Characterisation of neural progenitors from the adult retina and ciliary epithelium

The mammalian central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. However, retinal stem cells have been isolated from the ciliary epithelium of the mammalian retina. Chx10 is a paired-like homeobox transcription factor gene expressed in the presumptive neural retina of the invaginating optic vesicle. The Chx10 gene is expressed in the proliferating retinal progenitor cell population throughout retinal development hence is one of the earliest characterised RPC-specific markers. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, it was showed that in the ocular retardation mouse model lacking Chx10 (Chx10orJ/orJ), dividing cells persist in the adult CNR, suggesting the existence of a dormant stem/progenitor population. The neurosphere-forming assay is a tool which has allowed scientists to study the behaviour of neural stem/progenitor cells in vitro. Here, I show that cells deriving from the CNR of the adult microphthalmic retina are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Müller glial cell markers. My results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR possible deriving from the abnormal appearance of GFAPpos Müller glia in late embryonic stages of the Chx10orJ/orJ retina. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors. One of the cardinal features of stem cells is their differentiation potential and multipotency. My experiments illustrate that Chx10orJ/orJ CNR-derived neurospheres are able to differentiate in a similar fashion to wildtype CE-derived neurospheres. Furthermore, when neurospheres lacking Chx10 are placed in conditions that promote differentiation, they significantly up-regulate the expression of photoreceptor genes in comparison to wildtype. Hitherto, the developmental origin of CE-derived neurosphere-forming retinal stem cell is unclear. The ciliary body, where the CE is located in adult mammals, includes cells of mesodermal, neural crest and neural ectodermal origin. Here, data collected from lineage tracing analysis and in vivo BrdU-tagging experiments suggest that neurospheres are formed from BrdUpos cells observed in vivo, and that these cells originate from the embryonic anterior forebrain. The comparative analysis of the microphthalmic CNR retinal progenitors and CE-derived progenitors provides valuable information on cell properties relevant for potential cell-based replacement therapies, as well for retinal regeneration potential in mammals

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies

Mature Peripheral RPE Cells Have an Intrinsic Capacity to Proliferate; A Potential Regulatory Mechanism for Age-Related Cell Loss

Mammalian peripheral retinal pigmented epithelium (RPE) cells proliferate throughout life, while central cells are senescent. It is thought that some peripheral cells migrate centrally to correct age-related central RPE loss.We ask whether this proliferative capacity is intrinsic to such cells and whether cells located centrally produce diffusible signals imposing senescence upon the former once migrated. We also ask whether there are regional differences in expression patterns of key genes involved in these features between the centre and the periphery in vivo and in vitro. Low density RPE cultures obtained from adult mice revealed significantly greater levels of proliferation when derived from peripheral compared to central tissue, but this significance declined with increasing culture density. Further, exposure to centrally conditioned media had no influence on proliferation in peripheral RPE cell cultures at the concentrations examined. Central cells expressed significantly higher levels of E-Cadherin revealing a tighter cell adhesion than in the peripheral regions. Fluorescence-labelled staining for E-Cadherin, F-actin and ZO-1 in vivo revealed different patterns with significantly increased expression on central RPE cells than those in the periphery or differences in junctional morphology. A range of other genes were investigated both in vivo and in vitro associated with RPE proliferation in order to identify gene expression differences between the centre and the periphery. Specifically, the cell cycle inhibitor p27(Kip1) was significantly elevated in central senescent regions in vivo and mTOR, associated with RPE cell senescence, was significantly elevated in the centre in comparison to the periphery.These data show that the proliferative capacity of peripheral RPE cells is intrinsic and cell-autonomous in adult mice. These differences between centre and periphery are reflected in distinct patterns in junctional markers. The regional proliferation differences may be inversely dependent to cell-cell contact

Fundamental differences in patterns of retinal ageing between primates and mice

Photoreceptors have high metabolic demands and age rapidly, undermining visual function. We base our understanding mainly on ageing mice where elevated inflammation, extracellular deposition, including that of amyloid beta, and rod and cone photoreceptor loss occur, but cones are not lost in ageing primate although their function declines, revealing that primate and mouse age differently. We examine ageing primate retinae and show elevated stress but low inflammation. However, aged primates have a >70% reduction in adenosine triphosphate (ATP) and a decrease in cytochrome c oxidase. There is a shift in cone mitochondrial positioning and glycolytic activity increases. Bruch’s membrane thickens but unlike in mice, amyloid beta is absent. Hence, reduced ATP may explain cone functional decline in ageing but their retained presence offers the possibility of functional restoration if they can be fuelled appropriately to restore cellular function. This is important because as humans we largely depend on cone function to see and are rarely fully dark adapted. Presence of limited aged inflammation and amyloid beta deposition question some of the therapeutic approaches taken to resolve problems of retinal ageing in humans and the possible lack of success in clinical trials in macular degeneration that have targeted inflammatory agents

T Cells from Programmed Death-1 Deficient Mice Respond Poorly to Mycobacterium tuberculosis Infection

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.Here, we report that PD-1 deficient (PD-1(-/-)) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1(-/-) mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study

GABA Maintains the Proliferation of Progenitors in the Developing Chick Ciliary Marginal Zone and Non-Pigmented Ciliary Epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABAA receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABAA receptor system. To quantify the effects on proliferation by GABAA receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABAA receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABAA receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl–transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABAA receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABAA receptors. This supported the depolarising role for the GABAA receptors. Inhibition of L-type voltage-gated Ca2+ channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABAA receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27KIP1, along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27KIP1 after inhibition of either the GABAA receptors or the L-type VGCCs suggests a link between the GABAA receptors, membrane potential, and intracellular Ca2+ in regulating the cell cycle

Treatment with 670 nm light up regulates cytochrome C oxidase expression and reduces inflammation in an age-related macular degeneration model.

Inflammation is an umbrella feature of ageing. It is present in the aged retina and many retinal diseases including age-related macular degeneration (AMD). In ageing and in AMD mitochondrial function declines. In normal ageing this can be manipulated by brief exposure to 670 nm light on the retina, which increases mitochondrial membrane potential and reduces inflammation. Here we ask if 670 nm exposure has the same ability in an aged mouse model of AMD, the complement factor H knockout (CFH(-/-)) where inflammation is a key feature. Further, we ask whether this occurs when 670 nm is delivered briefly in environmental lighting rather than directly focussed on the retina. Mice were exposed to 670 nm for 6 minutes twice a day for 14 days in the form of supplemented environmental light. Exposed animals had significant increase in cytochrome c oxidase (COX), which is a mitochondrial enzyme regulating oxidative phosphorylation.There was a significant reduction in complement component C3, an inflammatory marker in the outer retina. Vimetin and glial fibrillary acidic protein (GFAP) expression, which reflect retinal stress in Muller glia, were also significantly down regulated. There were also significant changes in outer retinal macrophage morphology. However, amyloid beta (Aβ) load, which also increases with age in the outer retina and is pro-inflammatory, did not change. Hence, 670 nm is effective in reducing inflammation probably via COX activation in mice with a genotype similar to that in 50% of AMD patients even when brief exposures are delivered via environmental lighting. Further, inflammation can be reduced independent of Aβ. The efficacy revealed here supports current early stage clinical trials of 670 nm in AMD patients

Atención de enfermería en la reducción manual del prolapso de ostomía

Objetivo: relatar um caso sobre a redução manual do prolapso em um estoma intestinal. Método: trata-se de um estudo descritivo, tipo relato de caso clínico, sobre a redução manual do prolapso em um estoma intestinal de um paciente. Relata-se que o cenário foi um centro de referência no atendimento ao estomizado. Avaliaram-se a efetividade das intervenções de Enfermagem com base na comparação dos resultados iniciais e finais do procedimento. Resultados: nota-se a presença de lesões e permeabilidade na presença de prolapso, realizando a manobra de redução digital, até a sua total regressão, facilitando a limpeza da pele e a colocação do equipamento coletor, observando as possíveis lesões periestomais e na mucosa. Conclusão: informa-se que os pacientes com prolapso em estoma intestinal têm um desconforto devido ao volume dentro do equipamento coletor e ao pouco espaço para conter as suas eliminações. Utiliza-se a técnica de redução manual para facilitar a troca do equipamento coletor, esta deverá ser realizada por enfermeiro estomaterapeuta ou enfermeiro capacitado. Salienta-se que os casos em que se afeta o desenvolvimento de atividades cotidianas merecem uma avaliação da equipe cirúrgica.Objective: to report a case about manual reduction of prolapse in an intestinal stoma. Method: it is a descriptive, case report type study on the manual reduction of prolapse in a patient's intestinal stoma. It is reported that the scenario was a reference center in the care of the stoma. They evaluated the effectiveness of nursing interventions based on the comparison of the initial and final results of the procedure. Results: the presence of injuries and permeability in the presence of prolapse is noted, performing the digital reduction maneuver, until its total regression, facilitating the cleaning of the skin and the placement of the collector equipment, observing the possible periestomal and mucosal lesions. Conclusion: it is reported that patients with prolapse in the intestinal stoma have a discomfort due to the volume inside the collector equipment and the little space to contain their eliminations. The manual reduction technique is used to facilitate the exchange of the collector equipment, this should be performed by a stoma therapist or trained nurse. It is emphasized that the cases in which the development of daily activities is affected deserve an evaluation of the surgical team.Objetivo: reportar un caso de reducción manual del prolapso en un estoma intestinal. Método: se trata de un estudio descriptivo, a modo de reporte de caso clínico, sobre la reducción manual del prolapso en el estoma intestinal de un paciente. Se informa que el escenario fue un centro de referencia en la atención de pacientes ostomizados. Evaluaron la efectividad de las intervenciones de Enfermería a partir de una comparación de los resultados iniciales y finales del procedimiento. Resultados: se observa la presencia de lesiones y permeabilidad en presencia de prolapso, realizando la maniobra de reducción digital, hasta su total regresión, facilitando la limpieza de la piel y la colocación del equipo colector, observando las posibles lesiones periestomales y mucosas. Conclusión: se informa que los pacientes con prolapso en estoma intestinal presentan molestias por el volumen dentro del equipo de recolección y el poco espacio para contener sus eliminaciones. La técnica de reducción manual se utiliza para facilitar el intercambio del equipo colector, esto debe ser realizado por un estomaterapeuta o enfermero capacitado. Cabe destacar que los casos en los que se ve afectado el desarrollo de las actividades diarias merecen una valoración por parte del equipo quirúrgico