Stem Cells. 2015 Mar;33(3):751-61.

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

Abu-Hassan DW1, Li X, Ryan EI, Acott TS, Kelley MJ.
  • 1Department 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.

 

Abstract

Normally, trabecular meshwork (TM) and Schlemm’s canal inner wall endothelial cells within the aqueous humor outflow pathway maintain intraocular pressure within a narrow safe range. Elevation in intraocular pressure, because of the loss of homeostatic regulation by these outflow pathway cells, is the primary risk factor for vision loss due to glaucomatous optic neuropathy. A notable feature associated with glaucoma is outflow pathway cell loss. Using controlled cell loss in ex vivo perfused human outflow pathway organ culture, we developed compelling experimental evidence that this level of cell loss compromises intraocular pressure homeostatic function. This function was restored by repopulation of the model with fresh TM cells. 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. When transplanted, they were able to fully restore intraocular pressure homeostatic function. This successful transplantation of TM-like iPSCs establishes the conceptual feasibility of using autologous stem cells to restore intraocular pressure regulatory function in open-angle glaucoma patients, providing a novel alternative treatment option.

KEYWORDS: Autologous stem cell transplantation; Cell transplantation; Experimental models; Induced pluripotent stem cells; Somatic stem cells; Stem cell transplantation; Tissue regeneration; Transplantation

PMID: 25377070

 

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Trabecular meshwork cells and Schlemm’s canal inner wall endothelial cells are thought to act in concert to maintain the proper pressure within the eye. This pressure, the intraocular pressure or IOP, is caused by a fluid produced by the ciliary body called the aqueous humor, which transports nutrients to the avascular tissues of the anterior eye and collects cellular debris. It weaves throughout channels in the trabecular meshwork(TM) and out of the eye through Schlemm’s canal into the venous system to maintain a healthy eye. (Fig. 1a) IOP is normally tightly regulated by outflow pathway cells, which sense pressure changes and keep the IOP within a narrow range. However, in glaucoma, this mechanism has gone awry, and the primary risk factor for the disease is persistent elevated IOP. Elevated IOP eventually compromises the optic nerve; glaucomatous optic neuropathy is one of the leading causes of blindness. The normal adjustment in response to pressure changes is called the IOP homeostatic response1, and loss of this functional response is one of the hallmarks of glaucoma. Although glaucoma is currently treatable for a period of time, a long-lasting and effective treatment still remains elusive for many patients.

In our study, we reported a ground-breaking finding that has the exciting potential of a personalized stem cell therapy as an alternative treatment for glaucoma. This manuscript’s novelty derives from two major firsts achieved: 1) This is the first experimental evidence of a correlation between the loss of cellularity and functional impairment in the TM; and 2) This is the first report of successfully transplanting human induced pluripotent stem cells differentiated into TM-like cells which restores function.

It is known that there is decreased cell density in aged eyes and that glaucomatous eyes show even greater loss of cells. This observation, that outflow pathway cell loss is increased in glaucoma, was made by Alvarado, et. al.2, 3 Previously, whether cellular loss causes glaucoma, or if glaucoma exacerbates the drop in cell density was also unclear. Our investigation showed that the loss of cellularity in a model for glaucoma was critical to the loss of IOP homeostasis. With fewer cells available to regulate the IOP, function gradually declines. We determined that a crucial number of functional TM cells are required to maintain IOP homeostasis.

To develop a uniform cell loss model for glaucoma, we used a detergent, saponin. This partially denuded the TM resulting in approximately one-third of the cells being killed, which approximates the glaucomatous cell loss. In our intraocular pressure studies, we use perfused human anterior segment organ culture. (Fig. 1b) In this flow system, the anterior chamber of the eye is dissected from the posterior portion, and the trabecular meshwork, Schlemm’s canal, the cornea, and a rim of sclera of the anterior portion are retained. The structure is attached to a pedestal in a flow cell, with tubing extending to a perfusion reservoir holding media. This reservoir is set at 8mm of Hg (calculated distance) for 1X flow, which delivers 2.5-3.0 ml/min of flow (normal flow in a healthy eye) and 16 mm of Hg for 2X flow, which is the pressure challenge. The media flows through the TM and Schlemm’s canal and media reservoir changes are weighed to calculate the flow rate. Concurrently, we began to differentiate induced pluripotent stem cells (iPSCs) to a trabecular meshwork-like (TM-like iPSCs) cell using extracellular matrix and conditioned media from TM cell cultures, as well as a specialized media formula over a period of about 30 days. (Fig. 2 a-c)

We secure the anterior segment in place in the perfusion system, and stabilized the flow at the 1X level, prior to increasing to a 2x pressure challenge. We operate with constant pressure perfusion using the formula: C=F/P, where C=outflow facility, F= flow, and P=pressure. An increase in pressure to 2X in the normal eye results in a normal IOP homeostatic response, where, as the pressure is increased, the eye adjusts to restore normal pressure by decreasing the resistance to flow, and increasing the rate of flow. This happens over the course of a number of days. After flowing in the dilute saponin at 1X, we stopped the saponin flow in the trabecular meshwork for 7 minutes to allow time for cell contact, and then restarted and re-stabilized the 1X flow, resulting in partial cellular denudation in the TM. Upon initiating a 2X pressure challenge, we found that the saponin had obliterated the normal IOP homeostatic response, such that there was not a decrease in resistance, nor was there an increase in flow, as the eye could no longer adjust to pressure changes after partial TM cellular denudation. (Fig.3)

We first examined transplantation by repopulating the partially denuded anterior segments with an allogeneic transplantation of cultured TM cells. A 2x pressure challenge showed that the allogeneic TM cells successfully restored the IOP homeostatic response previously abolished by saponin treatment.  When we applied the same procedures to our differentiated TM-like iPSCs, they also restored the IOP homeostatic response after a 2X pressure challenge.(Fig.4) To rule out the possibility that any type of cell could produce this restoration of function, we repeated the experiment with another endothelial type of cell, human umbilical vein endothelial cells (HUVEC), fibroblasts, embryoid bodies, and no cells at all, but nothing else restored the IOP homeostatic response after saponin cell depletion.

The successful transplantation of TM-like iPSCs in partially denuded anterior segments demonstrates their potential as a future autologous therapeutic glaucoma treatment. When derived from the patient’s dermal fibroblasts, these cells can be patient-specific, thereby ushering in an era of personalized medicine in restoring control of intraocular pressure in open-angle glaucoma patients.

 

  1. Acott TS, Kelley MJ, Keller KE, et al. Intraocular pressure homeostasis: maintaining balance in a high-pressure environment. Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics 2014;30:94-101.
  2. Jaffe GJ, Alvarado JA, Juster RP. Age-related changes of the normal visual field. Arch Ophthalmol. 1986 Jul; 104(7):1021-5.
  3. Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology 1984; 91:564-579.

 

MK FIG1

Fig. 1 a) Whole eye, showing position of trabecular meshwork and Schlemm’s Canal in the small square, and expanded view of trabecular meshwork, juxtacanalicular TM (JCT), and Schlemm’s Canal. b) Perfused anterior segment organ culture, using constant pressure perfusion.

 

 

Differentiation of Human iPSC to TM-Like iPSC    

MK FIG2

Fig. 2- Embryoid bodies (EBs) are shown in b) at two magnifications where scale bars are 50 m. After differentiation of iPSC embryoid bodies using a combination of extracellular matrix and conditioned medium from TM cells to drive the process, the differentiated TM-like iPSCs exhibit morphology that resembles normal human TM cells (c).

 

 

Saponin Treatment Abolishes the IOP Homeostatic Response to increased Pressure

MK FIG3

Fig.3-Schematic showing 0.01% saponin treatment pattern for flow studies. Normalized flow rate for perfused human anterior segments treated with saponin or vehicle (normal) and then subjected to IOP homeostatic 2x pressure challenge. Mean and standard error of the mean are shown where n = 8 for normal and n = 17 for saponin treated anterior segments with significance determined by one-way ANOVA at P < 0.001 indicated by **.

 

 

Replacement of Saponin-Depleted Cells with TM-like iPS Cells in Anterior Segments

MK FIG4

Fig.4- (a) Confocal analysis of frontal section showing transplanted Q-dot (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. (c) After 1x perfusion, saponin was added as indicated, rinsed out, and perfusion resumed for 24 hours at 1x pressure. The 2x pressure challenge gave no IOP homeostatic response. TM-like iPSCs were added and allowed to attach for 24 hours; flow was resumed at 1x pressure and then increased to 2x 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<0.05 and **P<0.001.

 

 

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