Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20–60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (≤2.5 dyn/cm²) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies. Biotechnol. Bioeng. 2010; 105: 611–626. © 2009 Wiley Periodicals, Inc.     

Spatial Pattern Dynamics of 3D Stem Cell Loss of Pluripotency via Rules-Based Computational Modeling

Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.     

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.     

Loss of discordant cells during micro-mass differentiation of embryonic stem cells into the chondrocyte lineage

Embryonic stem cells (ESCs) have attracted particular interest in regenerative medicine because of their unlimited self-renewal and multipotentiality for differentiation. Spontaneous differentiated ESCs display heterogeneous multipotent cell populations and generate teratomas in vivo, with process by which ESCs differentiate into specific lineages remaining unclear. In this study, we focused on the in vitro chondrocyte differentiation of ESCs through micro-mass without using an embryoid body (EB) step and observed the unique characteristics of cartilage formation coupled with endochondral ossification in vivo. This approach resulted in an aggressive loss of discordant cells by apoptosis, which was accompanied by significant changes in gene expression during the course of ESC differentiation into chondrocytes. Unlike EB formation where discordant cells remain trapped within aggregates, micro-mass permits cells to die, leave the group and/or form a new group in response to changes in gene expression. Our observations suggest that the cell death that accompanies ESC micro-mass differentiation helps purify a terminally differentiated cell population and selects for targeted end points within a suitable microenvironment.     

Paramagnetic Beads and Magnetically Mediated Strain Enhance Cardiomyogenesis in Mouse Embryoid Bodies

Mechanical forces play an important role in proper embryologic development, and similarly such forces can directly impact pluripotency and differentiation of mouse embryonic stem cells (mESC) in vitro. In addition, manipulation of the embryoid body (EB) microenvironment, such as by incorporation of microspheres or microparticles, can similarly influence fate determination. In this study, we developed a mechanical stimulation regimen using permanent neodymium magnets to magnetically attract cells within an EB. Arginine-Glycine-Aspartic Acid (RGD)-conjugated paramagnetic beads were incorporated into the interior of the EBs during aggregation, allowing us to exert force on individual cells using short-term magnetization. EBs were stimulated for one hour at different magnetic field strengths, subsequently exerting a range of force intensity on the cells at different stages of early EB development. Our results demonstrated that following exposure to a 0.2 Tesla magnetic field, ESCs respond to magnetically mediated strain by activating Protein Kinase A (PKA) and increasing phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. The timing of stimulation can also be tailored to guide ESC differentiation: the combination of bone morphogenetic protein 4 (BMP4) supplementation with one hour of magnetic attraction on Day 3 enhances cardiomyogenesis by increasing contractile activity and the percentage of sarcomeric α-actin-expressing cells compared to control samples with BMP4 alone. Interestingly, we also observed that the beads alone had some impact on differentiation by increasingly slightly, albeit not significantly, the percentage of cardiomyocytes. Together these results suggest that magnetically mediated strain can be used to enhance the percentage of mouse ESC-derived cardiomyocytes over current differentiation protocols.     

Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) exhibit reduced efficiency and higher variability in neural differentiation compared to embryonic stem cells (ESCs). In this study, we showed that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation. We reported a novel approach which remarkably increases neural differentiation of mouse iPSCs. This novel approach initiated embryoid body (EB) formation directly from the whole cell clones isolated from the top of feeder cells. Compared to conventional neural induction methods such as single cell suspension or monolayer culture, the cell clone-derived EB method led to a pronounced increase in directed generation of various types of neural cells including neural stem cells, motoneurons and dopaminergic neurons in response to different inducers. Through gene expression microarray analysis, we identified 14 genes that were highly expressed in the cell clone-derived EBs. Among them, we found that Cdh2, also known as N-cadherin, played important roles in controlling the neural differentiation efficiency of mouse iPSCs. Forced expression of Cdh2 in iPSCs substantially enhanced the differentiation efficiency while knocking-down of Cdh2 by shRNA blocked the neural differentiation. Our results revealed a critical role of Cdh2 in the process of efficient neural differentiation of mouse iPS cells.     

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self‐organization into germ layers. In this work, we aimed to investigate how different three‐dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut‐off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification. Biotechnol. Bioeng. 2012; 109: 3119–3132. © 2012 Wiley Periodicals, Inc.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.     

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote™ as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote™, as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the silicon-coating of glass petri dishes by Sigmacote™ is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.     

Enrichment of cardiac differentiation of mouse embryonic stem cells by optimizing the hanging drop method

Hanging drop (HD) culture is used to induce differentiation of embryonic stem cells (ESCs) into other cell types including cardiomyocytes. However, the factors affecting cardiac differentiation of ESCs with this method remain incompletely understood. We have investigated the effects of the starting number of ESCs in embryoid bodies (EBs) and the time of EB adherence to gelatin-coated plates on cardiac differentiation: cardiac differentiation was increased in the EBs by a larger number of ESCs and was decreased by plating EBs at day 4 or earlier. These two factors can thus be optimized to enrich the cardiac differentiation in ESCs using the HD method.     

In vitro generation of germ cells from murine embryonic stem cells

The demonstration of germ cell and haploid gamete development from embryonic stem cells (ESCs) in vitro has engendered a unique set of possibilities for the study of germ cell development and the associated epigenetic phenomenon. The process of embryoid body (EB) differentiation, like teratoma formation, signifies a spontaneous differentiation of ESCs into cells of all three germ layers, and it is from these differentiating aggregates of cells that putative primordial germ cells (PGCs) and more mature gametes can be identified and isolated. The differentiation system presented here requires the differentiation of murine ESCs into EBs and the subsequent isolation of PGCs as well as haploid male gametes from EBs at various stages of differentiation. It serves as a platform for studying the poorly understood process of germ cell allocation, imprint erasure and gamete formation, with 4-6 weeks being required to isolate PGCs as well as haploid cells.     

Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors

Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.     

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 μm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.     

Inducing human induced pluripotent stem cell differentiation through embryoid bodies: A practical and stable approach

Human induced pluripotent stem cells (hiPSCs) are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use. They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research. However, significant limitations exist when using conventional flat culturing methods especially concerning cell expansion, differentiation efficiency, stability maintenance and multicellular 3D structure establishment, differentiation prediction. Embryoid bodies (EBs), the multicellular aggregates spontaneously generated from iPSCs in the suspension system, might help to address these issues. Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve, EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing, differentiation efficiency enhancement, ex vivo simulation, and organoid establishment. EBs can potentially also be used in early prediction of iPSC differentiation capability. To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs, critical factors including iPSC pluripotency maintenance, generation of uniform morphology using micro-pattern 3D culture systems, proper cellular density inoculation, and EB size control are discussed on the basis of both published data and our own laboratory experiences. Collectively, the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.     

Differentiation of mouse embryonic stem cells into dental epithelial-like cells induced by ameloblasts serum-free conditioned medium

Embryonic stem cells (ESCs) possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells; however, whether ESCs can differentiate toward the odontogenic lineage is still unknown. In this study, we developed an efficient culture strategy to induce the differentiation of murine ESCs (mESCs) into dental epithelial cells. By culturing mESCs in ameloblasts serum-free conditioned medium (ASF-CM), we could induce their differentiation toward dental epithelial cell lineages; however, similar experiments with the tooth germ cell-conditioned medium (TGC-CM) did not yield effective results. After culturing the cells for 14 days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.     

Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency

Murine embryonic stem cells (ESC) provide a unique homogeneous cell system for studying early vasculogenic cell differentiation in vitro. In this report, we characterized endothelial development of cultured E14 ESCs and mapped the effects of vascular endothelial growth factor (VEGF) on these cells. After removal of leukemia inhibitory factor undifferentiated state ESCs were precultured for 6 days and then cultured for up to 30 days in differentiation culture medium, with or without supplemental VEGF. ELISA analysis was used to detect endogenous VEGF levels. Early vasculogenic development and expression of selected genes were characterized using flow cytometry for specific antigens and quantitative RT-PCR. ELISA analysis showed no endogenous VEGF after preculture and at day 2 in unsupplemented culture, therafter VEGF levels rise. Directly after preculture a high proportion (36%) of the ESCs showed positivity for endothelial CD31. We describe characteristic endothelial differentiation patterns in embryoid bodies (EB) kept in culture for up to 30 days. VEGF supplementation lead to qualitative changes in the EB vessels, specific activation of vasculogenesis-related genes (CD31, CD144, and ERG) and temporary down-regulation of the VEGF receptor gene flk-1. VEGF supplementation did not produce measurable changes in the endothelial cell fractions as judged by surface antigen presence. We conclude that early ESCs may undergo endothelial differentiation through VEGF-independent pathways, whereas endothelial cell patterns in EBs are cytokine dependent and fully stimulated by endogenous cytokine levels.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

The Long-Term Differentiation of Embryonic Stem Cells into Cardiomyocytes: An Indirect Co-Culture Model

Background

Embryonic Stem Cells (ESCs) can differentiate into cardiomyocytes (CMs) in vitro but the differentiation level from ESCs is low. Here we describe a simple co-culture model by commercially available Millicell™ hanging cell culture inserts to control the long-term differentiation of ESCs into CMs.

Methodology/Principal Findings

Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with 0.1 mmol/L ascorbic acid to induce the differentiation of ESCs into CMs. In the indirect co-culture system, EBs were co-cultured with epidermal keratinocytes (EKs) or neonatal CMs (NCMs) by the hanging cell culture inserts (PET membranes with 1 µm pores). The molecular expressions and functional properties of ESC-derived CMs in prolonged culture course were evaluated. During time course of ESC differentiation, the percentages of EBs with contracting areas in NCMs co-culture were significantly higher than that without co-culture or in EKs co-culture. The functional maintenance of ESC-derived CMs were more prominent in NCMs co-culture model.

Conclusions/Significance

These results indicate that NCMs co-culture promote ESC differentiation and has a further effect on cell growth and differentiation. We assume that the improvement of the differentiating efficiency of ESCs into CMs in the co-culture system do not result from the effect of co-culture directly on cell differentiation, but rather by signaling effects that influence the cells in proliferation and long-term function maintenance.