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Embryonic stem cells (ESCs) are an important source of cardiomyocytes for regenerating injured myocardium. The successful use of ESC-derived cardiomyocytes in cardiac tissue engineering requires an understanding of the important scaffold properties and culture conditions to promote cell attachment, differentiation, organization, and contractile function. The goal of this work was to investigate how scaffold architecture and coculture with fibroblasts influences the differentiated phenotype of murine ESC-derived cardiomyocytes (mESCDCs). Electrospinning was used to process an elastomeric biodegradable polyurethane (PU) into aligned or unaligned fibrous scaffolds. Bioreactor produced mESCDCs were seeded onto the PU scaffolds either on their own or after pre-seeding the scaffolds with mouse embryonic fibroblasts (MEFs). Viable mESCDCs attached to the PU scaffolds and were functionally contractile in all conditions tested. Importantly, the aligned scaffolds led to the anisotropic organization of rod-shaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length-to-diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre-seeding the scaffolds with MEFs improved mESCDC sarcomere formation compared to mESCDCs cultured alone. These results suggest that both fiber alignment and pre-treatment of scaffolds with fibroblasts improve the differentiation of mESCDCs and are important parameters for developing engineered myocardial tissue constructs using ESC-derived cardiac cells., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

A successful regenerative therapy to treat damage incurred after an ischemic event in the heart will require an integrated approach including methods for appropriate revascularization of the infarct site, mechanical recovery of damaged tissue, and electrophysiological coupling with native cells. Cardiomyocytes are the ideal cell type for heart regeneration because of their inherent electrical and physiological properties, and cardiomyocytes derived from embryonic stem cells (ESCs) represent an attractive option for tissue-engineering therapies. An important step in developing tissue engineering-based approaches to cardiac cell therapy is understanding how scaffold architecture affects cell behavior. In this work, we generated large numbers of ESC-derived cardiomyocytes in bioreactors and seeded them on porous, 3-dimensional scaffolds prepared using 2 different techniques: electrospinning and thermally induced phase separation (TIPS). The effect of material macro-architecture on the adhesion, viability, and morphology of the seeded cells was determined. On the electrospun scaffolds, cells were elongated in shape, a morphology typical of cultured ESC-derived cardiomyocytes, whereas on scaffolds fabricated using TIPS, the cells retained a rounded morphology. Despite these gross phenotypic and physiological differences, sarcomeric myosin and connexin 43 expression was evident, and contracting cells were observed on both scaffold types, suggesting that morphological changes induced by material macrostructure do not directly correlate to functional differences.

Myocardial infarction is one of the leading causes of death in industrialized nations. Recent advances in tissue engineering and cell biology have changed our understanding of regenerative activities in the infarcted heart and have raised considerable hopes for novel therapeutic approaches to treat patients. Studies have shown that cell transplantation results in small improvements in the infarct area and while these therapies hold promise, significant challenges remain in order to increase both cellular engraftment efficiencies and transplanted cell function. Robust cardiac healing will require appropriate revascularization of the infarct site, mechanical recovery of damaged tissue and electrophysiological coupling with native tissue. Embryonic stem cells, uniquely, have the potential to generate bonafide cardiomyocytes and other derivatives which should contribute toward these multifaceted requirements. Guiding embryonic stem derived cells to support healing and regeneration of heart tissue using tissue engineered constructs may provide advantages over direct cell transplantation, including replacement of damaged infrastructure, temporary support for transplanted cells, and control of size, shape, strength and composition of the graft.

In the absence of leukemia inhibitory factor, murine embryonic stem cells cultured in vitro spontaneously aggregate to from three-dimensional embryoid bodies that differentiate to produce hematopoietic, endothelial, muscle, and neuronal cell lineages in a manner recapitulating the events of early embryogenesis. Cardiomyogenesis in embryoid bodies was recently demonstrated to be promoted by PYS-2-derived native SPARC (secreted protein, acidic and rich in cysteine), whose expression is upregulated in parietal endoderm at the onset of the epithelial to mesenchymal transition. Here, we confirm the stimulatory effects of mouse SPARC on cardiomyogenesis using a recombinant baculovirus-produced protein (rmSPARC). Embryoid bodies cultured in the presence of glycosylated rmSPARC, or an unglycosylated peptide spanning the C-terminal EF-hand domain, developed greater numbers of beating cardiomyocytes than did time-matched controls, with enhanced expression of cardiac marker genes including Nkx2.5, Troponin, BMP-2, and MHCα. Histochemical analysis revealed an expansion of the peripheral endoderm, with thicker layers of extracellular matrix (ECM) material observed atop underlying cells. Embryoid bodies treated with SPARC also displayed increased adherence to polystyrene culture dishes, with enhanced expression of ECM mRNAs including collagen IVα3, collagen IVα5, and laminin α1. These results indicate that, in addition to the promotion of cardiomyogenesis, SPARC may also help regulate the molecular composition and organization of ECM secreted by the mesenchymal parietal endoderm. En absence de la chimiokine LIF (« leukemia inhibitory factor »), les cellules souches embryonnaires de souris cultivées in vitro s’agrègent spontanément pour former des corps embryoïdes tridimensionnels qui se différencient pour produire des lignées cellulaires hématopoïétiques, musculaires et neuronales d’une façon qui récapitule les étapes de l’embryogenèse précoce. L’on a démontré récemment que la cardiomyogenèse est favorisée par la protéine native SPARC (« secreted protein, acidic and rich in cysteine ») produite par les cellules PYS-2, dont l’expression est stimulée lors de la transition épithélium à mésenchyme. Nous confirmons ici les effets stimulateurs de la SPARC de souris sur la cardiomyogenèse à l’aide d’une protéine recombinante produite chez le baculovirus (rsSPARC). Les corps embryoïdes cultivés en présence de rsSPARC glycosylée ou d’un peptide non glycosylé qui comprend le domaine EF-hand C-terminal, formaient un plus grand nombre de cardiomyocytes pulsatiles relativement aux contrôles appariés en fonction du temps. Ceci était accompagné d’une augmentation de l’expression de marqueurs cardiaques dont les gènes Nkx2,5, Troponin, BMP-2 et MHCα. Des analyses histochimiques ont révélé une expansion de l’endoderme périphérique et un épaississement de la couche de composantes de la matrice extracellulaire (MEC) observée sur les cellules sous-jacentes. Les corps embryoïdes traités à la SPARC adhéraient davantage au polystyrène des plats de culture et les ARNm des composantes de la MEC, dont le collagène IVα3, le collagène IV α5 et la laminine α1, étaient plus fortement exprimés. Ces résultats indiquent que, en plus de promouvoir la cardiomyogenèse, la SPARC peut aussi aider à réguler la composition moléculaire et l’organisation de la MEC secrétée par l’endoderme pariétal du mésenchyme. [ABSTRACT FROM AUTHOR]