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     

Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions

Human pluripotent stem cells (hPSCs) are conventionally grown in a mouse feeder cell-dependent manner. Chemically defined culture conditions are, however, desirable not only for potential medically oriented applications but also for investigating mechanisms of self-renewal and differentiation. In light of the rather high complexity and cost of existing defined hPSC culture systems, we have systematically evaluated over 20 potential media ingredients. Only components that reproducibly gave beneficial effects were ultimately combined to yield a simple and cost-effective formulation termed FTDA. This xeno-free medium is based on mimicking self-renewal factor activities present in mouse embryonic fibroblast-conditioned medium, at minimal dosages. Additionally, small molecule inhibitors of BMP and WNT signaling served to specifically suppress typical types of spontaneous differentiation seen in hPSC cultures. FTDA medium was suitable for the generation of human induced pluripotent stem cells and enabled robust long-term maintenance of diverse hPSC lines including hard-to-grow ones. Comparisons with existing defined media suggested reduced spontaneous differentiation rates in FTDA. Our results imply that using supportive factors at minimal concentrations may still promote robust self-renewal and preserve pluripotency of hPSCs.     

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.     

Dendritic cells derived from pluripotent stem cells: Potential of large scale production简

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells (DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension culture of embryoid bodies (EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

Dendritic cells derived from pluripotent stem cells:Potential of large scale production

Human pluripotent stem cells(hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells(DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension cultureof embryoid bodies(EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Suspended in culture--human pluripotent cells for scalable technologies

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively termed human pluripotent stem cells (hPSCs), are typically derived and maintained in adherent and semi-defined culture conditions. Recently a number of groups, including Chen et al., 2012, have demonstrated that hESCs can now be expanded efficiently and maintain pluripotency over long-term passaging as aggregates in a serum-free defined suspension culture system, permitting the preparation of scalable cGMP derived hPSC cultures for cell banking, high throughput research programs and clinical applications. In this short commentary we describe the utility and potential future uses of suspension culture systems for hPSCs.     

Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells

The survival, proliferation, self-renewal and differentiation of human pluripotent stem cells (hPSCs, including human embryonic stem cells and human induced pluripotent stem cells) involve a number of processes that require cell-cell and cell-matrix interactions. The cell adhesion molecules (CAMs), a group of cell surface proteins play a pivotal role in mediating such interactions. Recent studies have provided insights into the essential roles and mechanisms of CAMs in the regulation of hPSC fate decisions. Here, we review the latest research progress in this field and focus on how E-cadherin and several other important CAMs including classic cadherins, Ig-superfamily CAMs, integrins and heparin sulfate proteoglycans control survival and differentiation of hPSCs     

Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells

The survival, proliferation, self-renewal and differentiation of human pluripotent stem cells (hPSCs, including human embryonic stem cells and human induced pluripotent stem cells) involve a number of processes that require cell-cell and cell-matrix interactions. The cell adhesion molecules (CAMs), a group of cell surface proteins play a pivotal role in mediating such interactions. Recent studies have provided insights into the essential roles and mechanisms of CAMs in the regulation of hPSC fate decisions. Here, we review the latest research progress in this field and focus on how E-cadherin and several other important CAMs including classic cadherins, Ig-superfamily CAMs, integrins and heparin sulfate proteoglycans control survival and differentiation of hPSCs     

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.     

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.     

A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells

Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.