Assessment of differentiation aspects by the morphological classification of embryoid bodies derived from human embryonic stem cells

In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ～10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.     

Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.     

Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell–cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.     

Automated mechanical passaging: a novel and efficient method for human embryonic stem cell expansion

There is a need for more standardized methods of maintenance and propagation of human embryonic stem cell (hESC) cultures. Enzymatic passaging currently represents the most widely used method for expansion of hESCs. Although rapid and straightforward, this technique results in variable-sized cell clusters and significant cellular trauma, which may apply selective pressure in long-term culture. Mechanical passaging has the potential advantages of defined colony fragment sizes, reduced cellular trauma, and the possibility of selecting undifferentiated colonies for transfer. However, manual dissection of individual colonies is a prohibitively time-consuming process unsuitable for maintaining large numbers of hESCs without the use of additional chemical means. In this study we report an efficient automated method for mechanically passaging hESCs. We have used this method exclusively to maintain hESCs in long-term undifferentiated culture without the use of enzymatic digestion for longer than 100 days. This automated technique can thus be used routinely to culture hESCs in the laboratory.     

The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance

The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination.     

Establishment of clinically compliant human embryonic stem cells in an autologous feeder-free system

Applications of human embryonic stem cells (hESCs) are limited by the use of mouse embryonic fibroblasts feeder and animal-derived components during culture. In this study, we demonstrated the potential use of extracellular matrix (ECM) derived from the autologous feeders to support long-term undifferentiated growth of hESCs in xeno-free, serum-free, and feeder-free conditions. Autologous H9 ebF (feeder cells derived from outgrowth of embryoid body [EB] predifferentiated from H9 hESCs) was derived from EBs predifferentiated from H9 hESCs through a direct-plating outgrowth system. The ECM comprising collagen VI, laminin, and fibronectin was extracted from H9 ebF through a freeze-thaw procedure. The autologous ECM together with animal component-free TeSR?2 medium was used to support long-term growth of H1 and H9 hESC lines for up to 20 passages. The maintenance of hESC undifferentiated state by autologous ECM was confirmed by the positive staining of hESC-specific markers (Oct4, SSEA-4, and Tra-1-60) and the expression of pluripotency marker genes (Oct4, Nanog, and Sox2). Flow cytometry further showed that more than 99% of hESCs retained the expression of SSEA-3/4 after long-term culture on autologous ECM. Pluripotency of hESCs on ECM was further proven by in vitro EB formation and in vivo teratoma assay. Overall, this study suggested a strategy for efficient propagation of clinically compliant hESCs in an autologous feeder-free culture system.     

Modulation of Wnt/β-catenin signaling in human embryonic stem cells using a 3-D microwell array

Intercellular interactions in the cell microenvironment play a critical role in determining cell fate, but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates, including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell-cell contact on canonical Wnt/β-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs, and competition for β-catenin between the Wnt pathway and cadherin-mediated cell-cell interactions impacts various developmental processes, including the epithelial-mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling, as evidenced by the lack of nuclear β-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures, embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore, the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/β-catenin signaling in hESCs.     

Mass transfer limitations in embryoid bodies during human embryonic stem cell differentiation

Due to their ability to differentiate into cell types from all the three germ layers and their potential unlimited capacity for expansion, embryonic stem cells have tremendous potential to treat diseases and injuries. Spontaneous differentiation of human embryonic stem cells (hESCs) is influenced by the size of the differentiating embryoid bodies (EBs). To further understand the dynamics between nutrient mass transfer, EB size, and stem cell differentiation, a transient mass diffusion model of a single hESC EB was constructed. The results revealed that the oxygen concentration at the centers of large EBs (400-μm radius) was 50% lower when compared to that in smaller EBs (200-μm radius). In addition, the concentration profile of cytokines within an EB depended strongly on their depletion rate, with higher depletion rates resulting in cytokine concentrations that varied significantly throughout the EB. A comparison of the results of our model with published experimental data reveals a close correlation between the fraction of cells that differentiate to a given lineage and the fraction of cells exposed to different oxygen or cytokine concentrations. This, along with other data from the literature, suggests that diffusive mass transfer influences the differentiation of hESCs within EBs by controlling the spatial distribution of soluble factors. This has important implications for research involving the differentiation of embryonic stem cells in EBs, as well as for bioprocess design and the development of robust differentiation protocols where mass transfer could be altered to control the cell differentiation trajectory.     

Cultivation of human embryonic stem cells without the embryoid body step enhances osteogenesis in vitro

Osteogenic cultures of embryonic stem cells (ESCs) are predominately derived from three-dimensional cell spheroids called embryoid bodies (EBs). An alternative method that has been attempted and merits further attention avoids EBs through the immediate separation of ESC colonies into single cells. However, this method has not been well characterized and the effect of omitting the EB step is unknown. Herein, we report that culturing human embryonic stem cells (hESCs) without the EB stage leads to a sevenfold greater number of osteogenic cells and to spontaneous bone nodule formation after 10-12 days. In contrast, when hESCs were differentiated as EBs for 5 days followed by plating of single cells, bone nodules formed after 4 weeks only in the presence of dexamethasone. Furthermore, regardless of the inclusion of EBs, bone matrix formed, including cement line matrix and mineralized collagen, which displayed apatitic mineral (PO4) with calcium-to-phosphorous ratios similar to those of hydroxyapatite and human bone. Together these results demonstrate that culturing hESCs without an EB step can be used to derive large quantities of functional osteogenic cells for bone tissue engineering.     

Generation and differentiation of human embryonic stem cell-derived keratinocyte precursors

Human embryonic stem cells (hESC) hold tremendous potential in the future of tissue engineering, offering promise as a source of virtually unlimited quantities of desired cell and tissue types. We have identified soluble chemical and extracellular matrix factors that permit isolation of keratinocyte precursors from hESCs. Culturing embryoid bodies (EB) formed from hESCs in a defined serum-free keratinocyte growth medium on a gelatin matrix generated keratin 14 (K14) expressing cells with an epithelial morphology. These K14 expressing cells could be subcultured in medium supplemented with hydrocortisone and induced to stratify and terminally differentiate by addition of calcium. Optimum times for obtaining K14 expressing cells were found for EB formation and for differentiation and growth of cultures after EB plating. EB formation was not necessary to generate keratinocyte precursors; direct transfer of hESC colonies to keratinocyte growth medium permitted differentiation into the keratinocyte lineage. With further studies to optimize generation and purification of hESC-derived keratinocyte precursors, these cells could provide a source of epidermal cells for skin tissue engineering applications in vitro or in vivo.     

Human-serum matrix supports undifferentiated growth of human embryonic stem cells

One of the most frequently used matrices for feeder-free growth of undifferentiated human embryonic stem cells (hESCs) is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of mouse embryonic fibroblast-conditioned medium. Unfortunately, application of Matrigel or medium conditioned by mouse embryonic feeder cells is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions. We demonstrate here that human serum as matrix and medium conditioned by differentiated hESCs reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal-free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.     

Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of >or=30 AP(+) cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.     

Human Embryonic Stem Cells (hESCs) Cultured Under Distinctive Feeder-Free Culture Conditions Display Global Gene Expression Patterns Similar to hESCs from Feeder-Dependent Culture Conditions

Human embryonic stem cell (hESC)–based assay systems and genetically modified hESCs are very useful tools for screening drugs that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination. For these types of studies, feeder cell–dependent cultures of hESCs are often problematic because the physiology of the feeder cells is perturbed by the drug treatments or genetic modifications, which potentially obscures research outcomes. In this study, we evaluated three commonly used feeder-free culture conditions to determine whether they supported the undifferentiated growth of hESCs and to determine whether the hESCs grown in these conditions displayed gene expression patterns that were similar to the expression patterns of feeder cell-dependent hESCs. Our results demonstrate that hESCs grown in the three feeder-free conditions expressed undifferentiation marker genes as strongly as hESCs that were grown in the feeder-dependent cultures. Furthermore, genome-wide gene expression profiles indicated that the gene expression patterns of hESCs that were grown under feeder-free or feeder-dependent culture conditions were highly similar. These results indicate that the feeder-free culture conditions support the undifferentiated growth of hESCs as effectively as the feeder-dependent culture conditions. Therefore, feeder-free culture conditions are potentially suitable for drug screening and for the genetic manipulation of hESCs in basic research.     

Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.     

Notch signaling is inactive but inducible in human embryonic stem cells

The NOTCH signaling pathway performs a wide range of critical functions in a number of different cell types during development and differentiation. The role of NOTCH signals in human embryonic stem cells (hESCs) has not been tested. We measured the activity of canonical NOTCH signaling in undifferentiated embryonic stem (ES) cells and tested the requirement for NOTCH activity in hESC self-renewal or differentiation by growing hESCs in the presence of gamma-secretase inhibitors. Our results suggest that NOTCH signaling is not required for the propagation of undifferentiated human ES cells but instead is required for the maintenance of the differentiating cell types that accumulate in human ES cell cultures. Our studies suggest that NOTCH signaling is not required in human embryonic differentiation until the formation of extraembryonic, germ layer, or tissue-specific stem cells and progenitors.     

Human Embryonic Stem Cells: Mechanisms to Escape Replicative Senescence?

Human embryonic stem cells (hESCs) are unique in that they can proliferate indefinitely in culture in an undifferentiated state as well as differentiate into any somatic cells. Undifferentiated hESCs do not appear to undergo senescence and remain nontransformed over multiple passages. Culture hESCs maintain telomere length and exhibit high telomerase activity after prolonged in vitro culture. The ability of hESCs to bypass senescence is lost as hESCs differentiate into fully differentiated somatic cells. This loss of immortality upon differentiation may be due to a variety aging related factors such as reduction in telomere length, alteration of telomerase activity, changes in cell cycle regulation and decrease in DNA repair ability. Absence of such aging factors as well as the lack of genomic, mitochondrial and epigenetic changes, may contribute to the lack of senescence in hESCs. In this review, we will summarize recent advances in determining changes in these aspects in prolonged hESC cultures. We will in particular discuss the potential roles of several cellular pathways including the telomerase, p53, and Rb pathways in escaping senescence in hESCs. We will also discuss the genomic and epigenetic changes in long-term hESC culture and their potential roles in bypassing senescence, as well as alternative sources of pluripotent stem cells.