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Andrew J Lotery - One of the best experts on this subject based on the ideXlab platform.

  • adult limbal neurosphere cells a potential autologous cell resource for retinal cell generation
    PLOS ONE, 2014
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery, Jessica Cooke, Jennifer Scott
    Abstract:

    The Corneal Limbus is a readily accessible region at the front of the eye, separating the cornea and sclera. Neural colonies (neurospheres) can be generated from adult Corneal Limbus in vitro. We have previously shown that these neurospheres originate from neural crest stem/progenitor cells and that they can differentiate into functional neurons in vitro. The aim of this study was to investigate whether mouse and human limbal neurosphere cells (LNS) could differentiate towards a retinal lineage both in vivo and in vitro following exposure to a developing retinal microenvironment. In this article we show that LNS can be generated from adult mice and aged humans (up to 97 years) using a serum free culture assay. Following culture with developing mouse retinal cells, we detected retinal progenitor cell markers, mature retinal/neuronal markers and sensory cilia in the majority of mouse LNS experiments. After transplantation into the sub-retinal space of neonatal mice, mouse LNS cells expressed photoreceptor specific markers, but no incorporation into host retinal tissue was seen. Human LNS cells also expressed retinal progenitor markers at the transcription level but mature retinal markers were not observed in vitro or in vivo. This data highlights that mouse Corneal limbal stromal progenitor cells can transdifferentiate towards a retinal lineage. Complete differentiation is likely to require more comprehensive regulation; however, the accessibility and plasticity of LNS makes them an attractive cell resource for future study and ultimately therapeutic application.

  • characterisation of mouse limbal neurosphere cells a potential cell source of functional neurons
    British Journal of Ophthalmology, 2012
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Background/aims To characterise the origin, ultrastructure and functional properties of Corneal limbal neurospheres (LNS). Methods Limbal cells were isolated from the Corneal Limbus of adult mice and cultured in a serum-free sphere forming culture system. LNS were characterised by immunocytochemistry, Reverse-transcription-PCR and electron microscopy. LNS cells were also cocultured with neonatal mouse retinal cells. Phenotype and function were then assessed by immunofluorescence and a calcium influx/efflux assay. Results LNS cells displayed clonal growth and self-renewal, and expressed a wide range of stem cell and neural lineage markers. The acquisition of neural properties was concordant with expression of neural crest markers including CD34, Sca1, Sox9, Twist1, but not CD45. LNS exhibited similar morphology and microstructure to neurospheres derived from the central nervous system. Following culture in a conducive environment, the derived cells displayed mature neural markers and exhibited electrical excitability. Conclusions Corneal limbal stromal progenitor cells are a potential and convenient autologous cell source to generate functional neurons.

  • derivation of neural progenitor cells from adult mouse Corneal Limbus
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Purpose: cells from the adult rodent Corneal limbal epithelium basal layer have been demonstrated to display neuronal potential and differentiate towards functional neurons in vitro. A sphere forming culture system has also been used to expand stem cells of a neural crest origin from mouse Corneal stroma. The aim of this study was to investigate whether progenitors with neuronal potential could be cultured from adult murine Limbus to form neuronal characteristics. Methods: cells from adult (8 weeks) murine Corneal Limbus were isolated and cultured in a serum-free sphere-forming system in the presence of mitogens. Cells were maintained in the presence or absence of the bone morphogenetic protein 4 (BMP4) inhibitor (Noggin). Sphere derived cells and their progeny were characterized using immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). Results: adult mouse limbal cells formed sphere colonies with the ability to self renew. The frequency of sphere-forming cells was 0.45-1.0% subject to culture conditions. Noggin did not have an effect on the efficiency of sphere formation. In the presence and absence of Noggin, sphere cells expressed ABCG2, Sox2, Nestin and beta-III tubulin (early differentiated neuron) but not P63 (epithelial stem cell marker) as shown by immunocytochemistry. Expression of neuronal stem/progenitor cells markers (nestin, Sox2, Musashi, beta-III tubulin) and neural crest markers (Twist1 and Slug) increased with time in culture and passage, concomitant with a decrease in expression of the epithelium lineage markers P63 and cytokeratin12, as shown by RT-PCR. Conclusions: the adult mouse Corneal Limbus contains stem / precursor cells which express neural stem cell markers in vitro. These stem cells/ progenitor cells appear to be neural crest & are likely to be derived from the Corneal limbal stroma

Xiaoli Chen - One of the best experts on this subject based on the ideXlab platform.

  • adult limbal neurosphere cells a potential autologous cell resource for retinal cell generation
    PLOS ONE, 2014
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery, Jessica Cooke, Jennifer Scott
    Abstract:

    The Corneal Limbus is a readily accessible region at the front of the eye, separating the cornea and sclera. Neural colonies (neurospheres) can be generated from adult Corneal Limbus in vitro. We have previously shown that these neurospheres originate from neural crest stem/progenitor cells and that they can differentiate into functional neurons in vitro. The aim of this study was to investigate whether mouse and human limbal neurosphere cells (LNS) could differentiate towards a retinal lineage both in vivo and in vitro following exposure to a developing retinal microenvironment. In this article we show that LNS can be generated from adult mice and aged humans (up to 97 years) using a serum free culture assay. Following culture with developing mouse retinal cells, we detected retinal progenitor cell markers, mature retinal/neuronal markers and sensory cilia in the majority of mouse LNS experiments. After transplantation into the sub-retinal space of neonatal mice, mouse LNS cells expressed photoreceptor specific markers, but no incorporation into host retinal tissue was seen. Human LNS cells also expressed retinal progenitor markers at the transcription level but mature retinal markers were not observed in vitro or in vivo. This data highlights that mouse Corneal limbal stromal progenitor cells can transdifferentiate towards a retinal lineage. Complete differentiation is likely to require more comprehensive regulation; however, the accessibility and plasticity of LNS makes them an attractive cell resource for future study and ultimately therapeutic application.

  • characterisation of mouse limbal neurosphere cells a potential cell source of functional neurons
    British Journal of Ophthalmology, 2012
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Background/aims To characterise the origin, ultrastructure and functional properties of Corneal limbal neurospheres (LNS). Methods Limbal cells were isolated from the Corneal Limbus of adult mice and cultured in a serum-free sphere forming culture system. LNS were characterised by immunocytochemistry, Reverse-transcription-PCR and electron microscopy. LNS cells were also cocultured with neonatal mouse retinal cells. Phenotype and function were then assessed by immunofluorescence and a calcium influx/efflux assay. Results LNS cells displayed clonal growth and self-renewal, and expressed a wide range of stem cell and neural lineage markers. The acquisition of neural properties was concordant with expression of neural crest markers including CD34, Sca1, Sox9, Twist1, but not CD45. LNS exhibited similar morphology and microstructure to neurospheres derived from the central nervous system. Following culture in a conducive environment, the derived cells displayed mature neural markers and exhibited electrical excitability. Conclusions Corneal limbal stromal progenitor cells are a potential and convenient autologous cell source to generate functional neurons.

  • derivation of neural progenitor cells from adult mouse Corneal Limbus
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Purpose: cells from the adult rodent Corneal limbal epithelium basal layer have been demonstrated to display neuronal potential and differentiate towards functional neurons in vitro. A sphere forming culture system has also been used to expand stem cells of a neural crest origin from mouse Corneal stroma. The aim of this study was to investigate whether progenitors with neuronal potential could be cultured from adult murine Limbus to form neuronal characteristics. Methods: cells from adult (8 weeks) murine Corneal Limbus were isolated and cultured in a serum-free sphere-forming system in the presence of mitogens. Cells were maintained in the presence or absence of the bone morphogenetic protein 4 (BMP4) inhibitor (Noggin). Sphere derived cells and their progeny were characterized using immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). Results: adult mouse limbal cells formed sphere colonies with the ability to self renew. The frequency of sphere-forming cells was 0.45-1.0% subject to culture conditions. Noggin did not have an effect on the efficiency of sphere formation. In the presence and absence of Noggin, sphere cells expressed ABCG2, Sox2, Nestin and beta-III tubulin (early differentiated neuron) but not P63 (epithelial stem cell marker) as shown by immunocytochemistry. Expression of neuronal stem/progenitor cells markers (nestin, Sox2, Musashi, beta-III tubulin) and neural crest markers (Twist1 and Slug) increased with time in culture and passage, concomitant with a decrease in expression of the epithelium lineage markers P63 and cytokeratin12, as shown by RT-PCR. Conclusions: the adult mouse Corneal Limbus contains stem / precursor cells which express neural stem cell markers in vitro. These stem cells/ progenitor cells appear to be neural crest & are likely to be derived from the Corneal limbal stroma

Parwez Hossain - One of the best experts on this subject based on the ideXlab platform.

  • adult limbal neurosphere cells a potential autologous cell resource for retinal cell generation
    PLOS ONE, 2014
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery, Jessica Cooke, Jennifer Scott
    Abstract:

    The Corneal Limbus is a readily accessible region at the front of the eye, separating the cornea and sclera. Neural colonies (neurospheres) can be generated from adult Corneal Limbus in vitro. We have previously shown that these neurospheres originate from neural crest stem/progenitor cells and that they can differentiate into functional neurons in vitro. The aim of this study was to investigate whether mouse and human limbal neurosphere cells (LNS) could differentiate towards a retinal lineage both in vivo and in vitro following exposure to a developing retinal microenvironment. In this article we show that LNS can be generated from adult mice and aged humans (up to 97 years) using a serum free culture assay. Following culture with developing mouse retinal cells, we detected retinal progenitor cell markers, mature retinal/neuronal markers and sensory cilia in the majority of mouse LNS experiments. After transplantation into the sub-retinal space of neonatal mice, mouse LNS cells expressed photoreceptor specific markers, but no incorporation into host retinal tissue was seen. Human LNS cells also expressed retinal progenitor markers at the transcription level but mature retinal markers were not observed in vitro or in vivo. This data highlights that mouse Corneal limbal stromal progenitor cells can transdifferentiate towards a retinal lineage. Complete differentiation is likely to require more comprehensive regulation; however, the accessibility and plasticity of LNS makes them an attractive cell resource for future study and ultimately therapeutic application.

  • characterisation of mouse limbal neurosphere cells a potential cell source of functional neurons
    British Journal of Ophthalmology, 2012
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Background/aims To characterise the origin, ultrastructure and functional properties of Corneal limbal neurospheres (LNS). Methods Limbal cells were isolated from the Corneal Limbus of adult mice and cultured in a serum-free sphere forming culture system. LNS were characterised by immunocytochemistry, Reverse-transcription-PCR and electron microscopy. LNS cells were also cocultured with neonatal mouse retinal cells. Phenotype and function were then assessed by immunofluorescence and a calcium influx/efflux assay. Results LNS cells displayed clonal growth and self-renewal, and expressed a wide range of stem cell and neural lineage markers. The acquisition of neural properties was concordant with expression of neural crest markers including CD34, Sca1, Sox9, Twist1, but not CD45. LNS exhibited similar morphology and microstructure to neurospheres derived from the central nervous system. Following culture in a conducive environment, the derived cells displayed mature neural markers and exhibited electrical excitability. Conclusions Corneal limbal stromal progenitor cells are a potential and convenient autologous cell source to generate functional neurons.

  • derivation of neural progenitor cells from adult mouse Corneal Limbus
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Purpose: cells from the adult rodent Corneal limbal epithelium basal layer have been demonstrated to display neuronal potential and differentiate towards functional neurons in vitro. A sphere forming culture system has also been used to expand stem cells of a neural crest origin from mouse Corneal stroma. The aim of this study was to investigate whether progenitors with neuronal potential could be cultured from adult murine Limbus to form neuronal characteristics. Methods: cells from adult (8 weeks) murine Corneal Limbus were isolated and cultured in a serum-free sphere-forming system in the presence of mitogens. Cells were maintained in the presence or absence of the bone morphogenetic protein 4 (BMP4) inhibitor (Noggin). Sphere derived cells and their progeny were characterized using immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). Results: adult mouse limbal cells formed sphere colonies with the ability to self renew. The frequency of sphere-forming cells was 0.45-1.0% subject to culture conditions. Noggin did not have an effect on the efficiency of sphere formation. In the presence and absence of Noggin, sphere cells expressed ABCG2, Sox2, Nestin and beta-III tubulin (early differentiated neuron) but not P63 (epithelial stem cell marker) as shown by immunocytochemistry. Expression of neuronal stem/progenitor cells markers (nestin, Sox2, Musashi, beta-III tubulin) and neural crest markers (Twist1 and Slug) increased with time in culture and passage, concomitant with a decrease in expression of the epithelium lineage markers P63 and cytokeratin12, as shown by RT-PCR. Conclusions: the adult mouse Corneal Limbus contains stem / precursor cells which express neural stem cell markers in vitro. These stem cells/ progenitor cells appear to be neural crest & are likely to be derived from the Corneal limbal stroma

Kazuo Tsubota - One of the best experts on this subject based on the ideXlab platform.

  • melanocytes in the Corneal Limbus interact with k19 positive basal epithelial cells
    Experimental Eye Research, 2005
    Co-Authors: Kazunari Higa, Shigeto Shimmura, Hideyuki Miyashita, Jun Shimazaki, Kazuo Tsubota
    Abstract:

    Abstract The human Corneal Limbus is identified by the distinct features of the palisades of Vogt (POV), which contain pigment granules that are aligned with the microplicae of the epithelium. Although it is presumed that pigments are produced by melanocytes, the characterization of melanocytes in the Limbus has not been clearly documented. We examined human limbal tissues by whole mounts and serial histological sections to localize epithelial cells containing melanin granules. Most of the pigmented cells observed by immunohistochemistry were K19 (+) cells in the basal limbal epithelium. A superimposed image revealed that melanin granules were oriented towards the apex of each K19 (+) cell, acting as a pigmented cap facing the ocular surface. Melanocytes were identified by MART1, an antigen specific to melanocyte-lineage cells. Melanocytes were shown to exist as sporadic cells with dendritic processes that extend to surrounding epithelial cells. Melanocytes were also found in light-pigmented donor tissue when visualized by the tyrosinase assay using the enzyme substrate DOPA. Since tyrosinase activity was not found in epithelial cells, the production of melanin is exclusively the role of melanocytes that comprised 5·3±2·7% of the total cells in cytospin samples ( N =3). Melanocytes and K19 (+) epithelial cells may form a functional network similar to the melanin unit of the skin.

  • evidence of long term survival of donor derived cells after limbal allograft transplantation
    Investigative Ophthalmology & Visual Science, 1999
    Co-Authors: Jun Shimazaki, Shigeto Shimmura, Minako Kaido, Naoshi Shinozaki, Batmunkh Munkhbat, Masao Hagihara, Kimiyoshi Tsuji, Kazuo Tsubota
    Abstract:

    PURPOSE. Severe destruction of the Corneal Limbus causes conjunctival invasion and subsequent visual loss. Limbal allograft transplantation (LAT) was recently proposed for the treatment of these disorders. However, whether the method functions as a stem cell transplantation of the Corneal epithelium remains unclear. This study provided evidence that donor-derived Corneal epithelial cells survive long after LAT. METHODS. Epithelial cells on the paracentral cornea in patients who have undergone LAT were subjected to fluorescence in situ hybridization (FISH) and polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis. X and Y chromosomes were detected using sex chromosome‐specific probes in the FISH analysis, and HLA-DPB1 antigens were examined in the RFLP analysis. Eyes receiving conventional penetrating keratoplasty (PKP) served as controls. RESULTS. Donor-derived epithelial cells were detected in three of five eyes (60.0%) in the FISH analysis and in seven of nine eyes (77.8%) in the RFLP analysis. Among these eyes, one and three eyes in the FISH and RFLP analysis, respectively, had both donor- and recipient-derived cells. In control PKP eyes, none of the eyes in the FISH analysis and one of eight eyes (12.5%) in the RFLP analysis had donor-derived cells. CONCLUSIONS. These results suggest that donor-derived cells survive much longer after LAT than those after PKP, and that LAT may function as stem cell transplantation of the Corneal epithelium. (Invest Ophthalmol Vis Sci. 1999;40:1664 ‐1668)

Heather Thomson - One of the best experts on this subject based on the ideXlab platform.

  • adult limbal neurosphere cells a potential autologous cell resource for retinal cell generation
    PLOS ONE, 2014
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery, Jessica Cooke, Jennifer Scott
    Abstract:

    The Corneal Limbus is a readily accessible region at the front of the eye, separating the cornea and sclera. Neural colonies (neurospheres) can be generated from adult Corneal Limbus in vitro. We have previously shown that these neurospheres originate from neural crest stem/progenitor cells and that they can differentiate into functional neurons in vitro. The aim of this study was to investigate whether mouse and human limbal neurosphere cells (LNS) could differentiate towards a retinal lineage both in vivo and in vitro following exposure to a developing retinal microenvironment. In this article we show that LNS can be generated from adult mice and aged humans (up to 97 years) using a serum free culture assay. Following culture with developing mouse retinal cells, we detected retinal progenitor cell markers, mature retinal/neuronal markers and sensory cilia in the majority of mouse LNS experiments. After transplantation into the sub-retinal space of neonatal mice, mouse LNS cells expressed photoreceptor specific markers, but no incorporation into host retinal tissue was seen. Human LNS cells also expressed retinal progenitor markers at the transcription level but mature retinal markers were not observed in vitro or in vivo. This data highlights that mouse Corneal limbal stromal progenitor cells can transdifferentiate towards a retinal lineage. Complete differentiation is likely to require more comprehensive regulation; however, the accessibility and plasticity of LNS makes them an attractive cell resource for future study and ultimately therapeutic application.

  • characterisation of mouse limbal neurosphere cells a potential cell source of functional neurons
    British Journal of Ophthalmology, 2012
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Background/aims To characterise the origin, ultrastructure and functional properties of Corneal limbal neurospheres (LNS). Methods Limbal cells were isolated from the Corneal Limbus of adult mice and cultured in a serum-free sphere forming culture system. LNS were characterised by immunocytochemistry, Reverse-transcription-PCR and electron microscopy. LNS cells were also cocultured with neonatal mouse retinal cells. Phenotype and function were then assessed by immunofluorescence and a calcium influx/efflux assay. Results LNS cells displayed clonal growth and self-renewal, and expressed a wide range of stem cell and neural lineage markers. The acquisition of neural properties was concordant with expression of neural crest markers including CD34, Sca1, Sox9, Twist1, but not CD45. LNS exhibited similar morphology and microstructure to neurospheres derived from the central nervous system. Following culture in a conducive environment, the derived cells displayed mature neural markers and exhibited electrical excitability. Conclusions Corneal limbal stromal progenitor cells are a potential and convenient autologous cell source to generate functional neurons.

  • derivation of neural progenitor cells from adult mouse Corneal Limbus
    Investigative Ophthalmology & Visual Science, 2010
    Co-Authors: Xiaoli Chen, Heather Thomson, Parwez Hossain, Andrew J Lotery
    Abstract:

    Purpose: cells from the adult rodent Corneal limbal epithelium basal layer have been demonstrated to display neuronal potential and differentiate towards functional neurons in vitro. A sphere forming culture system has also been used to expand stem cells of a neural crest origin from mouse Corneal stroma. The aim of this study was to investigate whether progenitors with neuronal potential could be cultured from adult murine Limbus to form neuronal characteristics. Methods: cells from adult (8 weeks) murine Corneal Limbus were isolated and cultured in a serum-free sphere-forming system in the presence of mitogens. Cells were maintained in the presence or absence of the bone morphogenetic protein 4 (BMP4) inhibitor (Noggin). Sphere derived cells and their progeny were characterized using immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). Results: adult mouse limbal cells formed sphere colonies with the ability to self renew. The frequency of sphere-forming cells was 0.45-1.0% subject to culture conditions. Noggin did not have an effect on the efficiency of sphere formation. In the presence and absence of Noggin, sphere cells expressed ABCG2, Sox2, Nestin and beta-III tubulin (early differentiated neuron) but not P63 (epithelial stem cell marker) as shown by immunocytochemistry. Expression of neuronal stem/progenitor cells markers (nestin, Sox2, Musashi, beta-III tubulin) and neural crest markers (Twist1 and Slug) increased with time in culture and passage, concomitant with a decrease in expression of the epithelium lineage markers P63 and cytokeratin12, as shown by RT-PCR. Conclusions: the adult mouse Corneal Limbus contains stem / precursor cells which express neural stem cell markers in vitro. These stem cells/ progenitor cells appear to be neural crest & are likely to be derived from the Corneal limbal stroma