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.     

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.     

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.     

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.     

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

Primate embryonic stem cells create their own niche while differentiating in three-dimensional culture systems

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.     

Derivation of murine induced pluripotent stem cells (iPS) and assessment of their differentiation toward osteogenic lineage

Induced pluripotent stem cells (iPSCs) have generated hope and excitement because of the potential they possess for generating patient‐specific embryonic‐like stem cells (ESCs). Although many hurdles remain to be solved before the cells can be applied clinically; studies directed toward understanding factors that control differentiation of the cells toward various cell lineages are prerequisites for their future application. In the present study, we generated murine iPSC and assessed their differentiation toward osteogenic lineage. Murine tail tip fibroblasts were reprogrammed into embryonic‐like state by transduction with defined factors (Oct3/4, Sox2, c‐Myc, and klf4) carried in a retroviral vector. The reprogrammed cells expressed ESC markers, gave rise to three germ layers as demonstrated by teratoma formation and immunofluorescence staining. These data confirmed that the reprogrammed cells exhibited ESC‐like state. Treatment of iPSCs‐derived embryoid bodies (EBs) with transforming growth factor beta 1 (TGF‐β1) in the presence of retinoic acid enhanced generation of MSC‐like cells. The MSCs‐like cells expressed putative makers associated with MSCs; the cells deposited calcium in vitro when cultured in osteogenic medium. Interestingly MSCs‐like cells generated from iPSC directed EBs by treatment with retinoic acid and TGF‐β1 deposited more calcium in vitro than cells derived without TGF‐β1 treatment. Taken together, the data demonstrate that iPSC give rise to MSCs‐like state and that the cells have potential to differentiate toward osteoblasts. In addition, brief treatment of iPSC‐derived EBs with TGF‐β1 may be an approach for directing iPSC toward MSC‐like state. J. Cell. Biochem. 109: 643–652, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Cytotoxicity of the anticancer drug doxorubicin for human embryonic stem cells

The cytotoxic effect of the anticancer drug doxorubicin (DR) on human embryonic stem cells (ESCs) C910 and fibroblasts spontaneously differentiated from these cells has been examined. The fibroblasts retained a diploid karyotype. It was found that ESCs are more sensitive to DR than fibroblasts: the DR dose killing 20% of cells was 0.01 and 0.1 μg/mL, respectively. DR induced ESC apoptotic death and reduced both ESC and fibroblast proliferation. DR reversibly inhibited ESC, but not fibroblast, proliferation. Thus, we demonstrated that ESCs and differentiated derivatives thereof are distinguished by sensitivity and response to the genotoxic agent.     

Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.     

One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating

Controlled differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into cells that resemble adult mesenchymal stem cells (MSCs) is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.     

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.     

The fibroblast growth factor signaling axis controls cardiac stem cell differentiation through regulating autophagy

The fibroblast growth factor (FGF) signaling axis plays important roles in heart development. Yet, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. Using genetically engineered mouse and in vitro cultured embryoid body (EB) models, we demonstrate that FGF signaling suppresses premature differentiation of heart progenitor cells, as well as autophagy in outflow tract (OFT) myocardiac cells. The FGF also promotes mesoderm differentiation in embryonic stem cells (ESCs) but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, inhibition of FGF signaling increases myocardial differentiation and autophagy in both ex vivo cultured embryos and EBs, whereas activation of autophagy promotes myocardial differentiation. Thus, a link between FGF signals preventing premature differentiation of heart progenitor cells and suppression of autophagy has been established. These findings provide the first evidence that autophagy plays a role in heart progenitor differentiation, and suggest a new venue to regulate stem/progenitor cell differentiation.     

Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation

Embryonic stem cells (ESCs) are pluripotent cells capable of differentiating into all somatic and germ cell types. The intrinsic ability of pluripotent cells to generate a vast array of different cells makes ESCs a robust resource for a variety of cell transplantation and tissue engineering applications, however, efficient and controlled means of directing ESC differentiation is essential for the development of regenerative therapies. ESCs are commonly differentiated in vitro by spontaneously self‐assembling in suspension culture into 3D cell aggregates called embryoid bodies (EBs), which mimic many of the hallmarks of early embryonic development, yet the 3D organization and structure of EBs also presents unique challenges to effectively direct the differentiation of the cells. ESC differentiation is strongly influenced by physical and chemical signals comprising the local extracellular microenvironment, thus current methods to engineer EB differentiation have focused primarily on spatially controlling EB size, adding soluble factors to the media, or culturing EBs on or within natural or synthetic extracellular matrices. Although most such strategies aim to influence differentiation from the exterior of EBs, engineering the microenvironment directly within EBs enables new opportunities to efficiently direct the fate of the cells by locally controlling the presentation of morphogenic cues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The fibroblast growth factor signaling axis controls cardiac stem cell differentiation through regulating autophagy

The fibroblast growth factor (FGF) signaling axis plays important roles in heart development. Yet, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. Using genetically engineered mouse and in vitro cultured embryoid body (EB) models, we demonstrate that FGF signaling suppresses premature differentiation of heart progenitor cells, as well as autophagy in outflow tract (OFT) myocardiac cells. The FGF also promotes mesoderm differentiation in embryonic stem cells (ESCs) but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, inhibition of FGF signaling increases myocardial differentiation and autophagy in both ex vivo cultured embryos and EBs, whereas activation of autophagy promotes myocardial differentiation. Thus, a link between FGF signals preventing premature differentiation of heart progenitor cells and suppression of autophagy has been established. These findings provide the first evidence that autophagy plays a role in heart progenitor differentiation, and suggest a new venue to regulate stem/progenitor cell differentiation.     

Effects of mast cells on the behavior of isolated heart fibroblasts: modulation of collagen remodeling and gene expression

The extracellular matrix plays a critical role in the development and maintenance of the vertebrate heart. Changes in the accumulation, composition, or organization of the extracellular matrix are known to deleteriously affect heart function. Mast cells are thought to stimulate collagen expression and fibroblast proliferation accompanying fibrosis in some organs; however, the effects of mast cells on the heart interstitium are largely unexplored. The present studies were carried out to determine the effects of mast cells on isolated heart fibroblasts. Several in vitro assays were used including collagen gel contraction to examine the effects of mast cells on the function of isolated fibroblasts. Neonatal heart fibroblasts were cultured either with mast cells, mast cell-conditioned medium, or mast cell extracts, and their ability to contract collagen gels measured. Results from these experiments indicated that mast cells inhibit heart fibroblast migration and contraction of 3-dimensional collagen gels. Further experiments indicated that incubation of neonatal heart fibroblasts with extracts of mast cells altered the expression of collagen, matrix metalloproteases, and matrix receptors of the integrin family. These studies suggest that mast cells play an important role in the regulation of the cardiac interstitial matrix. Further studies are warranted to determine the mechanisms whereby mast cells modulate fibroblast activity.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Comparison of different protocols for neural differentiation of human induced pluripotent stem cells

Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.