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Summary The p21-activated kinase, Shk1, is required for the proper establishment of cell polarity in the fission yeast, Schizosaccharomyces pombe . We showed recently that loss of the essential Shk1 inhibitor, Skb15, causes significant spindle defects in fission yeast, thus implicating Shk1 as a potential regulator of microtubule dynamics. Here, we show that cells deficient in Shk1 function have malformed interphase microtubules and mitotic microtubule spindles, are hypersensitive to the microtubule-destabilizing drug thiabendazole (TBZ) and cold sensitive for growth. TBZ treatment causes a downregulation of Shk1 kinase activity, which increases rapidly after release of cells from the drug, thus providing a correlation between Shk1 kinase function and active microtubule polymerization. Consistent with a role for Shk1 as a regulator of microtubule dynamics, green fluorescent protein (GFP)–Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles, as well as to cell ends and septum-forming regions of fission yeast cells. We show that loss of Tea1, a cell end- and microtubule-localized protein previously implicated as a regulator of microtubule dynamics in fission yeast, exacerbates the growth and microtubule defects resulting from partial loss of Shk1 and that Shk1 localizes to illicit growth tips produced by tea1 mutant cells. Our results demonstrate that Shk1 is required for the proper regulation of microtubule dynamics in fission yeast and implicate Tea1 as a potential Shk1 regulator. [ABSTRACT FROM AUTHOR]

Elastin-associated vasculopathies are life-threatening conditions of blood vessel dysfunction. The extracellular matrix protein elastin endows the recoil and compliance required for physiologic arterial function, while disruption of function can lead to aberrant vascular smooth muscle cell proliferation manifesting through stenosis, aneurysm, or vessel dissection. Although research efforts have been informative, they remain incomplete as no viable therapies exist outside of a heart transplant. Induced pluripotent stem cell technology may be uniquely suited to address current obstacles as these present a replenishable supply of patient-specific material with which to study disease. The following review will cover the cutting edge in vascular smooth muscle cell modeling of elastin-associated vasculopathy, and aid in the development of human disease modeling and drug screening approaches to identify potential treatments. Vascular proliferative disease can affect up to 50% of the population throughout the world, making this a relevant and critical area of research for therapeutic development. [ABSTRACT FROM AUTHOR]

Familial cardiomyopathy is a precursor of heart failure and sudden cardiac death. Over the past several decades, researchers have discovered numerous gene mutations primarily in sarcomeric and cytoskeletal proteins causing two different disease phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathies. However, molecular mechanisms linking genotype to phenotype remain unclear. Here, we employ a systems approach by integrating experimental findings from preclinical studies (e.g. , murine data) into a cohesive signaling network to scrutinize genotype to phenotype mechanisms. We developed an HCM/DCM signaling network model utilizing a logic-based differential equations approach and evaluated model performance in predicting experimental data from four contexts (HCM, DCM, pressure overload, and volume overload). The model has an overall prediction accuracy of 83.8%, with higher accuracy in the HCM context (90%) than DCM (75%). Global sensitivity analysis identifies key signaling reactions, with calcium-mediated myofilament force development and calcium-calmodulin kinase signaling ranking the highest. A structural revision analysis indicates potential missing interactions that primarily control calcium regulatory proteins, increasing model prediction accuracy. Combination pharmacotherapy analysis suggests that downregulation of signaling components such as calcium, titin and its associated proteins, growth factor receptors, ERK1/2, and PI3K-AKT could inhibit myocyte growth in HCM. In experiments with patient-specific iPSC-derived cardiomyocytes (MLP-W4R;MYH7-R723C iPSC-CMs), combined inhibition of ERK1/2 and PI3K-AKT rescued the HCM phenotype, as predicted by the model. In DCM, PI3K-AKT-NFAT downregulation combined with upregulation of Ras/ERK1/2 or titin or Gq protein could ameliorate cardiomyocyte morphology. The model results suggest that HCM mutations that increase active force through elevated calcium sensitivity could increase ERK activity and decrease eccentricity through parallel growth factors, Gq-mediated, and titin pathways. Moreover, the model simulated the influence of existing medications on cardiac growth in HCM and DCM contexts. This HCM/DCM signaling model demonstrates utility in investigating genotype to phenotype mechanisms in familial cardiomyopathy. [Display omitted] • HCM/DCM signaling network model links mutations to cardiomyocyte morphological changes. • The computational model predicts higher efficiency for combination pharmacotherapies in familial cardiomyopathy. • Experiments on iPSC-CMs showed rescue of HCM phenotype by combined inhibition of ERK1/2 and PI3K-AKT predicted by the model. [ABSTRACT FROM AUTHOR]

Tissue engineered vascular grafts possess several advantages over synthetic or autologous grafts, including increased availability and reduced rates of infection and thrombosis. Engineered grafts constructed from human induced pluripotent stem cell derivatives further offer enhanced reproducibility in graft production. One notable obstacle to clinical application of these grafts is the lack of elastin in the vessel wall, which would serve to endow compliance in addition to mechanical strength. This study establishes the ability of the polyphenol compound epigallocatechin gallate, a principal component of green tea, to facilitate the extracellular formation of elastin fibers in vascular smooth muscle cells derived from human induced pluripotent stem cells. Further, this study describes the creation of a doxycycline-inducible elastin expression system to uncouple elastin production from vascular smooth muscle cell proliferative capacity to permit fiber formation in conditions conducive to robust tissue engineering. [Display omitted] • Epigallocatechin gallate addition leads to extracellular elastin formation in vitro. • Epigallocatechin gallate-treated tissue rings appear to display enhanced elasticity. • Doxycycline-inducible construct for ectopic expression of elastin mRNA and protein. • Use of doxycycline and epigallocatechin gallate produces elastin under high serum. • Applied to stem cell-derived culture for future elastin enriched vessel engineering. [ABSTRACT FROM AUTHOR]

Heart disease is one of the leading causes of death worldwide and the number of patients with the disease is likely to grow with the continual decline in health for most of the developed world. Heart transplantation is one of the only treatment options for heart failure due to an acute myocardial infarction, but limited donor supply and organ rejection limit its widespread use. Cellular cardiomyoplasty, or cellular implantation, combined with various tissue-engineering methods aims to regenerate functional heart tissue. This review highlights the numerous cell sources that have been used to regenerate the heart as well as cover the wide range of tissue-engineering strategies that have been devised to optimize the delivery of these cells. It will probably be a long time before an effective regenerative therapy can make a serious impact at the bedside. [ABSTRACT FROM AUTHOR]

Research in cell biology and the development of translational technologies are driven by competition, public expectations, and regulatory oversight, putting these fields at a critical juncture. Success in these fields is quickly becoming dependent on the ability of researchers to identify and isolate specific cell populations from heterogeneous mixtures accurately and efficiently. Many methods for cell purification have been developed, and each has advantages and disadvantages that must be considered in light of the intended application. Current cell separation strategies make use of surface proteins, genetic expression, and physics to isolate specific cells by phenotypic traits. Cell purification is also dependent on the cellular reagents available for use and the intended application, as these factors may preclude certain mechanisms used in the processes of labeling and sorting cells. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein, hiPSCs were differentiated into alpha-smooth muscle actin (α-SMA) and calponin-positive VSMCs, which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted, which contained cells largely positive for α-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover, when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts, they remained unruptured and patent with active vascular remodeling, and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs, and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) exist in different microenvironments in vivo , including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications. [ABSTRACT FROM AUTHOR]

As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs) could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs) could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs) may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP) expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of Isl1 expressing cardiac progenitors, which could lead to enhancement of cardiac differentiation and engraftment, holding a significant therapeutic value for cardiac repair in the future. [ABSTRACT FROM AUTHOR]

Summary: Bone morphogenetic proteins (BMPs) have diverse functions during development in vertebrates. We have recently shown that BMP2 signaling promotes venous-specific angiogenesis in zebrafish embryos. However, factors that confer a context-dependent proangiogenic function of BMP2 signaling within endothelial cells need to be identified. Here, we report that Disabled homolog 2 (Dab2), a cargo-specific adaptor protein for Clathrin, is essential to mediate the proangiogenic function of BMP2 signaling. We find that inhibition of Dab2 attenuates internalization of BMP receptors and abrogates the proangiogenic effects of BMP signaling in endothelial cells. Moreover, inhibition of Dab2 decreases phosphorylation of SMAD-1, 5, and 8, indicating that Dab2 plays an essential role in determining the outcome of BMP signaling within endothelial cells and may provide a molecular basis for a context-dependent proangiogenic function of BMP2 signaling. [ABSTRACT FROM AUTHOR]

Abstract: Murine embryonic stem cell (mESC)-derived cardiomyocytes represent a promising source of cells for use in the development of models for studying early cardiac development as well as cell-based therapies in postnatal pathologies. Here, we report a highly efficient cardiac differentiation system in which high density embryoid body (EB) cultures leads to a marked increase of cardiomyocytes production from multiple mESC lines without the addition of any cardiogenic growth factors. Our results show that high density EB cultures significantly increase the yield of functional cardiomyocytes, which express typical cardiac markers, exhibit normal rhythmic Ca2+ transients, and respond to both β-adrenergic and electric stimulations. During the differentiation period, the inhibition of bone morphogenetic protein (BMP) signaling significantly attenuates the increase of cardiac differentiation as well as the increased expression of cardiac-specific genes, NK2 transcription factor related 5 (Nkx2.5) and myosin light chain 2v (Mlc2v) by high density EB cultures. Therefore, we believe that we offer a novel and efficient means of cardiomyocyte production for practical use of mESCs in cardiac regenerative medicine. [Copyright &y& Elsevier]

Abstract: Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, existing approaches to cardiomyocyte production from human iPS cells are inefficient, limiting the application of iPS cells in basic and translational cardiac research. Furthermore, strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging, we showed that these induced cardiomyocytes expressed typical sarcomeric markers, exhibited normal rhythmic Ca2+ transients, and responded to both β-adrenergic and electric stimulation. Furthermore, human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus, modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct, rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics. [Copyright &y& Elsevier]

Conventional in vitro methods for biological evaluation of intra-arterial devices such as stents fail to accurately predict cytotoxicity and remodeling events. An ex vivo flow-tunable vascular bioreactor system (VesselBRx), comprising intra- and extra-luminal monitoring capabilities, addresses these limitations. VesselBRx mimics the in vivo physiological, hyperplastic, and cytocompatibility events of absorbable magnesium (Mg)-based stents in ex vivo stent-treated porcine and human coronary arteries, with in-situ and real-time monitoring of local stent degradation effects. Unlike conventional, static cell culture, the VesselBRx perfusion system eliminates unphysiologically high intracellular Mg2+ concentrations and localized O 2 consumption resulting from stent degradation. Whereas static stented arteries exhibited only 20.1% cell viability and upregulated apoptosis, necrosis, metallic ion, and hypoxia-related gene signatures, stented arteries in VesselBRx showed almost identical cell viability to in vivo rabbit models (~94.0%). Hyperplastic intimal remodeling developed in unstented arteries subjected to low shear stress, but was inhibited by Mg-based stents in VesselBRx, similarly to in vivo. VesselBRx represents a critical advance from the current static culture standard of testing absorbable vascular implants. [ABSTRACT FROM AUTHOR]

Summary: Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1 + precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1 + cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1 +/Nkx2.5 +/flk1 + defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1 + cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types. [Copyright &y& Elsevier]

Induced pluripotent stem cells (iPSC) represent an innovative, somatic cell-derived, easily obtained and renewable stem cell source without considerable ethical issues. iPSC and their derived cells may have enhanced therapeutic and translational potential compared with other stem cells. We previously showed that human iPSC-derived smooth muscle cells (hiPSC-SMC) promote angiogenesis and wound healing. Accordingly, we hypothesized that hiPSC-SMC may be a novel treatment for human patients with chronic limb-threatening ischemia who have no standard options for therapy. We determined the angiogenic potential of hiPSC-SMC in a murine hindlimb ischemia model. hiPSC-SMC were injected intramuscularly into nude mice after creation of hindlimb ischemia. Functional outcomes and perfusion were measured using standardized scores, laser Doppler imaging, microCT, histology and immunofluorescence. Functional outcomes and blood flow were improved in hiPSC-SMC-treated mice compared with controls (Tarlov score, p < 0.05; Faber score, p < 0.05; flow, p = 0.054). hiPSC-SMC-treated mice showed fewer gastrocnemius fibers (p < 0.0001), increased fiber area (p < 0.0001), and enhanced capillary density (p < 0.01); microCT showed more arterioles (<96 μm). hiPSC-SMC treatment was associated with fewer numbers of macrophages, decreased numbers of M1-type (p < 0.05) and increased numbers of M2-type macrophages (p < 0.0001). Vascular endothelial growth factor (VEGF) expression in ischemic limbs was significantly elevated with hiPSC-SMC treatment (p < 0.05), and inhibition of VEGFR-2 with SU5416 was associated with fewer capillaries in hiPSC-SMC-treated limbs (p < 0.0001). hiPSC-SMC promote VEGF-mediated angiogenesis, leading to improved hindlimb ischemia. Stem cell therapy using iPSC-derived cells may represent a novel and potentially translatable therapy for limb-threatening ischemia. [ABSTRACT FROM AUTHOR]

Wound healing is the physiologic response to a disruption in normal skin architecture and requires both spatial and temporal coordination of multiple cell types and cytokines. This complex process is prone to dysregulation secondary to local and systemic factors such as ischemia and diabetes that frequently lead to chronic wounds. Chronic wounds such as diabetic foot ulcers are epidemic with great cost to the healthcare system as they heal poorly and recur frequently, creating an urgent need for new and advanced therapies. Stem cell therapy is emerging as a potential treatment for chronic wounds, and adult-derived stem cells are currently employed in several commercially available products; however, stem cell therapy is limited by the need for invasive harvesting techniques, immunogenicity, and limited cell survival in vivo. Induced pluripotent stem cells (iPSC) are an exciting cell type with enhanced therapeutic and translational potential. iPSC are derived from adult cells by in vitro induction of pluripotency, obviating the ethical dilemmas surrounding the use of embryonic stem cells; they are harvested non-invasively and can be transplanted autologously, reducing immune rejection; and iPSC are the only cell type capable of being differentiated into all of the cell types in healthy skin. This review focuses on the use of iPSC in animal models of wound healing including limb ischemia, as well as their limitations and methods aimed at improving iPSC safety profile in an effort to hasten translation to human studies. [ABSTRACT FROM AUTHOR]