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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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Replacement of saponin-depleted cells with TM-like iPSCs in anterior segments. (A): Confocal analysis of frontal section showing transplanted QDot (red) labeled TM-like iPSCs at all levels of the outflow pathway. Blue shows TM beam collagen and elastic fiber autofluorescence; scale bars are 100 µm. (B): Frontal section after perfusion protocol shows TM beams (blue), cell surface CD44 immunostaining (green), and QDot-labeled transplanted TM-like iPSCs (red). Scale bar indicates 100 µm. White dashes outline individual transplanted cells attached to TM beams. Outlines were determined by scanning through the three-dimensional confocal Z-stacks. (C): After 1× perfusion, saponin was added as indicated, rinsed out, and perfusion resumed for 24 hours at 1× pressure. The 2× pressure challenge gave no intraocular pressure (IOP) homeostatic response. TM-like iPSCs were added and allowed to attach for 24 hours; flow was resumed at 1× pressure and then increased to 2× pressure. A typical IOP homeostatic response now occurred over several days. n = 6 experiments with separate anterior segments and significance by one-way ANOVA is *, p < .05 and **, p < .001. (D): Similar experiment with 300,000 DF transplanted as a control. They actually triggered a reduction in outflow but no IOP response to 2× pressure challenge. (E): Effects of mock-differentiated iPSC EB, which had been exposed only to 5% aqueous humor during a parallel differentiation period, (F) HUVECs, or (G) no cells added at all were compared. Only differentiated TM-like iPSCs produced an IOP homeostatic pressure response. Abbreviations: DF, dermal fibroblasts; EB, embryoid bodies; HTM, human trabecular meshwork; HUVEC, human umbilical vein endothelial cell; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.
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fig05: Replacement of saponin-depleted cells with TM-like iPSCs in anterior segments. (A): Confocal analysis of frontal section showing transplanted QDot (red) labeled TM-like iPSCs at all levels of the outflow pathway. Blue shows TM beam collagen and elastic fiber autofluorescence; scale bars are 100 µm. (B): Frontal section after perfusion protocol shows TM beams (blue), cell surface CD44 immunostaining (green), and QDot-labeled transplanted TM-like iPSCs (red). Scale bar indicates 100 µm. White dashes outline individual transplanted cells attached to TM beams. Outlines were determined by scanning through the three-dimensional confocal Z-stacks. (C): After 1× perfusion, saponin was added as indicated, rinsed out, and perfusion resumed for 24 hours at 1× pressure. The 2× pressure challenge gave no intraocular pressure (IOP) homeostatic response. TM-like iPSCs were added and allowed to attach for 24 hours; flow was resumed at 1× pressure and then increased to 2× pressure. A typical IOP homeostatic response now occurred over several days. n = 6 experiments with separate anterior segments and significance by one-way ANOVA is *, p < .05 and **, p < .001. (D): Similar experiment with 300,000 DF transplanted as a control. They actually triggered a reduction in outflow but no IOP response to 2× pressure challenge. (E): Effects of mock-differentiated iPSC EB, which had been exposed only to 5% aqueous humor during a parallel differentiation period, (F) HUVECs, or (G) no cells added at all were compared. Only differentiated TM-like iPSCs produced an IOP homeostatic pressure response. Abbreviations: DF, dermal fibroblasts; EB, embryoid bodies; HTM, human trabecular meshwork; HUVEC, human umbilical vein endothelial cell; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.

Mentions: Differentiated TM-like iPSCs were transplanted into saponin-treated human anterior segments (Fig. 5A–5C). The saponin treatment, transplantation process, and pressure challenges were the same as conducted earlier for cultured TM cells (Fig. 2) and detailed in Materials and Methods and outlined in Figure 2A. Many of the 300,000 QDot-labeled differentiated TM-like iPSCs attached and became integrated into the TM. Frontal sections (Fig. 5A, 5B) show that the transplanted cells were found throughout the TM, including the deepest JCT layer, which is thought to be the location of the outflow resistance 8,44. The dashed lines around the Qdot-containing transplanted cells (Fig. 5B) were drawn based on carefully scanning through the confocal Z-stacks to define the CD44-labeled cell surfaces.


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)

Replacement of saponin-depleted cells with TM-like iPSCs in anterior segments. (A): Confocal analysis of frontal section showing transplanted QDot (red) labeled TM-like iPSCs at all levels of the outflow pathway. Blue shows TM beam collagen and elastic fiber autofluorescence; scale bars are 100 µm. (B): Frontal section after perfusion protocol shows TM beams (blue), cell surface CD44 immunostaining (green), and QDot-labeled transplanted TM-like iPSCs (red). Scale bar indicates 100 µm. White dashes outline individual transplanted cells attached to TM beams. Outlines were determined by scanning through the three-dimensional confocal Z-stacks. (C): After 1× perfusion, saponin was added as indicated, rinsed out, and perfusion resumed for 24 hours at 1× pressure. The 2× pressure challenge gave no intraocular pressure (IOP) homeostatic response. TM-like iPSCs were added and allowed to attach for 24 hours; flow was resumed at 1× pressure and then increased to 2× pressure. A typical IOP homeostatic response now occurred over several days. n = 6 experiments with separate anterior segments and significance by one-way ANOVA is *, p < .05 and **, p < .001. (D): Similar experiment with 300,000 DF transplanted as a control. They actually triggered a reduction in outflow but no IOP response to 2× pressure challenge. (E): Effects of mock-differentiated iPSC EB, which had been exposed only to 5% aqueous humor during a parallel differentiation period, (F) HUVECs, or (G) no cells added at all were compared. Only differentiated TM-like iPSCs produced an IOP homeostatic pressure response. Abbreviations: DF, dermal fibroblasts; EB, embryoid bodies; HTM, human trabecular meshwork; HUVEC, human umbilical vein endothelial cell; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359625&req=5

fig05: Replacement of saponin-depleted cells with TM-like iPSCs in anterior segments. (A): Confocal analysis of frontal section showing transplanted QDot (red) labeled TM-like iPSCs at all levels of the outflow pathway. Blue shows TM beam collagen and elastic fiber autofluorescence; scale bars are 100 µm. (B): Frontal section after perfusion protocol shows TM beams (blue), cell surface CD44 immunostaining (green), and QDot-labeled transplanted TM-like iPSCs (red). Scale bar indicates 100 µm. White dashes outline individual transplanted cells attached to TM beams. Outlines were determined by scanning through the three-dimensional confocal Z-stacks. (C): After 1× perfusion, saponin was added as indicated, rinsed out, and perfusion resumed for 24 hours at 1× pressure. The 2× pressure challenge gave no intraocular pressure (IOP) homeostatic response. TM-like iPSCs were added and allowed to attach for 24 hours; flow was resumed at 1× pressure and then increased to 2× pressure. A typical IOP homeostatic response now occurred over several days. n = 6 experiments with separate anterior segments and significance by one-way ANOVA is *, p < .05 and **, p < .001. (D): Similar experiment with 300,000 DF transplanted as a control. They actually triggered a reduction in outflow but no IOP response to 2× pressure challenge. (E): Effects of mock-differentiated iPSC EB, which had been exposed only to 5% aqueous humor during a parallel differentiation period, (F) HUVECs, or (G) no cells added at all were compared. Only differentiated TM-like iPSCs produced an IOP homeostatic pressure response. Abbreviations: DF, dermal fibroblasts; EB, embryoid bodies; HTM, human trabecular meshwork; HUVEC, human umbilical vein endothelial cell; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.
Mentions: Differentiated TM-like iPSCs were transplanted into saponin-treated human anterior segments (Fig. 5A–5C). The saponin treatment, transplantation process, and pressure challenges were the same as conducted earlier for cultured TM cells (Fig. 2) and detailed in Materials and Methods and outlined in Figure 2A. Many of the 300,000 QDot-labeled differentiated TM-like iPSCs attached and became integrated into the TM. Frontal sections (Fig. 5A, 5B) show that the transplanted cells were found throughout the TM, including the deepest JCT layer, which is thought to be the location of the outflow resistance 8,44. The dashed lines around the Qdot-containing transplanted cells (Fig. 5B) were drawn based on carefully scanning through the confocal Z-stacks to define the CD44-labeled cell surfaces.

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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Related in: MedlinePlus