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Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma.

Abu-Hassan DW, Li X, Ryan EI, Acott TS, Kelley MJ - Stem Cells (2015)

Bottom Line: A notable feature associated with glaucoma is outflow pathway cell loss.We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model.These differentiated cells (TM-like iPSCs) became similar to TM cells in both morphology and expression patterns.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, USA; Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon, USA; Department of Biochemistry & Physiology, University of Jordan, Amman, Jordan.

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Saponin treatment of human anterior segments. (A): Experimental schematic for cell death assessment after a 7-minute treatment with 0.01% saponin. Frontal sections of normal vehicle-treated control aged anterior segments (B, C) compared to saponin-treated anterior segments (D, E) showing live cells (green) and nuclei of dead cells (red). White scale bars represent 100 µm. (F): Live cell values expressed as GFU per µm field volume, using total area of green fluorescence as a proxy and comparing saponin treated with control. Significance at p < .05 is indicated by *. (G): Dead cell count of red nuclei per three-dimensional (3D) field; both total and broken down into fields showing results for less than or more than 100 nuclei. Significance is indicated as **, p < .001 and *, p < .05. (H): Live cells/field showing comparisons of live cells for glaucoma eyes, saponin-treated, and normal control groups, all normalized to common 3D field volumes. (I): Schematic showing 0.01% saponin treatment pattern for flow studies. (J): Normalized flow rate for perfused human anterior segments treated with saponin or vehicle (normal) and then subjected to intraocular pressure homeostatic 2× pressure challenge. Mean and SEM are shown where n = 8 for normal and n = 17 for saponin-treated anterior segments with significance determined by one-way ANOVA at p < .001 indicated by **. Abbreviations: GFU, green fluorescence units; JCT, juxtacanalicular region; SC, Schlemm's canal; TM, trabecular meshwork.
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fig01: Saponin treatment of human anterior segments. (A): Experimental schematic for cell death assessment after a 7-minute treatment with 0.01% saponin. Frontal sections of normal vehicle-treated control aged anterior segments (B, C) compared to saponin-treated anterior segments (D, E) showing live cells (green) and nuclei of dead cells (red). White scale bars represent 100 µm. (F): Live cell values expressed as GFU per µm field volume, using total area of green fluorescence as a proxy and comparing saponin treated with control. Significance at p < .05 is indicated by *. (G): Dead cell count of red nuclei per three-dimensional (3D) field; both total and broken down into fields showing results for less than or more than 100 nuclei. Significance is indicated as **, p < .001 and *, p < .05. (H): Live cells/field showing comparisons of live cells for glaucoma eyes, saponin-treated, and normal control groups, all normalized to common 3D field volumes. (I): Schematic showing 0.01% saponin treatment pattern for flow studies. (J): Normalized flow rate for perfused human anterior segments treated with saponin or vehicle (normal) and then subjected to intraocular pressure homeostatic 2× pressure challenge. Mean and SEM are shown where n = 8 for normal and n = 17 for saponin-treated anterior segments with significance determined by one-way ANOVA at p < .001 indicated by **. Abbreviations: GFU, green fluorescence units; JCT, juxtacanalicular region; SC, Schlemm's canal; TM, trabecular meshwork.

Mentions: The most widely accepted experimental model for these studies is perfused anterior segment organ culture (Supporting Information Fig. S1B) 29. Some dead cells were detected in untreated porcine anterior segments, and this was designated basal cell death. However, saponin induced significantly more cell death (Supporting Information Fig. S3). Saponin (0.01%) was injected into the flow line of perfused human anterior segments mounted in the ocular perfusion system (Supporting Information Fig. S1B) and incubated in the TM for 7 minutes before being rinsed out. The live/dead assay was used to determine whether saponin affected the viability of human TM cells and this was viewed in frontal sections of anterior segments (Fig. 1B–1E). The TM has several zones of cells, and saponin was able to penetrate and kill cells in all zones down into the deepest zone, the juxtacanalicular region or JCT (Fig. 1D, 1E), which is the region thought to be responsible for IOP homeostasis 4,8,35. Saponin treatment induced cell death although more live than dead cells still populated the TM, indicating that saponin's effect at this concentration was partial (Fig. 1F, 1G). Saponin-treated anterior segments contained considerably more dead TM cells than vehicle-treated anterior segments (Fig. 1G). Furthermore, comparing the live cells per three-dimensional field in glaucomatous and saponin-treated eyes relative to normal eyes revealed a very similar pattern, that is, 67.42 and 66.42 live cells per field, respectively (Fig. 1H). Thus, this level of saponin treatment induced changes in cell counts that roughly approximate those found in glaucoma.


Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma.

Abu-Hassan DW, Li X, Ryan EI, Acott TS, Kelley MJ - Stem Cells (2015)

Saponin treatment of human anterior segments. (A): Experimental schematic for cell death assessment after a 7-minute treatment with 0.01% saponin. Frontal sections of normal vehicle-treated control aged anterior segments (B, C) compared to saponin-treated anterior segments (D, E) showing live cells (green) and nuclei of dead cells (red). White scale bars represent 100 µm. (F): Live cell values expressed as GFU per µm field volume, using total area of green fluorescence as a proxy and comparing saponin treated with control. Significance at p < .05 is indicated by *. (G): Dead cell count of red nuclei per three-dimensional (3D) field; both total and broken down into fields showing results for less than or more than 100 nuclei. Significance is indicated as **, p < .001 and *, p < .05. (H): Live cells/field showing comparisons of live cells for glaucoma eyes, saponin-treated, and normal control groups, all normalized to common 3D field volumes. (I): Schematic showing 0.01% saponin treatment pattern for flow studies. (J): Normalized flow rate for perfused human anterior segments treated with saponin or vehicle (normal) and then subjected to intraocular pressure homeostatic 2× pressure challenge. Mean and SEM are shown where n = 8 for normal and n = 17 for saponin-treated anterior segments with significance determined by one-way ANOVA at p < .001 indicated by **. Abbreviations: GFU, green fluorescence units; JCT, juxtacanalicular region; SC, Schlemm's canal; TM, trabecular meshwork.
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Related In: Results  -  Collection

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fig01: Saponin treatment of human anterior segments. (A): Experimental schematic for cell death assessment after a 7-minute treatment with 0.01% saponin. Frontal sections of normal vehicle-treated control aged anterior segments (B, C) compared to saponin-treated anterior segments (D, E) showing live cells (green) and nuclei of dead cells (red). White scale bars represent 100 µm. (F): Live cell values expressed as GFU per µm field volume, using total area of green fluorescence as a proxy and comparing saponin treated with control. Significance at p < .05 is indicated by *. (G): Dead cell count of red nuclei per three-dimensional (3D) field; both total and broken down into fields showing results for less than or more than 100 nuclei. Significance is indicated as **, p < .001 and *, p < .05. (H): Live cells/field showing comparisons of live cells for glaucoma eyes, saponin-treated, and normal control groups, all normalized to common 3D field volumes. (I): Schematic showing 0.01% saponin treatment pattern for flow studies. (J): Normalized flow rate for perfused human anterior segments treated with saponin or vehicle (normal) and then subjected to intraocular pressure homeostatic 2× pressure challenge. Mean and SEM are shown where n = 8 for normal and n = 17 for saponin-treated anterior segments with significance determined by one-way ANOVA at p < .001 indicated by **. Abbreviations: GFU, green fluorescence units; JCT, juxtacanalicular region; SC, Schlemm's canal; TM, trabecular meshwork.
Mentions: The most widely accepted experimental model for these studies is perfused anterior segment organ culture (Supporting Information Fig. S1B) 29. Some dead cells were detected in untreated porcine anterior segments, and this was designated basal cell death. However, saponin induced significantly more cell death (Supporting Information Fig. S3). Saponin (0.01%) was injected into the flow line of perfused human anterior segments mounted in the ocular perfusion system (Supporting Information Fig. S1B) and incubated in the TM for 7 minutes before being rinsed out. The live/dead assay was used to determine whether saponin affected the viability of human TM cells and this was viewed in frontal sections of anterior segments (Fig. 1B–1E). The TM has several zones of cells, and saponin was able to penetrate and kill cells in all zones down into the deepest zone, the juxtacanalicular region or JCT (Fig. 1D, 1E), which is the region thought to be responsible for IOP homeostasis 4,8,35. Saponin treatment induced cell death although more live than dead cells still populated the TM, indicating that saponin's effect at this concentration was partial (Fig. 1F, 1G). Saponin-treated anterior segments contained considerably more dead TM cells than vehicle-treated anterior segments (Fig. 1G). Furthermore, comparing the live cells per three-dimensional field in glaucomatous and saponin-treated eyes relative to normal eyes revealed a very similar pattern, that is, 67.42 and 66.42 live cells per field, respectively (Fig. 1H). Thus, this level of saponin treatment induced changes in cell counts that roughly approximate those found in glaucoma.

Bottom Line: A notable feature associated with glaucoma is outflow pathway cell loss.We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model.These differentiated cells (TM-like iPSCs) became similar to TM cells in both morphology and expression patterns.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, USA; Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon, USA; Department of Biochemistry & Physiology, University of Jordan, Amman, Jordan.

Show MeSH
Related in: MedlinePlus