Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease.

BACKGROUND: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells. RESULTS: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.

Drosophila taste neurons as an agonist-screening platform for P2X receptors.

The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC50 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.

Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells.

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K(+) but not outward Na(+) currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

The extracellular calcium-sensing receptor regulates human fetal lung development via CFTR.

Optimal fetal lung growth requires anion-driven fluid secretion into the lumen of the developing organ. The fetus is hypercalcemic compared to the mother and here we show that in the developing human lung this hypercalcaemia acts on the extracellular calcium-sensing receptor, CaSR, to promote fluid-driven lung expansion through activation of the cystic fibrosis transmembrane conductance regulator, CFTR. Several chloride channels including TMEM16, bestrophin, CFTR, CLCN2 and CLCA1, are also expressed in the developing human fetal lung at gestational stages when CaSR expression is maximal. Measurements of Cl(-)-driven fluid secretion in organ explant cultures show that pharmacological CaSR activation by calcimimetics stimulates lung fluid secretion through CFTR, an effect which in humans, but not mice, was also mimicked by fetal hypercalcemic conditions, demonstrating that the physiological relevance of such a mechanism appears to be species-specific. Calcimimetics promote CFTR opening by activating adenylate cyclase and we show that Ca(2+)-stimulated type I adenylate cyclase is expressed in the developing human lung. Together, these observations suggest that physiological fetal hypercalcemia, acting on the CaSR, promotes human fetal lung development via cAMP-dependent opening of CFTR. Disturbances in this process would be expected to permanently impact lung structure and might predispose to certain postnatal respiratory diseases.

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell-derived medium spiny neurons.

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma.

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

Oxygen sensing by the carotid body: is it all just rotten eggs?

SIGNIFICANCE: Ventilatory responses to hypoxia are initiated by the carotid body, where inhibition of specific K(+) channels causes cell depolarization, voltage-gated Ca(2+) influx, and neurotransmitter release. The identity of the upstream oxygen (O2) sensor is still controversial. RECENT ADVANCES: The activity of BKCa channels is regulated by O2, carbon monoxide (CO), and hydrogen sulfide (H2S), suggesting that integration of these signals may be crucial to the physiological response of this tissue. BKCa is colocalized with hemeoxygenase-2, an enzyme that generates CO in the presence of O2, and CO is a BKCa channel opener. Reduced CO during hypoxia results in channel closure, conferring a degree of O2 sensitivity to the BKCa channel. Conversely, H2S is a potent BKCa inhibitor. H2S is produced endogenously by cystathionine-ß-synthase and cystathionine-γ-lyase in the rat carotid body, and its intracellular concentration is dependent upon the balance between its enzymatic generation and its mitochondrial breakdown. During hypoxia, mitochondrial oxidation of H2S in many tissues is reduced, leading to hypoxia-evoked rises in its concentration. This may be sufficient to inhibit K(+) channels and lead to carotid body excitation. CRITICAL ISSUES: Carotid body function is heavily dependent upon regulated production and breakdown of CO and H2S and integration of signals from these newly emerging gasotransmitters, in combination with several other proposed mechanisms, may refine, or even define, responses of this tissue to hypoxia. FUTURE DIRECTIONS: Since several other sensors have been postulated, the challenge of future research is to begin to integrate each in a unifying mechanism, as has been attempted for the first time herein.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes.

AIMS: Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. METHODS AND RESULTS: We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). CONCLUSIONS: These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 µM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 µM amiloride and that recombinant αßγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 µM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the ß(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

Lung organogenesis.

Developmental lung biology is a field that has the potential for significant human impact: lung disease at the extremes of age continues to cause major morbidity and mortality worldwide. Understanding how the lung develops holds the promise that investigators can use this knowledge to aid lung repair and regeneration. In the decade since the "molecular embryology" of the lung was first comprehensively reviewed, new challenges have emerged-and it is on these that we focus the current review. Firstly, there is a critical need to understand the progenitor cell biology of the lung in order to exploit the potential of stem cells for the treatment of lung disease. Secondly, the current familiar descriptions of lung morphogenesis governed by growth and transcription factors need to be elaborated upon with the reinclusion and reconsideration of other factors, such as mechanics, in lung growth. Thirdly, efforts to parse the finer detail of lung bud signaling may need to be combined with broader consideration of overarching mechanisms that may be therapeutically easier to target: in this arena, we advance the proposal that looking at the lung in general (and branching in particular) in terms of clocks may yield unexpected benefits.

Mechanism of inhibition by hydrogen sulfide of native and recombinant BKCa channels.

Recent evidence suggests that H(2)S contributes to activation of the carotid body by hypoxia by inhibiting K(+) channels. Here, we determine both the molecular identity of the K(+) channel target within the carotid body and the biophysical characteristics of the H(2)S-evoked inhibition by analyzing native rat and human recombinant BK(Ca) channel activity in voltage-clamped, inside-out membrane patches. Rat glomus cells express the enzymes necessary for the endogenous generation of H(2)S, cystathionine-beta-synthase and cystathionine-gamma-lyase. H(2)S inhibits native carotid body and human recombinant BK(Ca) channels with IC(50) values of around 275 microM. Inhibition by H(2)S is rapid and reversible, works by a mechanism which is distinct from that suggested for CO gas regulation of this channel and does not involve an interaction with either the "Ca bowl" or residues distal to this Ca(2+)-sensing domain. These data show that BK(Ca) is a K(+) channel target of H(2)S, and suggest a mechanism to explain the H(2)S-dependent component of O(2) sensing in the carotid body.

Enzyme-linked oxygen sensing by potassium channels.

The ability of ion channels to respond to an acute perturbation in oxygen tension is a widespread phenomenon, which encompasses many of the major ion channel families. Integral to the ability of several ion channels to respond to acute hypoxic challenge is modulation by upstream enzymatic reactions, suggesting that many ion channels sense oxygen via enzyme-linked processes. Several enzyme-linked oxygen sensing systems have been proposed, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent production of hydrogen peroxide, hemoxygenase-dependent generation of carbon monoxide, adenosine monophosphate (AMP) kinase-dependent channel phosphorylation, and src-Lck protein tyrosine kinase, via a currently undetermined mechanism. Each of these enzymes has been shown to endow specific ion channels with the ability to respond to changes in oxygen, with hypoxia exclusively evoking channel inhibition. This article reviews these proposed mechanisms and presents new insights into how one system, hemeoxygenase-2, confers oxygen sensitivity to large conductance, voltage- and calcium-activated potassium channels.

Novel regulatory aspects of the extracellular Ca2+-sensing receptor, CaR.

The capacity to sense and adapt to changes in environmental cues is of paramount importance for every living organism. From yeast to man, cells must be able to match cellular activities to growth environment and nutrient availability. Key to this process is the development of membrane-bound systems that can detect modifications in the extracellular environment and to translate these into biological responses. Evidence gathered over the last 15 years has demonstrated that many of these cell surface "sensors" belong to the G protein-coupled receptor superfamily. Crucial to our understanding of nutrient sensing in mammalian species has been the identification of the extracellular Ca(2+)/cation-sensing receptor, CaR. CaR was the first ion-sensing molecule identified in man and genetic studies in humans have revealed the importance of the CaR in mineral ion metabolism. Latter, it has become apparent that the CaR also plays an important role outside the Ca(2+) homeostatic system, as an integrator of multiple environmental signals for the regulation of many vital cellular processes, from cell-to-cell communication to secretion and cell survival/cell death. Recently, novel aspects of receptor function reveal an unexpected role for the CaR in the regulation of growth and development in utero.

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.

Thiazide diuretics directly induce osteoblast differentiation and mineralized nodule formation by interacting with a sodium chloride co-transporter in bone.

Thiazide diuretics are used worldwide as a first-choice drug for patients with uncomplicated hypertension. In addition to their antihypertensive effect, thiazides increase bone mineral density and reduce the prevalence of fractures. Traditionally, these effects have been attributed to increased renal calcium reabsorption that occurs secondary to the inhibition of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal tubule. The aim of the current study was to determine whether thiazides exert a direct bone-forming effect independent of their renal action. We found that the osteoblasts of human and rat bone also express NCC, suggesting that these bone-forming cells may be an additional target for thiazides. In vitro, NCC protein was virtually absent in proliferating human and fetal rat osteoblasts, whereas its expression dramatically increased during differentiation. Thiazides did not affect osteoblast proliferation, but directly stimulated the production of the osteoblast differentiation markers runt-related transcription factor 2 (runx2) and osteopontin. Using overexpression/knockdown studies in fetal rat calvarial cells, we show that thiazides increase the formation of mineralized nodules, but loop diuretics do not. Overall, our study demonstrates that thiazides directly stimulate osteoblast differentiation and bone mineral formation independent of their effects in the kidney. Therefore, in addition to their use as antihypertensive drugs, our results suggest that thiazides may find a role in the prevention and treatment of osteoporosis.

Environmental signals regulate lineage choice and temporal maturation of neural stem cells from human embryonic stem cells.

Human embryonic stem cells (hESCs) are a potential source of defined tissue for cell-based therapies in regenerative neurology. In order for this potential to be realized, there is a need for the evaluation of the behaviour of human embryonic stem cell-derived neural stem cells (hES-NSCs) both in the normal and the injured CNS. Using normal tissue and two experimental models, we examined the response of clinically compatible hES-NSCs to physiological and pathological signals. We demonstrate that the phenotypic potential of a multipotent population of hES-NSCs is influenced by these cues both in vitro and in vivo. hES-NSCs display a temporal profile of neurogenic and gliogenic differentiation, with the generation of mature neurons and glia over 4 weeks in vitro, and 20 weeks in the uninjured rodent brain. However, transplantation into the pathological CNS accelerates maturation and polarizes hES-NSC differentiation potential. This study highlights the role of environmental signals in determining both lineage commitment and temporal maturation of human neural stem cells. Controlled manipulation of environmental signals appropriate to the pathological specificity of the targeted disease will be necessary in the design of therapeutic stem cell-based strategies.

Cloning of the human TASK-2 (KCNK5) promoter and its regulation by chronic hypoxia.

The tandem P domain potassium channel family includes five members of the acid-sensing subfamily, TASK. TASK channels are active at resting potential and are inhibited by extracellular protons, suggesting they function as acid sensors and control excitability/ion homeostasis. Indeed, TASK-2 (KCNK5) has been shown to control excitability, volume regulation, bicarbonate handling, and apoptosis in a variety of tissues. With such diverse functions being ascribed to TASK-2, it is important to understand long-term as well as short-term regulation of this important channel. Thus, we have cloned the TASK-2 promoter, demonstrated that its transcriptional activity is dependent upon pO(2), shown that deletion of overlapping consensus binding sites for NF-kappaB/Elk-1 ablates this O(2) sensitivity, and proved that Elk-1 binds preferentially to this site. Furthermore, the consequences of chronic hypoxia on natively expressed TASK-2 are decreased steady-state mRNA and cell depolarization showing that TASK-2 contributes to the excitability of this important lung cell type.

Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells.

BACKGROUND: Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. METHODS: Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. RESULTS: This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. CONCLUSION: This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease.