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  1. Article ; Online: Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro

    Vignesh Jayarajan / George T. Hall / Theodoros Xenakis / Neil Bulstrode / Dale Moulding / Sergi Castellano / Wei-Li Di

    Cells, Vol 12, Iss 346, p

    2023  Volume 346

    Abstract: Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes ... ...

    Abstract Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.
    Keywords keratinocyte stem cells ; ROCK inhibitor ; single-cell RNA sequencing ; gene therapy ; Y-27632 ; Biology (General) ; QH301-705.5
    Subject code 571
    Language English
    Publishing date 2023-01-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Automated computational analysis reveals structural changes in the enteric nervous system of nNOS deficient mice

    Ben R. Cairns / Benjamin Jevans / Atchariya Chanpong / Dale Moulding / Conor J. McCann

    Scientific Reports, Vol 11, Iss 1, Pp 1-

    2021  Volume 12

    Abstract: Abstract Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has ...

    Abstract Abstract Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has been shown in a range of enteric neuropathies. However, the effects of nNOS loss on the composition and structure of the ENS remain poorly understood. The aim of this study was to assess the structural and transcriptional consequences of loss of nNOS neurons within the murine ENS. Expression analysis demonstrated compensatory transcriptional upregulation of pan neuronal and inhibitory neuronal subtype targets within the Nos1 −/− colon, compared to control C57BL/6J mice. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1 −/− ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching. These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Further, we demonstrate the utility of machine learning approaches, and automated computational image analysis, in revealing previously undetected; yet potentially clinically relevant, changes in ENS structure which could provide improved understanding of pathological mechanisms across a host of enteric neuropathies.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2021-08-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Endosomal structure and APP biology are not altered in a preclinical mouse cellular model of Down syndrome

    Claudia Cannavo / Karen Cleverley / Cheryl Maduro / Paige Mumford / Dale Moulding / Elizabeth M. C. Fisher / Frances K. Wiseman

    PLoS ONE, Vol 17, Iss

    2022  Volume 5

    Abstract: Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer’s disease, characterised by the accumulation in the brain of amyloid-β plaques. Amyloid-β is a product of the processing of the amyloid precursor ... ...

    Abstract Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer’s disease, characterised by the accumulation in the brain of amyloid-β plaques. Amyloid-β is a product of the processing of the amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-β accumulation is within endosomes, and changes to endosome biology occur early in Alzheimer’s disease. Here, we determine if primary mouse embryonic fibroblasts isolated from a mouse model of Down syndrome can be used to study endosome and APP cell biology. We report that in this cellular model, endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in the model, despite three copies of App.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2022-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Endosomal structure and APP biology are not altered in a preclinical mouse cellular model of Down syndrome.

    Claudia Cannavo / Karen Cleverley / Cheryl Maduro / Paige Mumford / Dale Moulding / Elizabeth M C Fisher / Frances K Wiseman

    PLoS ONE, Vol 17, Iss 5, p e

    2022  Volume 0262558

    Abstract: Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer's disease, characterised by the accumulation in the brain of amyloid-β plaques. Amyloid-β is a product of the processing of the amyloid precursor ... ...

    Abstract Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer's disease, characterised by the accumulation in the brain of amyloid-β plaques. Amyloid-β is a product of the processing of the amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-β accumulation is within endosomes, and changes to endosome biology occur early in Alzheimer's disease. Here, we determine if primary mouse embryonic fibroblasts isolated from a mouse model of Down syndrome can be used to study endosome and APP cell biology. We report that in this cellular model, endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in the model, despite three copies of App.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2022-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Generation of 3D retinal tissue from human pluripotent stem cells using a directed small molecule-based serum-free microwell platform

    Hassan Rashidi / Yeh Chwan Leong / Kerrie Venner / Hema Pramod / Qi-Zhen Fei / Owen J. R. Jones / Dale Moulding / Jane C. Sowden

    Scientific Reports, Vol 12, Iss 1, Pp 1-

    2022  Volume 13

    Abstract: Abstract Retinal degenerative diseases are a leading cause of blindness worldwide with debilitating life-long consequences for the affected individuals. Cell therapy is considered a potential future clinical intervention to restore and preserve sight by ... ...

    Abstract Abstract Retinal degenerative diseases are a leading cause of blindness worldwide with debilitating life-long consequences for the affected individuals. Cell therapy is considered a potential future clinical intervention to restore and preserve sight by replacing lost photoreceptors and/or retinal pigment epithelium. Development of protocols to generate retinal tissue from human pluripotent stem cells (hPSC), reliably and at scale, can provide a platform to generate photoreceptors for cell therapy and to model retinal disease in vitro. Here, we describe an improved differentiation platform to generate retinal organoids from hPSC at scale and free from time-consuming manual microdissection steps. The scale up was achieved using an agarose mould platform enabling generation of uniform self-assembled 3D spheres from dissociated hPSC in microwells. Subsequent retinal differentiation was efficiently achieved via a stepwise differentiation protocol using a number of small molecules. To facilitate clinical translation, xeno-free approaches were developed by substituting Matrigel™ and foetal bovine serum with recombinant laminin and human platelet lysate, respectively. Generated retinal organoids exhibited important features reminiscent of retinal tissue including correct site-specific localisation of proteins involved in phototransduction.
    Keywords Medicine ; R ; Science ; Q
    Subject code 571
    Language English
    Publishing date 2022-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis

    Michael Taschner / Anna Lorentzen / André Mourão / Toby Collins / Grace M Freke / Dale Moulding / Jerome Basquin / Dagan Jenkins / Esben Lorentzen

    eLife, Vol

    2018  Volume 7

    Abstract: Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the Chlamydomonas IFT-B protein ... ...

    Abstract Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the Chlamydomonas IFT-B protein IFT80, which reveals the architecture of two N-terminal β-propellers followed by an α-helical extension. The N-terminal β-propeller tethers IFT80 to the IFT-B complex via IFT38 whereas the second β-propeller and the C-terminal α-helical extension result in IFT80 homo-dimerization. Using CRISPR/Cas to create biallelic Ift80 frameshift mutations in IMCD3 mouse cells, we demonstrate that IFT80 is absolutely required for ciliogenesis. Structural mapping and rescue experiments reveal that human disease-causing missense mutations do not cluster within IFT80 and form functional IFT particles. Unlike missense mutant forms of IFT80, deletion of the C-terminal dimerization domain prevented rescue of ciliogenesis. Taken together our results may provide a first insight into higher order IFT complex formation likely required for IFT train formation.
    Keywords Chlamydomonas reinhardtii ; Cilium ; Intraflagelllar transport ; protein structure ; ciliopathies ; IFT80 ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2018-04-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Spinal neural tube closure depends on regulation of surface ectoderm identity and biomechanics by Grhl2

    Evanthia Nikolopoulou / Caroline S. Hirst / Gabriel Galea / Christina Venturini / Dale Moulding / Abigail R. Marshall / Ana Rolo / Sandra C. P. De Castro / Andrew J. Copp / Nicholas D. E. Greene

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 17

    Abstract: Loss or over-expression of Grainyhead-like transcription factors (Grhl) prevents closure of the neural tube but the mechanism underlying this is unclear. Here, the authors show that Grhl2 regulates murine posterior-neuropore closure via changes in the ... ...

    Abstract Loss or over-expression of Grainyhead-like transcription factors (Grhl) prevents closure of the neural tube but the mechanism underlying this is unclear. Here, the authors show that Grhl2 regulates murine posterior-neuropore closure via changes in the identity and biomechanics of the non-neural, surface ectoderm cells.
    Keywords Science ; Q
    Language English
    Publishing date 2019-06-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Spinal neural tube closure depends on regulation of surface ectoderm identity and biomechanics by Grhl2

    Evanthia Nikolopoulou / Caroline S. Hirst / Gabriel Galea / Christina Venturini / Dale Moulding / Abigail R. Marshall / Ana Rolo / Sandra C. P. De Castro / Andrew J. Copp / Nicholas D. E. Greene

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 17

    Abstract: Loss or over-expression of Grainyhead-like transcription factors (Grhl) prevents closure of the neural tube but the mechanism underlying this is unclear. Here, the authors show that Grhl2 regulates murine posterior-neuropore closure via changes in the ... ...

    Abstract Loss or over-expression of Grainyhead-like transcription factors (Grhl) prevents closure of the neural tube but the mechanism underlying this is unclear. Here, the authors show that Grhl2 regulates murine posterior-neuropore closure via changes in the identity and biomechanics of the non-neural, surface ectoderm cells.
    Keywords Science ; Q
    Language English
    Publishing date 2019-06-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Vangl2 disruption alters the biomechanics of late spinal neurulation leading to spina bifida in mouse embryos

    Gabriel L. Galea / Oleksandr Nychyk / Matteo A. Mole / Dale Moulding / Dawn Savery / Evanthia Nikolopoulou / Deborah J. Henderson / Nicholas D. E. Greene / Andrew J. Copp

    Disease Models & Mechanisms, Vol 11, Iss

    2018  Volume 3

    Abstract: Human mutations in the planar cell polarity component VANGL2 are associated with the neural tube defect spina bifida. Homozygous Vangl2 mutation in mice prevents initiation of neural tube closure, precluding analysis of its subsequent roles in ... ...

    Abstract Human mutations in the planar cell polarity component VANGL2 are associated with the neural tube defect spina bifida. Homozygous Vangl2 mutation in mice prevents initiation of neural tube closure, precluding analysis of its subsequent roles in neurulation. Spinal neurulation involves rostral-to-caudal ‘zippering’ until completion of closure is imminent, when a caudal-to-rostral closure point, ‘Closure 5’, arises at the caudal-most extremity of the posterior neuropore (PNP). Here, we used Grhl3Cre to delete Vangl2 in the surface ectoderm (SE) throughout neurulation and in an increasing proportion of PNP neuroepithelial cells at late neurulation stages. This deletion impaired PNP closure after the ∼25-somite stage and resulted in caudal spina bifida in 67% of Grhl3Cre/+Vangl2Fl/Fl embryos. In the dorsal SE, Vangl2 deletion diminished rostrocaudal cell body orientation, but not directional polarisation of cell divisions. In the PNP, Vangl2 disruption diminished mediolateral polarisation of apical neuroepithelial F-actin profiles and resulted in eversion of the caudal PNP. This eversion prevented elevation of the caudal PNP neural folds, which in control embryos is associated with formation of Closure 5 around the 25-somite stage. Closure 5 formation in control embryos is associated with a reduction in mechanical stress withstood at the main zippering point, as inferred from the magnitude of neural fold separation following zippering point laser ablation. This stress accommodation did not happen in Vangl2-disrupted embryos. Thus, disruption of Vangl2-dependent planar-polarised processes in the PNP neuroepithelium and SE preclude zippering point biomechanical accommodation associated with Closure 5 formation at the completion of PNP closure.
    Keywords Neural tube ; Vangl2 ; Biomechanics ; F-actin ; Mouse ; Embryo ; Medicine ; R ; Pathology ; RB1-214
    Language English
    Publishing date 2018-03-01T00:00:00Z
    Publisher The Company of Biologists
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease

    Daniyal J Jafree / Dale Moulding / Maria Kolatsi-Joannou / Nuria Perretta Tejedor / Karen L Price / Natalie J Milmoe / Claire L Walsh / Rosa Maria Correra / Paul JD Winyard / Peter C Harris / Christiana Ruhrberg / Simon Walker-Samuel / Paul R Riley / Adrian S Woolf / Peter J Scambler / David A Long

    eLife, Vol

    2019  Volume 8

    Abstract: Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three- ... ...

    Abstract Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis; a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease.
    Keywords kidney ; lymphatics ; vessels ; development ; kidney disease ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 616
    Language English
    Publishing date 2019-12-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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