned by the system. From the cell photographs taken automatically during each task, it was confirmed that the cells proliferated smoothly over time. The cell expansion (n = 3) was calculated and shown in Figure 2. To confirm whether the undifferentiated state of iPSCs remains unchanged even after maintenance culture in the automated culture system, immunocytometoric analysis (Oct3/4 and SSEA4) was performed using the remaining samples at each passaging (Figure 3A). Furthermore, after five cycles, the three dishes were unloaded from the system, and immunohistochemical analysis (Oct3/4, SSEA4, and Tra1-81) was performed as well (Figure 3B). Both analyses using fluorescent immunostaining showed that the undifferentiated state of iPS cells was preserved. Karyotyping of iPSCs was also performed before and after maintenance culture in an automated culture system. Although an abnormality was observed in an allele of chromosome 17 before the start of the maintenance culture, no particular change was observed by the system after 5 maintenance cultures, including the abnormality (Figure 3C). The settings used for maintenance of iPSCs are summarized in Table 1.
Differentiation 
Cardiomyocytes 
The differentiation protocol from a previous publication was followed here10. The system seeded undifferentiated hiPSCs (KMUR001) at a density of 30,000 cells/cm2 on cell culture matrix-coated 6-well plates in hiPSC culture medium supplemented with 10 &#181;M of Y-27632. The system changed the medium to human pluripotent stem cell medium with 6 &#181;M of the GSK-3&#223; inhibitor CHIR-99021, 20 ng/mL Activin A, and 10 ng/mL BMP4 after 3 days (differentiation day 1). On differentiation day 3, the system changed to CDM3 medium: RPMI 1640, 500 &#181;g/mL recombinant human albumin, and 213 &#181;g/mL L-ascorbic acid 2-phosphate with 2 &#181;M Wnt-C59. On differentiation day 5, the system changed to fresh CDM3 medium; thereafter, it repeated changing to fresh CDM3 medium every other day. The settings used for cardiomyocyte differentiation of iPSCs are summarized in Table 2.
Hepatocytes 
The differentiation protocol from a previous publication was followed here11. The system seeded undifferentiated hiPSCs (RIKEN2F) at a density of 25,000 cells/cm2 on cell culture matrix-coated 6-well plates in hiPSC culture medium supplemented with 10 &#181;M of Y-27632. After 2 days (differentiation day 1), the system changed the medium to RPMI-1640 plus 2% B27 Minus Insulin (RPMI-B27) containing 100 ng/mL Activin A, 6 &#181;M CHIR-99021, and 1% glutamine supplement (e.g., GlutaMAX) for 24 h. On differentiation day 2, we changed the medium to RPMI-B27 with 50 ng/mL Activin A. On differentiation day 5, the system changed the medium to RPMI-B27, containing 1% glutamine supplement and 10 ng/mL BMP-4. On differentiation day 9, the system changed the medium to hepatocyte maturation medium: Leibovitz&#39;s L-15 medium containing 8.3% tryptose phosphate broth, 10 &#181;M hydrocortisone 21-hemisuccinate, 50 &#181;g/mL sodium L-ascorbate, 100 nM dexamethasone, 0.58% insulin-transferrin-selenium, 2 mM glutamine supplement, 8.3% fetal bovine serum, and 100 nM rac-1,2-dihexadecylglycerol. Thereafter, the system repeated changing the medium to a fresh medium every other day. The settings used for hepatocyte differentiation of iPSCs are summarized in Table 3.
Neuronal precursor cells 
The differentiation protocol from a previous publication was followed here12. The system seeded undifferentiated hiPSCs (RIKEN2F) at a density of 25,000 cells/cm2 on basement membrane matrix-coated 6-well plates in Dulbecco&#39;s modified Eagle medium (DMEM)/F12 and neurobasal medium (1:1) supplemented with 10% Knockout serum replacement, 0.1 mM non-essential amino acids, 1 mM glutamine supplement with 10 &#181;M TGF-beta receptor inhibitor (SB431542), 10 &#181;M BMP signal inhibitor (dorsomorphin), and 10 &#181;M Y-27632 (differentiation day 1). On differentiation day 5, the system changed the medium to DMEM/F12 and Neurobasal medium 1:1 supplemented with 0.1mM non-essential amino acids, 1 mM glutamine supplement, 1% N-2 supplement, 1% B-27 supplement, 50 &#181;g/mL ascorbic acid 2-phosphate, 10 &#181;M SB431542, and 10 &#181;M dorsomorphin. On differentiation day 9, the system changed the medium to DMEM/F12 and neurobasal medium 1:1 supplemented with 0.1 mM non-essential amino acids, 1 mM glutamine supplement, 1% N-2 supplement, 1% B-27 supplement, 50 &#181;g/mL ascorbic acid 2-phosphate, and 1 &#181;M all-trans retinoic acid. On differentiation day 13-16, the system changed the medium every day with DMEM/F12 and neurobasal medium 1:1 supplemented with 0.1 mM non-essential amino acids, 1 mM glutamine supplement, 1% N-2 supplement, 1% B-27 supplement, 50 &#181;g/mL ascorbic acid 2-phosphate, and 10 ng/mL bFGF and 10 ng/mL EGF. The settings used for neural precursor cell differentiation of iPSCs are summarized in Table 4.
Keratinocytes 
The differentiation protocol from a previous publication was followed here13. The system seeded undifferentiated hiPSCs (253G1) at a density of 15,000 cells/cm2 in cell culture matrix-coated 6-well plates in hiPSC culture medium with 10 &#181;M Y-27632. After 2 days later (differentiation day 1), the system changed the medium to Dulbecco&#39;s modified Eagle medium (DMEM)/F12 and Neurobasal medium (1:1) with 0.1 mM non-essential amino acids, 1 mM glutamine, 55 &#181;M 2-mercaptoethanol, 1% N-2 supplement, 2% B-27 supplement, 50 &#181;g/mL ascorbic acid 2-phosphate, 0.05% bovine serum albumin, and 100 ng/mL FGF-basic. On differentiation day 3, and thereafter, the medium was changed to human keratinocyte medium (without supplement for day 3 and with a supplement on/after day 5) with the addition of 0.5 &#181;g/mL hydrocortisone, 1 &#181;M all-trans retinoic acid, 25 ng/mL hBMP-4, 2.4 &#181;g/mL adenine, 1.37 ng/mL triiodothyronine, 0.3 mM ascorbic acid 2-phosphate, and 2 &#181;M forskolin, every 2 days by the system. The settings used for keratinocyte differentiation of iPSCs are summarized in Table 5.
As mentioned above, the entire process was performed using only automated culture equipment, from seeding iPS cells to the subsequent series of differentiation protocols. The expression of characteristic markers in each cell was evaluated (Figure 4). It was shown that differentiation could be induced using only an automated culture system.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65672/65672fig01.jpg" /> 
Figure 1: Summary of the automated culture system. (A) Picture of the system showing size and layout sketch 1. (B) Picture of the workbench and layout sketch 2. (C) Hand tools: dish, tube, and pipette. This figure has been modified with permission from Bando et al.9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/65672fig01large.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/65672/65672fig02.jpg" /> 
Figure 2: Passaging task and cell growth. (A) The square illustrates each process. (B) iPSC expansion calculated using every automated cell count in the three independent experiments. (C) Representative images captured automatically from passage to passage. Scale bars = 400 &#181;m and 100 &#181;m (inset). This figure has been modified with permission from Bando et al.9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/65672fig02large.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/65672/65672fig03.jpg" /> 
Figure 3: Automated long-term maintenance of iPSC. (A) All immunocytometoric analyses of the three independent experiments for Oct-3/4 and SSEA-4. Each blue histogram indicates no primary antibody control. Each red histogram represents the indicated antigen-specific signal. (B) Representative images of immunohistochemically stained cells for Oct-3/4, SSEA-4, and Tra 1-81. Scale bars = 50 &#181;m. (C) Karyotype of the RIKEN2F-iPSC after the fifth passage. This figure has been modified with permission from Bando et al.9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/65672fig03large.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/65672/65672fig04.jpg" /> 
Figure 4: Immunohistochemical staining. Immunohistochemical pictures of hepatocytes (Scale bars = 100 &#181;m), cardiomyocytes (Scale bars = 100 &#181;m), neuronal progenitor cells (Scale bars = 100 &#181;m), and keratinocytes (Scale bars = 200 &#181;m). This figure has been modified with permission from Bando et al.9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/65672fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Passage preparations and system settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/Table 1 (Passage) .xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Cardiomyocyte differentiation preparations and system settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/Table 2 (cardiomyocytes).xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Hepatocyte differentiation preparations and system settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/Table 3 (hepatocytes).xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Neural precursor cell differentiation preparations and system settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/Table 4 (Neural cells).xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Keratinocyte differentiation preparations and system settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65672/Table 5 (keratinocytes) .xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Automating iPSC Culture for Enhanced Reproducibility at JoVE.com

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

an-automated-culture-system-for-maintaining-differentiating-human

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.2K Views.  Kansai Medical University Graduate School of Medicine. Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Video: Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

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Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...