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 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.
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 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).
Single-cell based non-colony type monolayer (NCM) culture of hPSCs.
1. Preparations
2. Protocol 1 (Basic): Grow hPSC Colonies on Feeders
3. Protocol 2: Convert hPSC Colonies from Feeders to NCM
4. Protocol 3: Convert hPSC Colonies on Matrigel to NCM Culture
5. Protocol 4: NCM Culture of hPSCs on LN-521
6. Protocol 5: NCM Culture for Plasmid DNA Transfection
7. Protocol 6: NCM Culture for Transfection of MicroRNAs
8. Protocol 7: NCM Culture for Transduction of Lentiviral Vector
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 μM 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 μM to enhance single-cell plating. In this report, we confirmed that 10 μM Y-27632 significantly increase 24 hr single-cell plating efficiency of hiPSCs (P < 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 μM when compared with their controls (P < 0.05) (Figure 2). Moreover, the effects of the three ROCK inhibitors (at 1 μM) on single-cell plating efficiency were comparable to that of Y-27632 at 10 μM (P > 0.05) (Figure 2). Notably, the ROCK I inhibitor (at 5 μM) appears to show pronounced cytotoxicity compared with the drug at 1 μM (P < 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 (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.
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 μm. Please click here to view a larger version of this figure.
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 > 0.05), whereas the double asterisk signs (**) denotes the observed difference is of statistical significance (P < 0.05). Columns in the histograms designate the mean values of the quadruplicate determinants and bars represent standard deviations.
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 μM 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 μm. Please click here to view a larger version of this figure.
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 μm. Please click here to view a larger version of this figure.
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 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 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., < 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 α6β1-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 (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.
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 authors have nothing to disclose.
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.
Countess automated cell counter | Invitrogen Inc. | C10227 | Automatic cell counting |
Faxitron Cabinet X-ray System | Faxitron X-ray Corporation, Wheeling, IL | Model RX-650 | X-ray irradiation of MEFs |
MULTIWELL six-well plates | Becton Dickinson Labware | 353046 | Polystyrene plates |
DMEM | Invitrogen Inc. | 11965–092 | For MEF medium |
mitomycin C | Roche | 107 409 | Mitotic inhibitor |
Trypsin | Invitrogen Inc. | 25300-054 | For MEF dissociation |
DMEM/F12 | Invitrogen Inc. | 11330–032 | For hPSC medium |
Opti-MEM I Reduced Serum Medium | Invitrogen Inc. | 31985-062 | For hPSC transfection |
Heat-inactivated FBS | Invitrogen Inc. | 16000–044 | Component of MEF medium |
Knockout Serum Replacer | Invitrogen Inc. | 10828–028 | KSR, Component of hPSC medium |
Dulbecco’s Phosphate-Buffered Saline | Invitrogen Inc. | 14190-144 | D-PBS, free of Ca2+/Mg2+ |
Non-essential amino acids | Invitrogen | 11140–050 | NEAA, component of hPSC medium |
L-Glutamine | Invitrogen | 25030–081 | Component of hPSC medium |
mTeSR1 & Supplements | StemCell Technologies | 5850 | Animal protein-free |
medium | |||
TeSR2 & Supplements | StemCell Technologies | 5860 | Xeno-free medium |
β-mercaptoethanol | Sigma | 7522 | Component of hPSC medium |
MEF (CF-1) ATCC |
American Type Culture Collection (ATCC) | SCRC-1040 | For feeder culture of hPSCs |
hESC-qualified Matrigel | BD Bioscience | 354277 | For feeder-free culture of hPSCs |
Laminin-521 | BioLamina | LN521-02 | Human recombinant protein |
FGF-2 (recombinant FGF, basic) | R&D Systems, MN | 223-FB | Growth factor in hPSC medium |
CryoStor CA10 | StemCell Technologies | 7930 | |
Accutase | Innovative Cell Technologies | AT-104 | 1X mixed enzymatic solution |
JAK inhibitor I | EMD4 Biosciences | 420099 | An inhibitor of Janus kinase |
Y-27632 | EMD4 Biosciences | 688000 | ROCK inhibitor |
Y-27632 | Stemgent | 04-0012 | ROCK inhibitor |
Y-39983 | Stemgent | 04-0029 | ROCK I inhibitor |
Phenylbenzodioxane | Stemgent | 04-0030 | ROCK II inhibitor |
Thiazovivin | Stemgent | 04-0017 | A novel ROCK inhibitor |
BD Falcon Cell Strainer | BD Bioscience | 352340 | 40-µm cell strainer |
Nalgene 5100-0001 Cryo 1°C | Thermo Scientific | C6516F-1 | “Mr. Frosty” Freezing Container |
Lipofectamine 2000 | Invitrogen Inc. | 11668-027 | Transfection reagents |
DharmaFECT Duo | Thermo Scientific | T-2010-02 | Transfection reagent |
Non-targeting miRIDIAN miRNA Transfection Control | Thermo Scientific | IP-004500-01-05 | Labeled with Dy547, to monitor the delivery of microRNAs |
SMART-shRNA | Thermo Scientific | To be determined | Lentiviral vector |
pmaxGFP | amaxa Inc (Lonza) | Included in every transfection kit | Expression plasmid for transfection control |
4-Oct | Santa Cruz Biotechnology | sc-5279 | Mouse IgG2b, pluripotent marker |
SSEA-1 | Santa Cruz Biotechnology | sc-21702 | Mouse IgM, differentiation marker |
SSEA-4 | Santa Cruz Biotechnology | sc-21704 | Mouse IgG3, pluripotent marker |
Tra-1-60 | Santa Cruz Biotechnology | sc-21705 | Mouse IgM, pluripotent marker |
Tra-1-81 | Santa Cruz Biotechnology | sc-21706 | Mouse IgM, pluripotent marker |
CK8 (C51) | Santa Cruz Biotechnology | sc-8020 | Mouse IgG1, against cytokeratin 8 |
α-fetoprotein | Santa Cruz Biotechnology | sc-8399 | AFP, mouse IgG2a |
HNF-3β (P-19) | Santa Cruz Biotechnology | sc-9187 | FOXA2, goat polyclonal antibody |
Troponin T (Av-1) | Thermo Scientific | MS-295-P0 | Mouse IgG1 |
Desmin | Thermo Scientific | RB-9014-P1 | Rabbit IgG |
Anti-NANOG | ReproCELL Inc, Japan | RCAB0004P-F | Polyclonal antibody |
Rat anti-GFAP | Zymed | 13-0300 | Glial fibrillary acidic protein |
Albumin (clone HSA1/25.1.3) | Cedarlane Laboratories Ltd. ( | CL2513A | Mouse IgG1, |
Smooth muscle actin (clone 1A4) | DakoCytomation Inc | IR611/IS611 | Mouse IgG2a |
Nestin | Chemicon International | MAB5326 | Rabbit polyclonal antibody |
TUBB3 | Convance Inc | MMS-435P | Tuj1, mouse IgG2a |
HNF4α (C11F12) | Cell Signaling Technologies | 3113 | Rabbit monoclonal antibody |
Paraformaldehyde (solution) | Electron Microscopy Sciences | 15710 | PFA, fixative, diluted in D-PBS |