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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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Biomarker expression comparison between iPSCs, TM, and differentiated TM-like iPSCs. (A): Immunohistochemical comparison of levels of four stem cell markers, NANOG, OCT3/4, SOX2, and KLF4 by these three cell types. Cell nuclei are stained with DAPI (blue). All panels are exactly the same size with scale bar = 100 µm. (B): TM cell markers as expressed by iPSCs, HTM, and TM-like iPSC. DAPI nuclear stain is blue, all panels are the exact same size and scales are identical with the white scale bars = 100 µm. (C): Western immunoblot showing levels of these two groups of proteins and (d) gels from quantitative RT-PCR analysis showing levels of mRNA expression for these genes. Loading controls are α-tubulin for Western immunoblots and 18S ribosomal subunit for mRNA gels. Abbreviations: HTM, human trabecular meshwork; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.
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fig03: Biomarker expression comparison between iPSCs, TM, and differentiated TM-like iPSCs. (A): Immunohistochemical comparison of levels of four stem cell markers, NANOG, OCT3/4, SOX2, and KLF4 by these three cell types. Cell nuclei are stained with DAPI (blue). All panels are exactly the same size with scale bar = 100 µm. (B): TM cell markers as expressed by iPSCs, HTM, and TM-like iPSC. DAPI nuclear stain is blue, all panels are the exact same size and scales are identical with the white scale bars = 100 µm. (C): Western immunoblot showing levels of these two groups of proteins and (d) gels from quantitative RT-PCR analysis showing levels of mRNA expression for these genes. Loading controls are α-tubulin for Western immunoblots and 18S ribosomal subunit for mRNA gels. Abbreviations: HTM, human trabecular meshwork; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.

Mentions: To use the iPSCs, they were differentiated to embryoid bodies, and then to a TM-like cell. Since patient-specific TM cells could only be obtained by an invasive patient surgery, autologous skin iPSCs would be a much preferred source of replacement cells 15. After extensive evaluation of possible methods to differentiate iPSCs (manuscript in preparation), we found conditions that seemed usable. The EBs developed from iPSCs became differentiated or more “TM-like” in appearance after sustained exposure to ECM and conditioned medium, both produced by cultured primary human TM cells. Other treatment combinations were less effective. These TM-like iPSCs resembled human TM cells morphologically (Supporting Information Fig. S4), which have a relatively distinctive appearance 28. Expression of typical stem cell markers, NANOG, OCT3/4, SOX2, and KLF4, 22,23,35 was much higher for iPSCs and negligible for differentiated TM-like iPSCs or human TM cells (Fig. 3A, 3C, 3D). Although there is no specific cell marker for TM cells, CHI3L1, Wnt1, α3 integrin, and AQP1 are typically expressed by TM cells 36–41 and are not expressed or are only lightly expressed by iPSCs (Fig. 3B–3D). The TM-like iPSC expression pattern resembled that of human TM cells much more closely than that of iPSCs (Fig. 3A–3D).


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)

Biomarker expression comparison between iPSCs, TM, and differentiated TM-like iPSCs. (A): Immunohistochemical comparison of levels of four stem cell markers, NANOG, OCT3/4, SOX2, and KLF4 by these three cell types. Cell nuclei are stained with DAPI (blue). All panels are exactly the same size with scale bar = 100 µm. (B): TM cell markers as expressed by iPSCs, HTM, and TM-like iPSC. DAPI nuclear stain is blue, all panels are the exact same size and scales are identical with the white scale bars = 100 µm. (C): Western immunoblot showing levels of these two groups of proteins and (d) gels from quantitative RT-PCR analysis showing levels of mRNA expression for these genes. Loading controls are α-tubulin for Western immunoblots and 18S ribosomal subunit for mRNA gels. Abbreviations: HTM, human trabecular meshwork; 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

fig03: Biomarker expression comparison between iPSCs, TM, and differentiated TM-like iPSCs. (A): Immunohistochemical comparison of levels of four stem cell markers, NANOG, OCT3/4, SOX2, and KLF4 by these three cell types. Cell nuclei are stained with DAPI (blue). All panels are exactly the same size with scale bar = 100 µm. (B): TM cell markers as expressed by iPSCs, HTM, and TM-like iPSC. DAPI nuclear stain is blue, all panels are the exact same size and scales are identical with the white scale bars = 100 µm. (C): Western immunoblot showing levels of these two groups of proteins and (d) gels from quantitative RT-PCR analysis showing levels of mRNA expression for these genes. Loading controls are α-tubulin for Western immunoblots and 18S ribosomal subunit for mRNA gels. Abbreviations: HTM, human trabecular meshwork; iPSCs, induced pluripotent stem cells; TM, trabecular meshwork.
Mentions: To use the iPSCs, they were differentiated to embryoid bodies, and then to a TM-like cell. Since patient-specific TM cells could only be obtained by an invasive patient surgery, autologous skin iPSCs would be a much preferred source of replacement cells 15. After extensive evaluation of possible methods to differentiate iPSCs (manuscript in preparation), we found conditions that seemed usable. The EBs developed from iPSCs became differentiated or more “TM-like” in appearance after sustained exposure to ECM and conditioned medium, both produced by cultured primary human TM cells. Other treatment combinations were less effective. These TM-like iPSCs resembled human TM cells morphologically (Supporting Information Fig. S4), which have a relatively distinctive appearance 28. Expression of typical stem cell markers, NANOG, OCT3/4, SOX2, and KLF4, 22,23,35 was much higher for iPSCs and negligible for differentiated TM-like iPSCs or human TM cells (Fig. 3A, 3C, 3D). Although there is no specific cell marker for TM cells, CHI3L1, Wnt1, α3 integrin, and AQP1 are typically expressed by TM cells 36–41 and are not expressed or are only lightly expressed by iPSCs (Fig. 3B–3D). The TM-like iPSC expression pattern resembled that of human TM cells much more closely than that of iPSCs (Fig. 3A–3D).

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