Biomaterializing the promise of cardiac tissue engineering.

During an average individual's lifespan, the human heart pumps nearly 200 million liters of blood delivered by approximately 3 billion heartbeats. Therefore, it is not surprising that native myocardium under this incredible demand is extraordinarily complex, both structurally and functionally. As a result, successful engineering of adult-mimetic functional cardiac tissues is likely to require utilization of highly specialized biomaterials representative of the native extracellular microenvironment. There is currently no single biomaterial that fully recapitulates the architecture or the biochemical and biomechanical properties of adult myocardium. However, significant effort has gone toward designing highly functional materials and tissue constructs that may one day provide a ready source of cardiac tissue grafts to address the overwhelming burden of cardiomyopathic disease. In the near term, biomaterial-based scaffolds are helping to generate in vitro systems for querying the mechanisms underlying human heart homeostasis and disease and discovering new, patient-specific therapeutics. When combined with advances in minimally-invasive cardiac delivery, ongoing efforts will likely lead to scalable cell and biomaterial technologies for use in clinical practice. In this review, we describe recent progress in the field of cardiac tissue engineering with particular emphasis on use of biomaterials for therapeutic tissue design and delivery.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.

Stem Cell Surface Marker Expression Defines Late Stages of Reprogramming to Pluripotency in Human Fibroblasts.

UNLABELLED: Our current understanding of the induction of pluripotency by defined factors indicates that this process occurs in discrete stages characterized by specific alterations in the cellular transcriptome and epigenome. However, the final phase of the reprogramming process is incompletely understood. We sought to generate tools to characterize the transition to a fully reprogramed state. We used combinations of stem cell surface markers to isolate colonies emerging after transfection of human fibroblasts with reprogramming factors and then analyzed their expression of genes associated with pluripotency and early germ lineage specification. We found that expression of a subset of these genes, including the cell-cell adhesion molecule CDH3, characterized a late stage in the reprogramming process. Combined live-cell staining with the antibody GCTM-2 and anti-CDH3 during reprogramming identified colonies of cells that showed gene expression patterns very similar to those of embryonic stem cell or established induced pluripotent stem cell lines, and gave rise to stable induced pluripotent stem cell lines at high frequency. Our findings will facilitate studies of the final stages of reprogramming of human cells to pluripotency and will provide a simple means for prospective identification of fully reprogrammed cells. SIGNIFICANCE: Reprogramming of differentiated cells back to an embryonic pluripotent state has wide ranging applications in understanding and treating human disease. However, how cells traverse the barriers on the journey to pluripotency still is not fully understood. This report describes tools to study the late stages of cellular reprogramming. The findings enable a more precise approach to dissecting the final phases of conversion to pluripotency, a process that is particularly poorly defined. The results of this study also provide a simple new method for the selection of fully reprogrammed cells, which could enhance the efficiency of derivation of cell lines for research and therapy.

Comparison of reprogramming efficiency between transduction of reprogramming factors, cell-cell fusion, and cytoplast fusion.

Reprogramming human somatic cells into pluripotent cells opens up new possibilities for transplantation therapy, the study of disease, and drug screening. In addition to somatic cell nuclear transfer, several approaches to reprogramming human cells have been reported: transduction of defined transcription factors to generate induced pluripotent stem cell (iPSC), human embryonic stem cell (hESC)-somatic cell fusion, and hESC cytoplast-somatic cell fusion or exposure to extracts of hESC. Here, we optimized techniques for hESC-human fibroblast fusion and enucleation and cytoplast fusion, and then compared the reprogramming efficiency between iPSC generation, cell-fusion and cytoplast-fusion. When compared with iPSC, hESC-fusion provided much faster and efficient reprogramming of somatic cells. The reprogramming required more than 4 weeks and the efficiency was less than 0.001% in iPSC generation, and it was less than 10 days and more than 0.005% in hESC-fusion. In addition, fusion yielded almost no partially reprogrammed cell colonies. However, the fused cells were tetraploid or aneuploid. hESC cytoplast fusion could initiate reprogramming but was never able to complete reprogramming. These data indicate that in cell fusion, as in nuclear transfer, reprogramming through direct introduction of a somatic nucleus into the environment of a pluripotent cell provides relatively efficient reprogramming. The findings also suggest that the nucleus of the host pluripotent cell may contain components that accelerate the reprogramming process.

Current technology for the derivation of pluripotent stem cell lines from human embryos.

Technology for the derivation, propagation, and characterization of pluripotent stem cell lines from the human embryo has undergone considerable refinement and improvement since the first published description of human embryonic stem cells in 1998. In particular, there has been extensive effort to optimize protocols and develop defined culture systems with a view toward future clinical applications of embryonic stem cell-derived products. Here, we review the current status of methodology for human embryonic stem cell derivation and culture, and we highlight the challenges that remain for workers in the field.

Manipulation of small-molecule inhibitory kinetics modulates MCH-R1 function.

The capacity of novel benzopyridazinone-based antagonists to inhibit MCH-R1 function, relative to their affinity for the receptor, has been investigated. Three compounds that differ by the addition of either a chlorine atom, or trifluoromethyl group, have nearly identical receptor affinities; however their abilities to inhibit receptor elicited signaling events, measured as a function of time, are dramatically altered. Both the chlorinated and trifluoromethyl modified compounds have a very slow on-rate to maximal functional inhibition relative to the unmodified base compound. A similar impact on inhibitory capacity can be achieved by modifying the side-chain composition at position 2.53 of the receptor; replacement of the native phenylalanine with alanine significantly reduces the amount of time required by the chlorinated compound to attain maximal functional inhibition. The primary attribute responsible for this alteration in inhibitory capacity appears to be the overall bulk of the amino acid at this position-substitution of the similarly sized amino acids leucine and tyrosine results in phenotypes that are indistinguishable from the wild type receptor. Finally, the impact of these differential inhibitory kinetics has been examined in cultured rat neurons by measuring the ability of the compounds to reverse MCH mediated inhibition of calcium currents. As observed using the cell expression models, the chlorinated compound has a diminished capacity to interfere with receptor function. Collectively, these data suggest that differential inhibitory on rates between a small-molecule antagonist and its target receptor can impact the ability of the compound to modify the biological response(s) elicited by the receptor.

Indiplon is a high-affinity positive allosteric modulator with selectivity for alpha1 subunit-containing GABAA receptors.

Indiplon (NBI 34060) is a novel pyrazolopyrimidine currently in development for the treatment of insomnia. We have previously shown that indiplon exhibits high-affinity binding to native GABA(A) receptors from rat brain and acts as a positive allosteric modulator of GABA(A) receptor currents in cultured rat neurons (Sullivan et al., 2004). In this study, we examined the GABA(A) receptor alpha subunit selectivity of indiplon using electrophysiological techniques to record GABA-activated chloride currents from recombinant rodent GABA(A) receptors expressed in human embryonic kidney 293 cells. Indiplon potentiated the GABA-activated chloride current in recombinant GABA(A) receptors in a dose-dependent and reversible manner and was approximately 10-fold selective for alpha1 subunit-containing receptors over GABA(A) receptors containing alpha2, alpha3, or alpha5 subunits. The EC(50) values were 2.6, 24, 60, and 77 nM for alpha1beta2gamma2, alpha2beta2gamma2, alpha3beta3gamma2, and alpha5beta2gamma2 receptors, respectively. Indiplon was approximately 10 times more potent than zolpidem and zopiclone and >100 times more potent than zaleplon. Moreover, indiplon, up to 1 microM, did not potentiate GABA(A) receptors composed of alpha4beta2gamma2 and alpha6beta2gamma2 subunits. This mechanism of action is proposed to underlie the sedative-hypnotic effects of indiplon in animals and humans.

Role of the GLT-1 subtype of glutamate transporter in glutamate homeostasis: the GLT-1-preferring inhibitor WAY-855 produces marginal neurotoxicity in the rat hippocampus.

Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock-out animals and antisense techniques have been used to study their specific roles. Here we used WAY-855, a GLT-1-preferring blocker, to assess the role of GLT-1 in rat hippocampus. GLT-1 was the most abundant transporter in the hippocampus at the mRNA level. According to [(3)H]-l-glutamate uptake data, GLT-1 was responsible for approximately 80% of the GLAST-, GLT-1-, and EAAC1-mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY-855 (100 microm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad-spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 microm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY-855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY-855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1-4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT-1 is insufficient to provoke glutamate build-up, leading to NMDA receptor-mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.