Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells

Human pluripotent (embryonic or induced) stem cells (hPSCs) have many potential applications, not only for research purposes but also for clinical and industrial uses. While culturing these cells as undifferentiated lines, an adherent cell culture based on supportive layers or matrices is most often used. However, the use of hPSCs for industrial or clinical applications requires a scalable, reproducible and controlled process. Here we present a suspension culture system for undifferentiated hPSCs, based on a serum-free medium supplemented with interleukins and basic fibroblast growth factor, suitable for the mass production of these cells. The described system supports a suspension culture of hPSC lines, in both static and dynamic cultures. Results showed that hPSCs cultured with the described dynamic method maintained all hPSC features after 20 passages, including stable karyotype and pluripotency, and increased in cell numbers by 25-fold in 10 d. Thus, the described suspension method is suitable for large-scale culture of undifferentiated hPSCs.     

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.     

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have gained enormous interest as potential sources for regenerative biomedical therapies and model systems for studying early development. Traditionally, mouse embryonic fibroblasts have been used as a supportive feeder layer for the sustained propagation of hPSCs. However, the use of nonhuman‐derived feeders presents concerns about the possibility of xenogenic contamination, labor intensiveness, and variability in experimental results in hPSC cultures. Toward addressing some of these concerns, we report the propagation of three different hPSCs on feeder‐free extracellular matrix (ECM)‐based substrates derived from human fibroblasts. hPSCs propagated in this setting were indistinguishable by multiple criteria, including colony morphology, expression of pluripotency protein markers, trilineage in vitro differentiation, and gene expression patterns, from hPSCs cultured directly on a fibroblast feeder layer. Further, hPSCs maintained a normal karyotype when analyzed after 15 passages in this setting. Development of this ECM‐based culture system is a significant advance in hPSC propagation methods as it could serve as a critical component in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are important resources for cell-based therapies and pharmaceutical applications. In order to realize the potential of hPSCs, it is critical to develop suitable technologies required for specific applications. Most hPSC technologies depend on cell culture, and are critically influenced by culture medium composition, extracellular matrices, handling methods, and culture platforms. This review summarizes the major technological advances in hPSC culture, and highlights the opportunities and challenges in future therapeutic applications.     

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.     

Optimization of slow cooling cryopreservation for human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However, the current conventional method leads to poor recovery of hPSCs after thawing. Here, we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single‐cell dissociation. After confirming hPSC survivability after freeze‐thawing, we found that hPSCs that were freeze‐thawed as colonies showed markedly decreased survival, whereas freeze‐thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze‐thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post‐thawing. The improved method was also adapted to a xeno‐free culture system. Moreover, the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin‐521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high‐quality hPSCs. genesis 52:49–55, 2014. © 2013 Wiley Periodicals, Inc.     

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.     

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.     

Process engineering of human pluripotent stem cells for clinical application

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, constitute an extremely attractive tool for cell therapy. However, flexible platforms for the large-scale production and storage of hPSCs in tightly controlled conditions are necessary to deliver high-quality cells in relevant quantities to satisfy clinical demands. Here we discuss the main principles for the bioprocessing of hPSCs, highlighting the impact of environmental factors, novel 3D culturing approaches and integrated bioreactor strategies for controlling hPSC culture outcome. Knowledge on hPSC bioprocessing accumulated during recent years provides important insights for the establishment of more robust production platforms and should potentiate the implementation of novel hPSC-based therapies.     

Multisite studies for validation and improvement of a highly efficient culture assay for detection of undifferentiated human pluripotent stem cells intermingled in cell therapy products

Background aimsThe Multisite Evaluation Study on Analytical Methods for Non-Clinical Safety Assessment of Human-Derived Regenerative Medical Products (MEASURE) is a Japanese experimental public–private partnership initiative, which aims to standardize methodology for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). Undifferentiated hPSCs possess tumorigenic potential, and thus residual undifferentiated hPSCs are one of the major hazards for the risk of tumor formation from hPSC-derived CTPs. Among currently available assays, a highly efficient culture (HEC) assay is reported to be one of the most sensitive for the detection of residual undifferentiated hPSCs.MethodsMEASURE first validated the detection sensitivity of HEC assay and then investigated the feasibility of magnetic-activated cell sorting (MACS) to improve sensitivity.ResultsThe multisite experiments confirmed that the lower limit of detection under various conditions to which the human induced pluripotent stem cell lines and culture medium/substrate were subjected was 0.001%. In addition, MACS concentrated cells expressing undifferentiated cell markers and consequently achieved a detection sensitivity of 0.00002%.ConclusionsThese results indicate that HEC assay is highly sensitive and robust and that the application of MACS on this assay is a promising tool for further mitigation of the potential tumorigenicity risk of hPSC-derived CTPs.     

Development of a simple, repeatable, and cost-effective extracellular matrix for long-term xeno-free and feeder-free self-renewal of human pluripotent stem cells

Given the potential importance of human pluripotent stem cells (hPSCs) in translational research and regenerative medicine, the aim of the present study was to develop a simple, safe, and cost-effective substrate to expand hPSCs. We report the development of an extracellular matrix (ECM), designated “RoGel,” based on conditioned medium (CM) of human fibroblasts under serum- and xeno-free culture conditions. The long-term self-renewal of hPSCs on RoGel was also assessed. The results showed that self-renewal, pluripotency, plating efficiency, and cloning efficiency of hPSCs on this newly developed ECM were similar to those of Matrigel, the conventional mouse-cell line-derived ECM. The cells had the capability to passage mechanically on a cold surface, which resulted in their long-term maintenance with normal karyotype. We have demonstrated that CM-coated plates preserved for 1 year at room temperature maintained the capability of hPSC expansion. This ECM provides an attractive hPSC culture platform for both research and future therapeutic applications.     

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.     

Engineering the human pluripotent stem cell microenvironment to direct cell fate

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell–cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial–temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types.     

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.Download video file.^{(107M, mp4)}     

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.