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Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

Lakowski J, Gonzalez-Cordero A, West EL, Han YT, Welby E, Naeem A, Blackford SJ, Bainbridge JW, Pearson RA, Ali RR, Sowden JC - Stem Cells (2015)

Bottom Line: Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment.As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures.Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation.

View Article: PubMed Central - PubMed

Affiliation: Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health, University College London, London, United Kingdom.

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Majority of CD73 positive cells in embryonic stem cell (ESC)‐derived retinal cultures express photoreceptor markers. (A): Immunocytochemical analysis of cells from dissociated ESC retinal cultures costained with cell surface biomarker CD73 and photoreceptor markers Rhodopsin or Recoverin. Representative confocal tile scan of plated cells. Inset show high magnification view of indicated area. (B): Image analysis software Cellprofiler (Broad Institute, http://www.cellprofiler.org/) was used to determine the number of single or double positive cells from three independent experiments and confirmed by manual counting. Total number of marker positive cells is shown as light bars while darkly shaded bars indicate the percentage of cells costaining for the respective second marker. About 44% of all cells express rhodopsin, 29% CD73, and 16% show Recoverin staining. The majority of CD73 positive cells (∼82%) label with rod photoreceptor marker rhodopsin and ∼40% label with Recoverin. (C): Analysis of cells selected from ESC‐derived day 27 retinal cultures using photoreceptor precursor biomarker panel (CD+). Immunocytochemical analysis of CD+ selected cells, CD− and unsorted ESC‐derived cells with photoreceptor markers CRX, Rhodopsin, or Recoverin was used to determine the number of cells expressing each marker (n = 3 independent experiments); 82.64 ± 7.53%, 76.83 ± 9.6%, and 17.3 ± 4.9% of CD+ cells labelled with photoreceptor markers CRX, Rhodopsin, and Recoverin, respectively; mean ± SD. Scale bars: 40 µm.
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stem2051-fig-0004: Majority of CD73 positive cells in embryonic stem cell (ESC)‐derived retinal cultures express photoreceptor markers. (A): Immunocytochemical analysis of cells from dissociated ESC retinal cultures costained with cell surface biomarker CD73 and photoreceptor markers Rhodopsin or Recoverin. Representative confocal tile scan of plated cells. Inset show high magnification view of indicated area. (B): Image analysis software Cellprofiler (Broad Institute, http://www.cellprofiler.org/) was used to determine the number of single or double positive cells from three independent experiments and confirmed by manual counting. Total number of marker positive cells is shown as light bars while darkly shaded bars indicate the percentage of cells costaining for the respective second marker. About 44% of all cells express rhodopsin, 29% CD73, and 16% show Recoverin staining. The majority of CD73 positive cells (∼82%) label with rod photoreceptor marker rhodopsin and ∼40% label with Recoverin. (C): Analysis of cells selected from ESC‐derived day 27 retinal cultures using photoreceptor precursor biomarker panel (CD+). Immunocytochemical analysis of CD+ selected cells, CD− and unsorted ESC‐derived cells with photoreceptor markers CRX, Rhodopsin, or Recoverin was used to determine the number of cells expressing each marker (n = 3 independent experiments); 82.64 ± 7.53%, 76.83 ± 9.6%, and 17.3 ± 4.9% of CD+ cells labelled with photoreceptor markers CRX, Rhodopsin, and Recoverin, respectively; mean ± SD. Scale bars: 40 µm.

Mentions: The distribution of CD markers is usually not restricted to one particular tissue or cell type. We, therefore, confirmed the identity of the PPr biomarker panel‐positive cells generated in the 3D retinal culture system using immuno‐cytochemistry (Fig. 4A, 4B). At day 27, retina were dissociated and plated on coverslips to allow investigation of colocalization of CD73 with Rhodopsin or Recoverin on a single cell basis. Rhodopsin and Recoverin were selected as indicators of photoreceptor cell identity with robust available antibodies. We observed that 36.3% ± 14.4 (n = 3) of the cells labelled with CD73. As expected, the majority of CD73 positive cells were also strongly colabelled with the rod pigment rhodopsin (78.3% ± 10.5; n = 3), confirming the rod photoreceptor identity of PPr biomarker labelled cells generated in the day 27 ESC‐derived retina. Similar analysis conducted using Recoverin showed colabelling of 41% ± 13.6 (n = 3) of the CD73 positive cells.


Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

Lakowski J, Gonzalez-Cordero A, West EL, Han YT, Welby E, Naeem A, Blackford SJ, Bainbridge JW, Pearson RA, Ali RR, Sowden JC - Stem Cells (2015)

Majority of CD73 positive cells in embryonic stem cell (ESC)‐derived retinal cultures express photoreceptor markers. (A): Immunocytochemical analysis of cells from dissociated ESC retinal cultures costained with cell surface biomarker CD73 and photoreceptor markers Rhodopsin or Recoverin. Representative confocal tile scan of plated cells. Inset show high magnification view of indicated area. (B): Image analysis software Cellprofiler (Broad Institute, http://www.cellprofiler.org/) was used to determine the number of single or double positive cells from three independent experiments and confirmed by manual counting. Total number of marker positive cells is shown as light bars while darkly shaded bars indicate the percentage of cells costaining for the respective second marker. About 44% of all cells express rhodopsin, 29% CD73, and 16% show Recoverin staining. The majority of CD73 positive cells (∼82%) label with rod photoreceptor marker rhodopsin and ∼40% label with Recoverin. (C): Analysis of cells selected from ESC‐derived day 27 retinal cultures using photoreceptor precursor biomarker panel (CD+). Immunocytochemical analysis of CD+ selected cells, CD− and unsorted ESC‐derived cells with photoreceptor markers CRX, Rhodopsin, or Recoverin was used to determine the number of cells expressing each marker (n = 3 independent experiments); 82.64 ± 7.53%, 76.83 ± 9.6%, and 17.3 ± 4.9% of CD+ cells labelled with photoreceptor markers CRX, Rhodopsin, and Recoverin, respectively; mean ± SD. Scale bars: 40 µm.
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stem2051-fig-0004: Majority of CD73 positive cells in embryonic stem cell (ESC)‐derived retinal cultures express photoreceptor markers. (A): Immunocytochemical analysis of cells from dissociated ESC retinal cultures costained with cell surface biomarker CD73 and photoreceptor markers Rhodopsin or Recoverin. Representative confocal tile scan of plated cells. Inset show high magnification view of indicated area. (B): Image analysis software Cellprofiler (Broad Institute, http://www.cellprofiler.org/) was used to determine the number of single or double positive cells from three independent experiments and confirmed by manual counting. Total number of marker positive cells is shown as light bars while darkly shaded bars indicate the percentage of cells costaining for the respective second marker. About 44% of all cells express rhodopsin, 29% CD73, and 16% show Recoverin staining. The majority of CD73 positive cells (∼82%) label with rod photoreceptor marker rhodopsin and ∼40% label with Recoverin. (C): Analysis of cells selected from ESC‐derived day 27 retinal cultures using photoreceptor precursor biomarker panel (CD+). Immunocytochemical analysis of CD+ selected cells, CD− and unsorted ESC‐derived cells with photoreceptor markers CRX, Rhodopsin, or Recoverin was used to determine the number of cells expressing each marker (n = 3 independent experiments); 82.64 ± 7.53%, 76.83 ± 9.6%, and 17.3 ± 4.9% of CD+ cells labelled with photoreceptor markers CRX, Rhodopsin, and Recoverin, respectively; mean ± SD. Scale bars: 40 µm.
Mentions: The distribution of CD markers is usually not restricted to one particular tissue or cell type. We, therefore, confirmed the identity of the PPr biomarker panel‐positive cells generated in the 3D retinal culture system using immuno‐cytochemistry (Fig. 4A, 4B). At day 27, retina were dissociated and plated on coverslips to allow investigation of colocalization of CD73 with Rhodopsin or Recoverin on a single cell basis. Rhodopsin and Recoverin were selected as indicators of photoreceptor cell identity with robust available antibodies. We observed that 36.3% ± 14.4 (n = 3) of the cells labelled with CD73. As expected, the majority of CD73 positive cells were also strongly colabelled with the rod pigment rhodopsin (78.3% ± 10.5; n = 3), confirming the rod photoreceptor identity of PPr biomarker labelled cells generated in the day 27 ESC‐derived retina. Similar analysis conducted using Recoverin showed colabelling of 41% ± 13.6 (n = 3) of the CD73 positive cells.

Bottom Line: Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment.As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures.Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation.

View Article: PubMed Central - PubMed

Affiliation: Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health, University College London, London, United Kingdom.

Show MeSH
Related in: MedlinePlus