An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.

• Keywords
• biomimetic, cardiomyogenesis, fluorescence signal, human-induced pluripotent stem cells, surfaces,
• MeSH
• Biocompatible Materials chemistry   MeSH
• Cell Differentiation * MeSH
• Fluorescence * MeSH
• Induced Pluripotent Stem Cells cytology   MeSH
• Myocytes, Cardiac cytology   MeSH
• Humans MeSH
• Polystyrenes chemistry   MeSH
• Cell Proliferation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biocompatible Materials MeSH
• Polystyrenes MeSH

Polyaniline cryogel is a new unique form of polyaniline combining intrinsic electrical conductivity and the material properties of hydrogels. It is prepared by the polymerization of aniline in frozen poly(vinyl alcohol) solutions. The biocompatibility of macroporous polyaniline cryogel was demonstrated by testing its cytotoxicity on mouse embryonic fibroblasts and via the test of embryotoxicity based on the formation of beating foci within spontaneous differentiating embryonic stem cells. Good biocompatibility was related to low contents of low-molecular-weight impurities in polyaniline cryogel, which was confirmed by liquid chromatography. The adhesion and growth of embryonic stem cells, embryoid bodies, cardiomyocytes, and neural progenitors prove that polyaniline cryogel has the potential to be used as a carrier for cells in tissue engineering or bio-sensing. The surface energy as well as the elasticity and porosity of cryogel mimic tissue properties. Polyaniline cryogel can therefore be applied in bio-sensing or regenerative medicine in general, and mainly in the tissue engineering of electrically excitable tissues.

• MeSH
• Algorithms MeSH
• Aniline Compounds chemistry   MeSH
• Biocompatible Materials chemistry   MeSH
• Cell Culture Techniques MeSH
• Electric Conductivity MeSH
• Fibroblasts MeSH
• Cryogels chemistry   MeSH
• Mechanical Phenomena MeSH
• Elastic Modulus MeSH
• Mice MeSH
• Porosity MeSH
• Models, Theoretical MeSH
• Materials Testing MeSH
• Tissue Engineering MeSH
• Cell Survival MeSH
• Chromatography, High Pressure Liquid MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aniline Compounds MeSH
• Biocompatible Materials MeSH
• Cryogels MeSH
• polyaniline MeSH Browser

The potentiation of the naturally limited regenerative capacity of the heart is dependent on an understanding of the mechanisms that are activated in response to pathological conditions such as hypoxia. Under these conditions, the expression of genes suggested to support cardiomyocyte survival and heart adaptation is triggered. Particularly important are changes in the expression of myosin heavy chain (MHC) isoforms. We propose here that alterations in the expression profiles of MHC genes are induced in response to hypoxia and are primarily mediated by hypoxia inducible factor (HIF). In in vitro models of mouse embryonic stem cell-derived cardiomyocytes, we showed that hypoxia (1% O2) or the pharmacological stabilization of HIFs significantly increased MHCbeta (Myh7) gene expression. The key role of HIF-1alpha is supported by the absence of these effects in HIF-1alpha-deficient cells, even in the presence of HIF-2alpha. Interestingly, ChIP analysis did not confirm the direct interaction of HIF-1alpha with putative HIF response elements predicted in the MHCalpha and beta encoding DNA region. Further analyses showed the significant effect of the mTOR signaling inhibitor rapamycin in inducing Myh7 expression and a hypoxia-triggered reduction in the levels of antisense RNA transcripts associated with the Myh7 gene locus. Overall, the recognized and important role of HIF in the regulation of heart regenerative processes could be highly significant for the development of novel therapeutic interventions in heart failure.

• Keywords
• fetal gene program, heart, hypoxia, mouse, myosin heavy chain,
• Publication type
• Journal Article MeSH

The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

• MeSH
• Acetylcysteine administration & dosage   MeSH
• Actinin genetics   MeSH
• Embryonic Stem Cells cytology   MeSH
• Homeobox Protein Nkx-2.5 genetics   MeSH
• Myocytes, Cardiac cytology   MeSH
• Culture Media, Serum-Free * MeSH
• Cells, Cultured MeSH
• Myosins genetics   MeSH
• Mice MeSH
• In Vitro Techniques MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcysteine MeSH
• Actinin MeSH
• Homeobox Protein Nkx-2.5 MeSH
• Culture Media, Serum-Free * MeSH
• Myosins MeSH
• Nkx2-5 protein, mouse MeSH Browser

Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.

• MeSH
• Actinin metabolism   MeSH
• Cell Differentiation MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit genetics   metabolism   MeSH
• Gene Knockout Techniques MeSH
• Cell Hypoxia MeSH
• Myocytes, Cardiac cytology   metabolism   MeSH
• Oxygen chemistry   MeSH
• Myosin Light Chains metabolism   MeSH
• Mouse Embryonic Stem Cells cytology   metabolism   MeSH
• Mice MeSH
• Regeneration MeSH
• Heart embryology   MeSH
• Gene Expression Profiling MeSH
• Myosin Heavy Chains metabolism   MeSH
• Troponin T metabolism   MeSH
• Muscle Development * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Actinin MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit MeSH
• Hif1a protein, mouse MeSH Browser
• Oxygen MeSH
• Myosin Light Chains MeSH
• Mlc2a protein, mouse MeSH Browser
• Myosin Heavy Chains MeSH
• Troponin T MeSH