This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

Single-cell based non-colony type monolayer (NCM) culture of hPSCs.
1. Preparations
<ol>
	<li>Make 500 ml&#160;of medium for culture of mouse embryonic fibroblasts (MEFs): DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (NEAA).</li>
	<li>Isolate mouse embryonic fibroblasts (MEFs) cells derived from the CF1 strain following a routine protocol16 and culture MEFs on 0.1% gelatin-coated 6-well cell culture plate in DMEM medium. Alternatively, purchase MEF stocks at passage 3 from commercial resources.</li>
	<li>Prepare Matrigel plates.
	<ol>
		<li>Dilute 5 ml&#160;of hESC-qualified Matrigel stock with 5 ml&#160;of DMEM/F12 medium (chilled at ~4 &#176;C) and store as 50% aliquots in a -20 &#176;C freezer. Thaw the frozen Matrigel stock in a refrigerator (~4 &#176;C) O/N and further dilute the Matrigel in cold DMEM/F12 medium (to give rise to a 2.5% working concentration).</li>
		<li>Coat 6-well cell culture plates with 1.5 ml&#160;of 2.5% Matrigel per well, store the coated plates in refrigerator O/N (Note: use the Matrigel-coated plate within 2 weeks).</li>
		<li>Remove the Matrigel plate from the refrigerator, allow the plate to warm at RT in the cell culture hood for 10 to 30 min (Note: do not warm the plate in a cell culture incubator), and aspirate DMEM medium prior to plating hPSCs.</li>
	</ol>
	</li>
	<li>Make 500 ml&#160;hESC medium: 80% DMEM/F12 medium, 20% KSR, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM &#946;-mercaptoethanol, and 4 ng/ml&#160;of FGF-2.</li>
	<li>Prepare 10 ml&#160;of 2X&#160;hPSC freezing medium: 60% FBS, 20% DMSO, and 20 &#181;M Y-27632 in mTeSR1 medium, sterilize by filtration, and use the medium within 1 week.</li>
</ol>
2. Protocol 1 (Basic): Grow hPSC Colonies on Feeders
<ol>
	<li>Use MEF passage numbers at 5 or 6 (designated as p5 and p6) for hPSC culture in order to get consistent results. Mitotically inactivate MEFs by treating the cells with 10 &#956;g/ml&#160;mitomycin C for 3 hr at 37 &#176;C, wash the cells 3x with 1X Dulbecco&#8217;s Phosphate-Buffered Saline (D-PBS), and then dissociate MEFs with 0.05% Trypsin in 0.53 mM EDTA. Alternatively, irradiate MEFs at a dose of 8,000 rads with an X-ray irradiator.</li>
	<li>Count cell numbers using the Trypan Blue stain exclusion method under microscope. Alternatively, use an automatic cell counter.</li>
	<li>Plate irradiated MEFs on 6-well polystyrene plates coated with 0.1% gelatin at a density of 1.88 x 105 cells per well (i.e., 1.96 x 104 cells/cm2). Incubate the cells at 37 &#176;C and 5% CO2 for 24 hr.</li>
	<li>Remove the MEF culture medium by aspiration with a Pasteur pipette (Note: no wash steps needed at this time).</li>
	<li>Triturate hPSC colonies from previous culture into small clumps, examine the clumps under microscope to ensure their sizes ranging from 50 to 100 &#956;m in diameters, and plate hPSC clumps on the top of MEF feeder layers in one well containing 2 ml&#160;of hPSC medium.</li>
	<li>Change hESC medium daily for 3 to 5 days, record colony growth by photograph, mark any morphologically altered or differentiated colonies (~5%), and manually remove the marked colonies by gently aspiration with a Pasteur pipette.</li>
	<li>Rinse the remaining colonies on MEFs twice (2 min each with D-PBS), and incubate the colonies with 2 ml&#160;of 1 mg/ml&#160;collagenase IV in hPSC medium for 10 to 30 min), and examine the detachment of hPSC colonies from the MEF-coated surface (Note: use a scraper to help the detachment process only if the colonies are tightly attached to the plate).</li>
	<li>Add 5 ml&#160;of hPSC medium to each well to minimize&#160;enzymatic reactions, transfer colonies to a 15 ml&#160;tube, allow hPSC colonies to sediment for 3 to 5 min at RT, and ensure the sedimentation of colonies by direct visualization of the cells at the bottom of the tube (Note: do not centrifuge the tube at this step).</li>
	<li>Remove the supernatants containing residual MEFs and dissociated single cells, resuspend the colonies with 5 ml&#160;of hESC medium, and repeat the sedimentation step twice.</li>
	<li>Remove the medium, triturate hPSC colonies into small clumps, store in 1X hPSC freezing medium (or CryoStore CS10 freezing medium) for cryopreservation (1 confluent well per frozen vial) or plate on a 6-well plate for cell passaging.</li>
</ol>
3. Protocol 2: Convert hPSC Colonies from Feeders to NCM
<ol>
	<li>Rinse hPSC pellets in D-PBS once, incubate the pellets with 1 ml&#160;of 1X Accutase for 10 min, and examine the enzymatic reaction under a microscope to ensure single-cell dissociation.</li>
	<li>Terminate the enzymatic reactions by gently resuspending the cells in 5 ml&#160;of mTeSR1 medium containing 10 &#956;M Y-27632 followed by centrifugation at 200 x&#160;g&#160;at RT for 5 min (Note, do not use excessive centrifugation forces which cause cell damage).</li>
	<li>Add 5 ml&#160;of mTeSR1 medium to the pellet, resuspend the pellet as single cells, and filter dissociated cells through a 40&#160;&#181;m cell strainer (to remove any residual cell aggregates).</li>
	<li>Seed 1.3-2 x 106&#160;hPSCs into one well (1.4-2.1 x 105&#160;cells/cm2) of a 6-well plate coated with 2.5% hESC-qualified Matrigel, add mTeSR1 medium up to 2.5 ml, and include 10 &#181;M Y-27632 in the medium (to facilitate the initial 24&#160;hr single-cell plating). Alternatively, utilize the following small molecules to replace 10 &#956;M Y-27632 for enhancing single-cell plating: 1 &#181;M Y-39983 (ROCK I inhibitor), 1 &#181;M phenylbenzodioxane (ROCK II inhibitor), 1 &#181;M thiazovivin (a novel ROCK inhibitor), and 2 &#956;M JAK inhibitor 1.</li>
	<li>Replace the medium with drug-free mTeSR1 medium on the next day (within 24 hr), dissociate cells from one extra well, and count cell number to determine single-cell plating efficiency (Note: approximately, 50 to 90% of single-cell plating efficiency that can be achieved at this stage).</li>
	<li>Allow the cells to grow as a single-cell-formed monolayer for a few days with 3 ml&#160;of mTeSR1 medium. Change medium daily.</li>
	<li>Empirically determine the schedule for passaging adapted NCM depending on the cell density. Passage when cell growth reaches confluence at day 3 or day 4, or at the 4&#160;hr&#160;time point after the disappearance of cell-to-cell borders. Dissociate the cells in 1 ml&#160;of Accutase, use a 1 to&#160;3 splitting ratio (i.e., 1 confluent well of cells plated in 3 wells of 6-well plate) for cell passaging, and stabilize adapted NCM by 5 passages.</li>
	<li>Cell freezing
	<ol>
		<li>Utilize one confluent well (~ 5-6 x106&#160;cells) for one frozen vial: dissociate cells from one confluent well with 1 ml&#160;of Accutase for 10 min.</li>
		<li>Dilute with 5 ml&#160;of mTeSR1 medium and centrifuge cells as described above.</li>
		<li>Resuspend the pellet in 500 &#956;l&#160;of mTeSR1 medium and then slowly add 500 &#956;l&#160;of 2X hPSC freezing medium (containing 20 &#956;M Y-27632) in a cryopreservation vial. Alternatively, resuspend the cells in 1X hPSC freezing medium containing 2 &#956;M JAK inhibitor 1 or 1X CryoStor CS10 medium in the presence of either 10 &#181;M Y-27632 or 2 &#956;M JAK inhibitor 1.</li>
		<li>Place frozen vials into an ice-chilled cryocontainer (bottom-filled with isopranol) and transfer the cryocontainer to a -80 &#176;C freezer immediately.</li>
		<li>Transfer cells from the -80 &#176;C freezer to a liquid nitrogen tank (on the next day) for long-term cryopreservation.</li>
	</ol>
	</li>
	<li>Cell thawing
	<ol>
		<li>Utilize one cryopreservation vial for plating 1 well of a 6-well plate.</li>
		<li>Pre-warm mTeSR1 medium in a 37 &#176;C water bath for 10 min, thaw cells in the same water bath for 2 min, drop-wise add cells to 5 ml&#160;of pre-warmed mTeSR1 medium containing 10 &#181;M Y-27632, and centrifuge as described above.</li>
		<li>Gently resuspend the cell pellets with 2 ml&#160;of mTeSR1 medium containing 10 &#181;M Y-27632 and slowly transfer cells to a well pre-coated with Matrigel (Note: avoid producing air bubbles).</li>
		<li>Change medium daily and expect hPSC growth to reach confluence at day 3 or 4.</li>
	</ol>
	</li>
</ol>
4. Protocol 3: Convert hPSC Colonies on Matrigel to NCM Culture
<ol>
	<li>Remove a Matrigel-coated plate from refrigerator, place the plate in the tissue culture hood for 10 to 30 min, remove the medium from each well of the plate, and add 2 ml&#160;of pre-warmed mTeSR1 medium to each well.</li>
	<li>Transfer triturated hPSC clumps from the feeder culture (Step 2.10 of the basic protocol) to the above Matrigel plate. Add mTeSR1 medium up to 2.5 ml&#160;final volume in each well.</li>
	<li>Change medium daily for 3 to 4 days until colonies reach 80 to 90% confluence.</li>
	<li>For cell passaging: rinse the colonies with D-PBS twice, treat the cells with 1 ml&#160;of 2 mg/ml&#160;dispase at 37 &#176;C for 15 to 20 min, and sediment and passage cells as clumps.</li>
	<li>For NCM culture: repeat Steps 3.1 to 3.9 described in Protocol&#160;2.</li>
</ol>
5. Protocol 4: NCM Culture of hPSCs on LN-521
<ol>
	<li>Thaw the recombinant LN-521 solution at 4 &#176;C before the day of use and make the laminin coating solution (LCS) by diluting the thawed laminin with 1X D-PBS (containing Ca2+/Mg2+) to a final concentration of 10 &#956;g/ml.</li>
	<li>Add 1 ml&#160;of the LCS to one well in 6-well plate (note: avoid dry-out during the coating process).</li>
	<li>Seal the coated plate with Parafilm to prevent evaporation and store the plate in refrigerator (4 &#176;C) O/N (note: use the plate within 1 week).</li>
	<li>Gently remove the LCS with a Pasteur pipette without touching the coated surface and add 2 ml&#160;of mTeSR1 medium to one well.</li>
	<li>Warm all culture solutions (including D-PBS) in a 37 &#176;C water bath for 25 min.</li>
	<li>Convert hPSC colonies to NCM
	<ol>
		<li>Dissociate cell aggregates to single cells with Accutase as described in Protocol 2.</li>
		<li>Seed 1.3-2 x 106&#160;hPSCs into one LN-521-coated well (1.4-2.1 x 105&#160;cells/cm2) without the presence of ROCK inhibitors.</li>
		<li>Change medium daily for 3 to 4 days.</li>
		<li>Dissociate the cells in 1 ml&#160;of Accutase at day 3 or 4 for the next cell passaging using a 1 to 3 splitting ratio.</li>
	</ol>
	</li>
</ol>
6. Protocol 5: NCM Culture for Plasmid DNA Transfection
<ol>
	<li>Adapt hPSC colonies to NCM culture as described in Protocols 2 to 4.</li>
	<li>Plate dissociated hPSCs in 12-well plate with a cell density of 7.5 x 105 cells/well in mTeSR1 medium in the presence of 10 &#956;M Y-27632.</li>
	<li>Replace mTeSR1 medium with drug-free mTeSR1 medium between 4-8 hr after plating the cells.</li>
	<li>For each transfection, dilute 2.5 &#956;g of expression plasmids (e.g., pmaxGFP) and 5 &#956;l of Lipofectamine 2000 in separate Eppendorf tubes in 125 &#956;l&#160;each of Opti-MEM Reduced Serum Medium.</li>
	<li>After 5 min, mix the diluted reagents and incubate for 20 min at RT (to form transfection complexes).</li>
	<li>Add the 250&#160;&#956;l&#160;transfection complexes to each well containing hPSCs in 1 ml mTeSR1 medium and incubate the cells at 37 &#176;C in a CO2 incubator for 24 hr.</li>
	<li>Examine the transfection efficiency under a fluorescence microscope and photograph randomly for calculation of the actual transfection efficiency.</li>
</ol>
7. Protocol 6: NCM Culture for Transfection of MicroRNAs
<ol>
	<li>Dissociate semi-confluent hPSCs at day 2 under NCM conditions by adding 1 ml&#160;of Accutase per well in 6-well plate.</li>
	<li>Add 10 ml&#160;of mTeSR1 medium to dilute enzymatic reactions and centrifuge at 200 x g for 5 min.</li>
	<li>Resuspend the cell pellet with mTeSR1 medium, seed the cells at a density of 7.5 x 105 cells per well in a 12-well plate in mTeSR1 medium containing 10 &#956;M Y-27632, and let the cells attach for 4 hr.</li>
	<li>Replace the medium with Y27632-free mTeSR1 medium.</li>
	<li>Use commercially available microRNAs (e.g., a non-targeting miRIDIAN miRNA Transfection Control labeled with Dy547). Titrate concentrations ranging from 0-160 nM using the provided reagents and follow the manufacturers&#8217; instructions.</li>
	<li>Optimize transfection efficiency for each concentration in hPSC lines by imaging the Dy547 fluorescence of the live cells under a microscope at 24 hr after the transfection.</li>
	<li>Calculate the transfection efficiencies based on the percentage of Dy547 positive cells over all imaged cells.</li>
</ol>
8. Protocol 7: NCM Culture for Transduction of Lentiviral Vector
<ol>
	<li>Repeat steps 7.1 to 7.4 of Protocol 6.</li>
	<li>Calculate the amounts of viral particles to be used for transduction: Use the following formula: TU = (MOI x CN) / VT, whereas TU denotes the total numbers of transforming units, MOI equals the desired multiplicity of infection in the well (MOI or TU/cell), CN specifies the number of cells in each well, and VT indicates stock viral titers (TU/ml). For example, given that stock viral titers for SMART-shRNA vector equals 1 x 109 TU/ml&#160;(transforming units per ml), 7.5 x 105 cells per well at the time of transduction, and an expected MOI of 20: use 15 &#956;l&#160;of the stock viral particles for each well (note: this condition is recommended for transient lentiviral induction).</li>
	<li>Pre-warm 300 &#956;l&#160;of mTeSR1 medium with 10 mg/ml&#160;of polybrene for 30 min.</li>
	<li>Add 15 &#956;l&#160;of stock viral particles into the pre-warmed mTeSR1 medium containing polybrene and gently mix the solution.</li>
	<li>Replace the cell culture medium with 300&#160;&#956;l&#160;of prewarmed medium containing the viral particles.</li>
	<li>After 4 hr incubation, examine turboGFP fluorescence (a maker for shRNA expression) to determine the transduction efficiency.</li>
	<li>Add 300 &#956;l&#160;of additional mTeSR1 medium to the transducing well when the cells begin to express turboGFP.</li>
	<li>After 12-16 hr incubation, reexamine turbo-GFP expression.</li>
	<li>Perform desired follow-up experiments with these transduced cells within 72 hr.</li>
</ol>

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The authors declare that they have no competing financial interests.

There are two major ways to culture hPSCs in vitro: conventional colony-type culture (of cells on feeders or extracellular matrices) and suspension culture of hPSCs as aggregates without feeders6. The limitations of both colony-type and suspension culture methods include accumulated heterogeneity and inheritable epigenetic changes. NCM culture, based on both single-cell passaging and high-density cell plating, represents a new culture method for hPSC growth6,18. Although various single-cell passaging methods have been documented in the literature, but none of them are used for routine propagation. For example, Wu and colleagues employed a single-cell passaging method to study the effects of various small molecules cocktails on hPSC growth19. However, their final culture products are colonies. So, low-density-based single-cell passaging methods still belong to colony-type culture. With regard to NCM culture, the high-density plating transforms this culture to a new method, since the final cell product is a homogenous monolayer. Recently, Dvorak and colleagues used similar method to comprehensively analyze hPSC properties under this growth condition18. Their results also support our major conclusions. It is clear now that NCM culture is an emerging method that possesses several new properties and can be further modified for many potential applications in pluripotent stem cell biology.
Basic properties of hPSCs from NCM culture
It is conceivable that hPSCs under NCM culture share many characteristics with the same types of cells grown as colonies9,18. The expression levels of hESC markers in hPSCs, which include Oct-4, NANOG, SSEA-3/4, Tra-1-60, Tra-1-81, and SSEA-1, are similar in both NCM and colony-type culture. NCM-adapted cells also retain similar global mRNA expression patterns to hPSC colonies grown on MEF feeder layers9. Clearly, NCM-adapted cells sustain the pluripotent state as determined by teratoma assay9,18. However, unlike hPSC colonies, cells under NCM conditions have a predictable growth rate that is characterized by consistent growth curves, cell cycles, and cell numbers9. Due to the high-density single-cell plating, hPSCs under NCM conditions exhibit exponential growth between days 2 and 4 (Figure 1B), thereby increasing cell number by 4-fold compared with hPSC colonies grown on MEFs. Prolonged culture does not increase cell production, but rather increases apoptotic and differentiation&#160;stress9. NCM also enables optimal cryopreservation, thus allowing rapid cell recovery upon plating thawed cells (Protocol 1). Moreover, NCM facilitates the adaptation of hPSC culture to various xeno-free protocols9. In general, NCM supports the growth of hPSCs, maintains the pluripotent state, and sustains the potential of hPSCs to differentiate into adult tissues of the three germ layers.
Chromosomal stability in hPSCs under NCM
In terms of chromosomal stability, there is no direct comparison between the cells from different culture methods. The majority of hPSCs under our NCM culture conditions have normal karyotypes and gene copy numbers as determined by G-banding, array-based comparative genomic hybridization (aCGH), and fluorescence in situ hybridization (FISH)9. Two lines (~13%) showed abnormal karyotypes, in which one line (i.e., WA09) exhibited elevated polyploidy and another one (ES01) had 14% of cells with trisomy 20&#160;9. It is unclear whether these abnormal karyotypes were derived from the selection of preexisting mutated cells prior to NCM culture or induced during NCM adaption. It is important that the starting hPSC colonies or subset of colonies used for NCM culture should be well-characterized in terms of their homogeneity and chromosomal stability at the time for NCM adaptation. Notably, the rate of abnormal karyotypes under NCM conditions is much lower than that reported in a recent cohort analysis, in which the authors reveal 34% abnormal karyotypes under predominant colony-type culture conditions20. Hence, our study indicates that we can grow dissociated single-cells and maintain their chromosomal stability under NCM conditions. Nevertheless, we also need to use more sensitive methods to examine chromosomal abnormalities of hPSCs in the coming years. Particularly, we need to scan chromosomes 1, 12, 17, and 20 using higher resolution probes (i.e., &#60; 50 kb), as some minor lesions (e.g., the 20q11.21 amplicon) at these frequently altered chromosomes cannot be detected by conventional karyotyping and FISH20-22. Furthermore, the development of diverse NCM protocols would enable us to identify robust and safe methods for future applications.
NCM culture under diverse modifications
The use of the ROCK inhibitor, Y-27632, has opened the door for single-cell based assays. The availability of diverse ROCK inhibitors would provide additional choices for NCM-based expansion and cryopreservation of hPSCs (Figure 2). In theory, any small molecule, that can significantly increase single-cell plating efficiency, can be used to facilitate NCM culture. JAK inhibitor 1, which functions differently from those ROCK inhibitors, represents such an example9. The use of single molecules or combinatorial approaches may provide us optimal cell growth, cellular assays, and cryopreservation of hPSCs. However, alternative NCM culture of hPSCs on defined extracellular matrix proteins, such as the laminin isoform LN-521, normally expressed in hESCs23-25, may eliminate the interference of small molecules (Figure 3). LN-521 might enhance dissociated-hPSC survival and sustains pluripotency through the activation of the &#945;6&#946;1-PI3K/AKT pathway24. The simplicity of passaging hPSCs with LN-521 reduces the heterogeneity of the cells, compatible with various feeder-free and xeno-free cell culture systems using completely defined medium (Protocol 4). It also provides an additional module for high-throughput assays without the influence of small molecules. Although the iPSCs under NCM culture on LN-521 retained the expression of a panel of hPSC markers, further characterization of these cells using teratoma assay and embryoid body-mediated multilineage differentiation may be necessary to confirm the pluripotent states in these cells. Of note, hiPSCs cultured on the laminin isoform LN-521 are reportedly cytogenetically stable&#160;(http://biolamina.com/). However, we also need to apply higher-resolution and sensitive methods (as discussed above) to examine the chromosomal stability in these cells.&#160;
Versatility of NCM culture for genetic engineering
NCM-based methods represent a simple, robust, and economical system that may be particularly useful for genetic manipulation of hPSCs (Protocols 5-7). It is known that hESC colonies are difficult to transfect or transduce, with great variability in transfection/transduction efficiencies among different laboratories26-28. For example, transfection efficiency in hESCs ranges from 3 to 35% under colony culture conditions26. Lentiviral transduction signals in BG01 cells under colony conditions were found to be extremely low9. However, the transfection efficiency mediated by NCM was greater than 75%9 and modification of transduction methods can increase lentivirus-mediated transduction efficiency up to 90%28. A tight comparative study has revealed that it is the multicellular associations in hESC colonies that contribute to the low transfection efficiency9. Furthermore, we have recently modified a microRNA transfection protocol and are able to achieve high transfection efficiency (~91%) without the use of lentiviruses (Protocol 6 and Figure 4). This protocol is easy to use and particularly useful for transient transfection experiments within a 3- to 5-day time frame. As we delineated in above protocols, multiple factors should be taken into considerations when we optimize transfection/transduction efficiency in hPSCs. These factors include cell density, plasmid concentrations, lentiviral titers, multiplicity of infection (MOI), duration of transfection/transduction, cytotoxicity of the reagents, and methods used for monitoring transfection/transduction efficiency.
We elaborate and extend NCM methods to culture hPSCs on Matrigel and on defined extracellular matrices. This culture method is an efficient way to eliminate the heterogeneity commonly found in hPSC colony and aggregated cultures. Human pluripotent stem cells under NCM growth conditions are pluripotent and chromosomally stable. This novel culture system is simple and versatile for hPSC maintenance, large-scale expansion, and genetic manipulations.

The capacity of hPSCs to differentiate toward multilineage adult tissues has opened new avenues to treating patients who suffer from severe diseases that involve cardiovascular, hepatic, pancreatic, and neurological systems1-4. Various cell types derived from hPSCs would also provide robust cellular platforms for disease modeling, genetic engineering, drug screening, and toxicological testing1,4. The key issue that ensures their future clinical and pharmacological applications is the generation of large numbers of clinical-grade hPSCs through in vitro cell culture. However, current culture systems are either insufficient or inherently variable, involving various feeder and feeder-free cultures of hPSCs as colonies5,6.
Colony-type growth of hPSCs shares many structural features of the inner cell mass (ICM) of early mammalian embryos. The ICM is prone to differentiate into the three germ-layers in a multicellular environment because of the existence of heterogeneous signaling gradients. Thus, the acquisition of heterogeneity in early embryonic development is considered as a required process for differentiation, but an unwanted feature of hPSC culture. The heterogeneity in hPSC culture is often induced by excessive apoptotic signals and spontaneous differentiation due to suboptimal growth conditions. Thus, in colony-type culture, the heterogeneous cells are often observed in the periphery of the colonies7,8. It has been also shown that the cells in human embryonic stem cell (hESC) colonies exhibit differential responses to signaling molecules such as BMP-4&#160;9. Moreover, colony culture methods produce low cell yields as well as very low cell recovery rates from cryopreservation due to uncontrollable growth rates and apoptotic signaling pathways6,9. In recent years, various suspension cultures have been developed for culturing hPSCs, particularly for expansion of large amounts of hPSCs in feeder- and matrix-free conditions6,10-13. Obviously, different culture systems have their own advantages and disadvantages. In general, the heterogeneous nature of hPSCs represents one of the major drawbacks in colony-type and aggregated culture methods, which are suboptimal for delivering DNA and RNA materials into hPSCs for genetic engineering6.
Clearly, there is an imperative need to develop new systems that circumvent some shortcomings of current culture methods. The discoveries of small molecule inhibitors (such as the ROCK inhibitor Y-27632 and JAK inhibitor 1) that improve single-cell survival pave the way for dissociated-hPSC culture14,15. With the use of these small molecules, we have recently developed a culture method based on non-colony type (NCM) growth of dissociated-hPSCs9. This novel culture method combines both single-cell passaging and high-density plating methods, allowing us to produce large amounts of homogeneous hPSCs under consistent growth cycles without major chromosomal abnormalities9. Alternatively, NCM culture might be implemented with different small molecules and defined matrices (such as laminins) in order to optimize the culture method for wide applications. Here, we present several detailed protocols based on NCM culture and delineate detailed procedures for genetic engineering. To demonstrate the versatility of NCM protocols, we also tested NCM culture with diverse ROCK inhibitors and with the single laminin isoform 521 (i.e., LN-521).

<table><tbody><tr><td>Countess automated cell counter</td><td>Invitrogen Inc.</td><td>C10227</td><td>Automatic cell counting</td></tr><tr><td>Faxitron Cabinet X-ray System</td><td>Faxitron X-ray Corporation, Wheeling, IL&amp;nbsp;</td><td>Model RX-650</td><td>X-ray irradiation of MEFs</td></tr><tr><td>MULTIWELL 6-well plates</td><td>Becton Dickinson Labware</td><td>353046</td><td>Polystyrene plates</td></tr><tr><td>DMEM</td><td>Invitrogen Inc.</td><td>11965&amp;ndash;092</td><td>For MEF medium</td></tr><tr><td>Mitomycin C</td><td>Roche</td><td>107409</td><td>Mitotic inhibitor</td></tr><tr><td>Trypsin</td><td>Invitrogen Inc.</td><td>25300-054</td><td>For MEF dissociation</td></tr><tr><td>DMEM/F12</td><td>Invitrogen Inc.</td><td>11330&amp;ndash;032</td><td>For hPSC medium</td></tr><tr><td>Opti-MEM I Reduced Serum Medium&amp;nbsp;</td><td>Invitrogen Inc.</td><td>31985-062</td><td>For hPSC transfection</td></tr><tr><td>Heat-inactivated FBS</td><td>Invitrogen Inc.</td><td>16000&amp;ndash;044</td><td>Component of MEF medium</td></tr><tr><td>Knockout Serum Replacement</td><td>Invitrogen Inc.</td><td>10828&amp;ndash;028</td><td>KSR, Component of hPSC medium</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline</td><td>Invitrogen Inc.</td><td>14190-144</td><td>D-PBS, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Non-essential amino acids</td><td>Invitrogen</td><td>11140&amp;ndash;050</td><td>NEAA, component of hPSC medium</td></tr><tr><td>L-Glutamine</td><td>Invitrogen&amp;nbsp;</td><td>25030&amp;ndash;081</td><td>Component of hPSC medium</td></tr><tr><td>mTeSR1 &amp;amp; Supplements</td><td>StemCell Technologies</td><td>5850</td><td>Animal protein-free</td></tr><tr><td>TeSR2 &amp;amp; Supplements</td><td>StemCell Technologies</td><td>5860</td><td>Xeno-free medium</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma&amp;nbsp;</td><td>M7522</td><td>Component of hPSC medium</td></tr><tr><td>&lt;br/&gt; MEF (CF-1) ATCC</td><td>American Type Culture Collection (ATCC)&amp;nbsp;</td><td>SCRC-1040</td><td>For feeder culture of hPSCs</td></tr><tr><td>hESC-qualified Matrigel</td><td>BD Bioscience</td><td>354277</td><td>For feeder-free culture of hPSCs</td></tr><tr><td>Laminin-521</td><td>BioLamina</td><td>LN521-02</td><td>Human recombinant protein</td></tr><tr><td>FGF-2 (recombinant FGF, basic)</td><td>R&amp;amp;D Systems, MN</td><td>223-FB</td><td>Growth factor in hPSC medium</td></tr><tr><td>CryoStor CS10</td><td>StemCell Technologies</td><td>7930</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td>1X mixed enzymatic solution</td></tr><tr><td>JAK inhibitor I</td><td>EMD4 Biosciences</td><td>420099</td><td>An inhibitor of Janus kinase</td></tr><tr><td>Y-27632</td><td>EMD4 Biosciences</td><td>688000</td><td>ROCK inhibitor</td></tr><tr><td>Y-27632</td><td>Stemgent</td><td>04-0012</td><td>ROCK inhibitor</td></tr><tr><td>Y-39983</td><td>Stemgent</td><td>04-0029</td><td>ROCK I inhibitor</td></tr><tr><td>Phenylbenzodioxane</td><td>Stemgent</td><td>04-0030</td><td>ROCK II inhibitor</td></tr><tr><td>Thiazovivin</td><td>Stemgent</td><td>04-0017</td><td>A novel ROCK inhibitor</td></tr><tr><td>BD Falcon Cell Strainer</td><td>BD Bioscience</td><td>352340</td><td>40 &amp;micro;m cell strainer</td></tr><tr><td>Nalgene 5100-0001 Cryo 1 &amp;deg;C</td><td>Thermo Scientific&amp;nbsp;</td><td>C6516F-1</td><td>&amp;ldquo;Mr. Frosty&amp;rdquo; Freezing Container</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen Inc.</td><td>11668-027</td><td>Transfection reagents</td></tr><tr><td>DharmaFECT Duo</td><td>Thermo Scientific</td><td>T-2010-02</td><td>Transfection reagent</td></tr><tr><td>Non-targeting miRIDIAN miRNA Transfection Control</td><td>Thermo Scientific</td><td>IP-004500-01-05</td><td>Labeled with Dy547, to monitor the delivery of microRNAs&amp;nbsp;</td></tr><tr><td>SMART-shRNA</td><td>Thermo Scientific&amp;nbsp;</td><td>To be determined</td><td>Lentiviral vector</td></tr><tr><td>pmaxGFP</td><td>amaxa Inc (Lonza)</td><td>Included in every transfection kit</td><td>Expression plasmid for transfection control</td></tr><tr><td>Oct-4</td><td>Santa Cruz Biotechnology</td><td>sc-5279</td><td>Mouse IgG2b, pluripotent marker</td></tr><tr><td>SSEA-1</td><td>Santa Cruz Biotechnology</td><td>sc-21702</td><td>Mouse IgM, differentiation marker</td></tr><tr><td>SSEA-4</td><td>Santa Cruz Biotechnology</td><td>sc-21704</td><td>Mouse IgG3, pluripotent marker</td></tr><tr><td>Tra-1-60</td><td>Santa Cruz Biotechnology</td><td>sc-21705&amp;nbsp;</td><td>Mouse IgM, pluripotent marker</td></tr><tr><td>Tra-1-81</td><td>Santa Cruz Biotechnology</td><td>sc-21706</td><td>Mouse IgM, pluripotent marker</td></tr><tr><td>CK8 (C51)</td><td>Santa Cruz Biotechnology</td><td>sc-8020</td><td>Mouse IgG1, against cytokeratin 8</td></tr><tr><td>&amp;alpha;-fetoprotein</td><td>Santa Cruz Biotechnology</td><td>sc-8399</td><td>AFP, mouse IgG2a</td></tr><tr><td>HNF-3&amp;beta; (P-19)</td><td>Santa Cruz Biotechnology</td><td>sc-9187</td><td>FOXA2, goat polyclonal antibody</td></tr><tr><td>Troponin T (Av-1)</td><td>Thermo Scientific</td><td>MS-295-P0</td><td>Mouse IgG1</td></tr><tr><td>Desmin</td><td>Thermo Scientific</td><td>RB-9014-P1</td><td>Rabbit IgG</td></tr><tr><td>Anti-NANOG</td><td>ReproCELL Inc, Japan</td><td>RCAB0004P-F</td><td>Polyclonal antibody&amp;nbsp;</td></tr><tr><td>Rat anti-GFAP</td><td>Zymed</td><td>13-0300</td><td>Glial fibrillary acidic protein</td></tr><tr><td>Albumin (clone HSA1/25.1.3)</td><td>Cedarlane Laboratories Ltd.</td><td>CL2513A</td><td>Mouse IgG1</td></tr><tr><td>Smooth muscle actin (clone 1A4)</td><td>DakoCytomation Inc</td><td>IR611/IS611</td><td>Mouse IgG2a</td></tr><tr><td>Nestin</td><td>Chemicon International</td><td>MAB5326</td><td>Rabbit polyclonal antibody</td></tr><tr><td>TUBB3</td><td>Convance Inc</td><td>MMS-435P</td><td>Tuj1, mouse IgG2a</td></tr><tr><td>HNF4&amp;alpha; (C11F12)</td><td>Cell Signaling Technologies</td><td>3113</td><td>Rabbit monoclonal antibody</td></tr><tr><td>Paraformaldehyde (solution)</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA, fixative, diluted in D-PBS</td></tr></tbody></table>

alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

A general schema of NCM culture
Figure 1 represents a typical NCM culture schema showing the dynamic changes of hPSCs after high-density single-cell plating in the presence of the ROCK inhibitor Y-27632. These morphological changes include intercellular connections after plating, cellular clusters formation, and exponential cell growth followed by cell condensation (Figure 1A). A representative experiment indicates WA01 (H1) hESCs, plated as single-cells at a density of 1.9 x 105 cells/cm2 in the presence of 10 &#956;M Y-27632 at day 1 (Figure 1B, left panel), further propagated without the formation of colonies (Figure 1B, middle panel) at day 2, and condensed as a homogeneous monolayer that is suitable for desired experiments or for cell passaging at day 3 (Figure 1B, right panel).
Various ROCK inhibitors support NCM culture
A 96-well plate assay was used for proof-of-concept of high-throughput drug screening. It was also designed to optimize the use of various ROCK inhibitors to support NCM culture. Approximately, 31,000 dissociated SCU-i10 cells, human induced pluripotent cells (hiPSCs)17, were plated on one Matrigel-coated well in the presence of different concentrations of ROCK inhibitors. After 24 hr, the cells were subjected to the CCK-8 based survival assay to determine cell survival under these conditions. We have previously shown that the NCM method requires the use of the ROCK inhibitor, Y-27632, at 10 &#956;M to enhance single-cell plating. In this report, we confirmed that 10 &#956;M Y-27632 significantly increase 24&#160;hr single-cell plating efficiency of hiPSCs (P &#60; 0.05) (Figure 2). We also found that Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor) significantly modulate single-cell plating efficiency and promote NCM growth at 1 &#956;M when compared with their controls (P &#60; 0.05) (Figure 2). Moreover, the effects of the three ROCK inhibitors (at 1 &#956;M) on single-cell plating efficiency were comparable to that of Y-27632 at 10 &#956;M (P &#62; 0.05) (Figure 2). Notably, the ROCK I inhibitor (at 5 &#956;M) appears to show pronounced cytotoxicity compared with the drug at 1 &#956;M (P &#60; 0.05), implicating a more specific interaction than other molecules. Thus, various ROCK inhibitors may be used for supporting NCM culture in the future. However, a complete characterization of both hESCs and hiPSCs under NCM with these new inhibitors would be required for future use.
LN-521 supports NCM culture without the use of ROCK inhibitors
To determine the role of a specific laminin isoform in supporting hESC growth, we cultured SCU-i30 hiPSCs on LN-521-coated plates in the xeno-free medium TeSR2. Interestingly, LN-521 alone, without the presence of ROCK inhibitors, supports single-cell plating and subsequent NCM growth for 15 passages under this condition (Figure 3). Immunostaining of SCU-i30 cells with an anti-NANOG polyclonal antibody indicated that the cells under this condition had high NANOG expression in the nuclei (Figure 3A). Flow cytometric analysis showed that the hESC marker expression profile was similar to the cells grown as NCM using the ROCK inhibitor Y-27632&#160;(Figure 3B).
High efficiency of microRNA delivery without the use of lentiviral particles
Transfection with Dy547-labeled oligonucleotide microRNAs was carried out in WA01 (H1) cells under NCM conditions. WA01 hESCs were grown as NCM on 2.5% Matrigel in mTeSR1 for 2 (Figure 4A) and 18 (Figure 4B) passages respectively. These cells showed high transfection efficiency 24 hr post-transfection (Figures 4A and 4B). Generally, we can obtain transfection efficiency up to 91% in hESCs at 24 hr after the transfection.
<img alt="Figure 1" fo:content-width="6.5in" src="/files/ftp_upload/51519/51519fig1highres.jpg" width="600" /> 
Figure 1. (A) Schema of NCM culture. The line graph delineates the dynamic changes of multicellular association in a typical 3-day culture under NCM conditions. (B) Representative phase images of WA01 (H1) hESCs propagated under an NCM condition on 2.5% Matrigel in mTeSR1 medium for 16 passages (designated as WA01, mcp16). The lower panel is the enlarged view of the upper panel. Scale bars indicate 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51519/51519fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="7in" src="/files/ftp_upload/51519/51519fig2highres.jpg" width="600" /> 
Figure 2. Single-cell survival assays using a 96-well format. Approximately, 31,000 SCU-i10 hiPSCs17 were plated on 2.5% Matrigel in the presence of various small molecule inhibitors related to Rho-associated kinase (ROCK) pathways at indicated concentrations. After 24 hr, the cells were subjected to the CCK-8 survival assay by measuring the absorbance at 450 nm (A450). The student t-test was used to determine whether the differences in single-cell plating efficiency between various ROCK inhibitors are of statistical significance. The singular asterisk sign (*) indicates no significant differences between the inhibitors (P &#62; 0.05), whereas the double asterisk signs (**) denotes the observed difference is of statistical significance (P &#60; 0.05). Columns in the histograms designate the mean values of the quadruplicate determinants and bars represent standard deviations.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51519/51519fig3highres.jpg" width="600" /> 
Figure 3. NCM culture of hiPSCs on laminin-521 (LN-521) without the presence of ROCK inhibitors. The SCU-i30 hiPSC line was established at the NIH Stem Cell Unit. SCU-i30 cells were grown on LN-521-coated 6-well plates in the xeno-free medium TeSR2 for 15 passages without the use of small molecule inhibitors such as ROCK inhibitors. (A) Immunostaining of SCU-i30 cells with an anti-NANOG polyclonal antibody and counterstained with Hoechst 33342 (Hoechst). Notably, some cells that have a negative NANOG staining are the cells under mitosis. (B) Flow cytometric analysis of hPSC marker expression in SCU-i30 cells grown as NCM on 2.5% Matrigel with the use of 10 &#956;M Y-27632 (Upper panel) or NCM on LN-521 without Y-27632 (Lower panel). The procedures for both immunostaining and flow cytometric analysis were described previously5. Scale bars depict 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51519/51519fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="7in" src="/files/ftp_upload/51519/51519fig4highres.jpg" width="600" /> 
Figure 4. NCM culture of hPSCs for microRNA transfection. WA01 hESCs were grown as NCM on 2.5% Matrigel in mTeSR1 for 2 (A) and 18 (B) passages, respectively. Representative phase and fluorescence images of WA01 cells transfected with Dy547-labeled control microRNAs for monitoring transfection efficiency at 24 hr after the transfection. Scale bars represent 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51519/51519fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis | Biology | Video

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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12.3K Views.  National Institutes of Health. Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Video: Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

Developmental Biology

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

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

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

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

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.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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

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

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

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

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

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.

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.

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

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...

Neuroscience

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.

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 ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

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