maturation-human-stem-cell-derived-cardiomyocytes-biowires-using

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

University Health Network

Basic-helix-loop-helix-PAS transcription factors play important roles in diverse biological processes including cellular differentiation and specification, oxygen tension regulation and dioxin metabolism. Drosophila tango is orthologous to mammalian Arnt and acts as a common dimerization partner for bHLH-PAS proteins during embryogenesis. A transient transfection assay using Drosophila S2 tissue culture cells and wild-type and mutant Drosophila tango cDNAs was used to localize the activation domain of the Tango protein. An activation domain was identified in the C-terminus of TGO consisting of poly-glutamine and histidine-proline repeats. Transcriptional activation of the fibroblast growth factor receptor (breathless) gene required an intact TGO C-terminus, in vitro. Co-expression assays of trachealess and tgo in the developing eye imaginal disc showed a requirement for the C-terminal transactivation domain of TGO for a cellular response. Genetic analysis of tgo(3) shows that the paired repeat is necessary for tracheal tubule formation in all branches. Lastly, expression of a C-terminal truncated tgo transgene specifically in the CNS midline and trachea resulted in reductions in the number of breathless-expressing cells. These results together identify TGO's transactivation domain and establish its importance for proper target gene regulation and cellular specification.

Analysis of the transcriptional activation domain of the Drosophila tango bHLH-PAS transcription factor.

Neuronal-glial communication is essential for constructing the orthogonal axon scaffold in the developing Drosophila central nervous system (CNS). Longitudinal glia (LG) guide extending commissural and longitudinal axons while pioneer and commissural neurons maintain glial survival and positioning. However, the transcriptional regulatory mechanisms controlling these processes are not known. Previous studies showed that the midline function of the jing C2H2-type zinc-finger transcription factor was only partially required for axon scaffold formation in the Drosophila CNS. We therefore screened for gain-of-function enhancers of jing gain of function in the eye and identified the Drosophila homolog of the disease gene of human alpha-thalassemia/mental retardation X-linked (ATR-X) as well as other genes with potential roles in gene expression, translation, synaptic transmission, and cell cycle. jing and DATR-X reporter genes are expressed in both CNS neurons and glia, including the LG. Coexpression of jing and DATR-X in embryonic neurons synergistically affects longitudinal connective formation. During embryogenesis, jing and DATR-X have autonomous and nonautonomous roles in the lateral positioning of LG, neurons, and longitudinal axons as shown by cell-specific knockdown of gene expression. jing and DATR-X are also required autonomously for glial survival. jing and DATR-X mutations show synergistic effects during longitudinal axon formation suggesting that they are functionally related. These observations support a model in which downstream gene expression controlled by a potential DATR-X-Jing complex facilitates cellular positioning and axon guidance, ultimately allowing for proper connectivity in the developing Drosophila CNS.

Glial and neuronal functions of the Drosophila homolog of the human SWI/SNF gene ATR-X (DATR-X) and the jing zinc-finger gene specify the lateral positioning of longitudinal glia and axons.

In the developing Drosophila trachea, extensive cell migration lays the foundation for an elaborate network of tubules to form. This process is controlled by the Drosophila fibroblast growth factor receptor, known as Breathless (Btl), whose expression is activated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix (bHLH)-PAS transcription factors. We previously identified the jing zinc finger transcription factor as a gene sensitive to the dosage of bHLH-PAS transcriptional activity and showed that its mutations interact genetically with those of trh and btl. Here, we demonstrate that jing is required for btl expression in the branching trachea and dominantly interacts with known regulators of btl expression, including the ETS and POU transcription factors, pointed, and drifter/ventral veinless, respectively. Furthermore, the zinc finger-containing C-terminus of Jing associates with a btl tracheal enhancer in a Trh/Tgo-dependent manner in chromatin immunoprecipitation assays in vitro and interferes with btl in vitro and in vivo. Together, our results support a model by which Jing/Trh/Tgo complexes regulate btl transcript levels during primary tracheal branching.

Drosophila Jing is part of the breathless fibroblast growth factor receptor positive feedback loop.

The E2F4-p130 transcriptional repressor complex is a cell-cycle inhibitor in mitotic cells. However, the role of E2F4/p130 in differentiated cells is largely unknown. We investigated the role of E2F4/p130 in the regulation of apoptosis in postmitotic cardiomyocytes. Here we demonstrate that E2F4 can inhibit hypoxia-induced cell death in isolated ventricular cardiomyocytes. As analyzed by chromatin immunoprecipitation, the E2F4-p130-repressor directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1). In contrast, diminution of the E2F4-p130-HDAC1-repressor and recruitment of E2F1 and histone acetylase activity to these E2F-regulated promoters is required for the execution of cell death. Expression of kinase-dead HDAC1.H141A or HDAC-binding deficient p130ΔHDAC1 abolishes the antiapoptotic effect of E2F4. Moreover, histological examination of E2F4(-/-) hearts revealed a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data delineate an essential negative function of E2F4 in cardiac myocyte apoptosis.

Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes.

p27(Kip1) (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo.

p27 protein protects metabolically stressed cardiomyocytes from apoptosis by promoting autophagy.

Cardiovascular diseases remain the leading cause of death globally. Since the adult heart lacks the capacity to regenerate, loss of myocardium following myocardial infarction is irreversible and ultimately leads to failure to maintain cardiac function. In order to repopulate the areas of cell loss in the damaged hearts for restoration of cardiac function, cell transplantation/replacement has been extensively investigated. Recently, biomaterials have emerged as an approach to improve delivery and viability of cells for the regeneration of the damaged heart. Here we review the most common approaches in hydrogel-based cardiac tissue regeneration with particular focus on the implementation of hydrogels to improve cell delivery.

Overview of hydrogel-based strategies for application in cardiac tissue regeneration.

The primary function of vascular networks is to transport blood and deliver oxygen and nutrients to tissues, which occurs at the interface of the microvasculature. Therefore, the formation of the vessels at the microcirculatory level, or angiogenesis, is critical for tissue regeneration and repair. Current strategies for vascularization of engineered tissues have incorporated multi-disciplinary approaches including engineered biomaterials, cells and angiogenic factors. Pre-vascularization of scaffolds composed of native matrix, synthetic polymers, or other biological materials can be achieved through the use of single cells in mono or co-culture, in combination or not with angiogenic factors or by the use of isolated vessels. The advance of these methods, together with a growing understanding of the biology behind vascularization, has facilitated the development of vascularization strategies for engineered tissues with therapeutic potential for tissue regeneration and repair. Here, we review the different cell-based strategies utilized to pre-vascularize engineered tissues and in making more complex vascularized cardiac tissues for regenerative medicine applications.

Vascularization strategies of engineered tissues and their application in cardiac regeneration.

Human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CMs) represent a potential indefinite cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, these cells are immature compared with adult ventricular cardiomyocytes. In order to overcome this limitation, an engineered platform, called biowire, was devised to provide cultured cardiomyocytes important biomimetic cues present during embryo development, such as three-dimensional cell culture, extracellular matrix composition, soluble factors and pacing through electrical stimulation, to induce the maturation of hPSC-CMs in vitro.

Biowire platform for maturation of human pluripotent stem cell-derived cardiomyocytes.

Engineered blood vessels have often been found to be immature and unstable. Similarly, numerous pathologies such as diabetic retinopathy and cancer are characterized by highly abnormal, defective, hypervascular networks, consisting of immature, leaky, and irregular vessels with a marked loss of perivascular cell coverage. An emerging therapeutic concept in treatment of such vascular diseases and their management is the potential to normalize blood vessels by strengthening the cellular components that form the vascular network. Vessel normalization is characterized by the reduction in the number and size of immature vessels, a decrease in interstitial fluid pressure, and increase in perivascular cell coverage. Understanding the molecular and cellular defects associated with abnormal blood vessels will allow us to find appropriate treatment options that can promote normal blood vessel development. These, in turn, can be applied to improve vessel maturation in engineered tissues. In this review, we describe the major perivascular abnormalities associated with various human diseases and engineered vasculatures and the major advances in obtaining mature vasculatures for translational applications.

Blood Vessel Maturation in Health and Disease and its Implications for Vascularization of Engineered Tissues.

Cell-based tissue engineering is a potential treatment alternative for organ replacement. However, the lack of a robust vasculature, especially in the context of diseases such as diabetes, is a major hindrance to its success. Despite extensive research on the effects of diabetes in angiogenic sprouting, its effects on vessel arterio-venous (AV) specification have not been addressed. Using an engineered tissue that yields functional vessels with characteristic AV identities, we demonstrate that type 1 diabetes negatively affects vessel AV specification and perivascular cell (PVC) coverage. Blockage of PVC recruitment in normoglycemia does not affect blood flow parameters, but recapitulates the vascular immaturity found in diabetes, suggesting a role for PVCs in AV specification. The downregulation of Jagged1 and Notch3, key modulators of endothelial-perivascular interaction, observed in diabetes support this assertion. Co-culture assays indicate that PVCs induce arterial identity specification by inducing EphrinB2 and downregulating EphB4. This is antagonized by high glucose or blockage of endothelial Jagged1. Engineered tissues composed of microvessels from diabetic mice display normal PVC coverage and Jagged1/Notch3 gene expression when implanted into non-diabetic hosts. These indicate a lack of legacy effect and support the use of a more aggressive treatment of diabetes in patients undergoing revascularization therapies.

Diabetes impairs arterio-venous specification in engineered vascular tissues in a perivascular cell recruitment-dependent manner.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, hPSC-CMs produced by current protocols are not representative of native adult human cardiomyocytes as they display immature gene expression profile, structure and function. In order to improve hPSC-CM maturity and function, various approaches have been developed, including genetic manipulations to induce gene expression, delivery of biochemical factors, such as triiodothyronine and alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues, mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a combination of these. In this mini review, we discuss biomimetic strategies for the maturation for hPSC-CMs with a particular focus on electromechanical conditioning methods.

Article	Year
Analysis of the transcriptional activation domain of the Drosophila tango bHLH-PAS transcription factor. Development genes and evolution\| PubMed ID: 15818484	2005
Glial and neuronal functions of the Drosophila homolog of the human SWI/SNF gene ATR-X (DATR-X) and the jing zinc-finger gene specify the lateral positioning of longitudinal glia and axons. Genetics\| PubMed ID: 16648585	2006
Drosophila Jing is part of the breathless fibroblast growth factor receptor positive feedback loop. Development genes and evolution\| PubMed ID: 21061018	2010
Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes. Journal of molecular and cellular cardiology\| PubMed ID: 22985930	2012
p27 protein protects metabolically stressed cardiomyocytes from apoptosis by promoting autophagy. The Journal of biological chemistry\| PubMed ID: 24794871	2014
Overview of hydrogel-based strategies for application in cardiac tissue regeneration. Biomedical materials (Bristol, England)\| PubMed ID: 26040708	2015
Vascularization strategies of engineered tissues and their application in cardiac regeneration. Advanced drug delivery reviews\| PubMed ID: 26056716	2016
Biowire platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Methods (San Diego, Calif.)\| PubMed ID: 26546730	2016
Blood Vessel Maturation in Health and Disease and its Implications for Vascularization of Engineered Tissues. Critical reviews in biomedical engineering\| PubMed ID: 27480585	2015
Diabetes impairs arterio-venous specification in engineered vascular tissues in a perivascular cell recruitment-dependent manner. Biomaterials\| PubMed ID: 27988406	2017
Bioengineering Approaches to Mature Human Pluripotent Stem Cell-Derived Cardiomyocytes. Frontiers in cell and developmental biology\| PubMed ID: 28337437	2017

Xuetao Sun

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