PEER REVIEWED

Reactional Retinal Progenitor Neuronal Cells in the Human Ciliary Epithelium Are a Potential Autologous Source for Retinal Repair

Claude Boscher, MD • Yvette Ducournau, MD • Ron A. Adelman, MD • Didier Ducournau, MD.

Retinal stem cell transplantation has received attention with the recent publication of encouraging results in two patients with geographic atrophy and Stargardt disease.¹ The patients seemed to benefit from cultured human embryonic stem cell-derived pigment epithelial cell transplantation.

Notably, there was no graft rejection and no teratoma formation. Pigmented tissue was detectable in retinal photos and on spectral-domain optical coherence tomography. In January 2012, a third patient was transplanted with embryonic stem cells in the United Kingdom.

Embryonic stem cell transplantation is an allogenic (healthy donor-derived) nonhomologous transplantation technique that requires immunosuppressive treatment. A clinical trial testing human allogenic homologous transplantation (tissue derived from the same organ) of neural nonembryonic stem cells will be initiated in the near future for dry AMD. An alternate avenue of research is the search for stem cell candidates for autologous transplantation: hematopoietic stem cells and mesenchymal stem cells (both nonhomologous) are currently being investigated.

Claude Boscher, MD, is on the faculty of the American Hospital of Paris. Yvette Ducournau, MD, is on the faculty of the Service d'Anatomie et Cytologie Pathologiques of l'Hôpital G et R Laënnec in Nantes, France. Ron A Adelman, MD, is associate professor of ophthalmology and visual science at the Yale University School of Medicine in New Haven, CT. Didier Ducournau, MD, is the CEO of European VitreoRetinal Services Company and a retinal physician in private practice. None of the authors reports any financial interests in any products mentioned in this article. Dr. Boscher can be reached via e-mail at cboscher@wanadoo.fr.

CILIARY BODY STEM CELLS

A potential source for both autologous and homologous transplantation might be a quiescent population of cells in the adult human ciliary body. A similar population has been reported in adult fish and amphibians, in a zone called the ciliary margin, which lies between the anterior retina and the pars plana epithelium.² Two groups described similar findings in 2000 in adult murine and chicken eyes.^3-5

Since then, these cells have also been described in mammals and, more recently, in mature primates in vivo.⁶ Such populations have also been identified in the ciliary body itself and in the iris. There is currently a debate regarding the true nature of this ciliary body cell contingent. They may represent either transdifferentiated ciliary epithelial cells^7-10 or true stem cells.¹¹

Our Study

In this context, we studied the nature of the materials observed intraoperatively in contact with the ciliary surface in the anterior part of the vitreous base, during endoscopy-assisted vitrectomy in patients with retinal detachments (RDs) and anterior proliferative vitreoretinopathy.¹² The only means of identifying these structures may be to study human eyes eviscerated for RDs complicated by PVR.

This study was published in the February 2012 issue of Graefe's Archives of Clinical and Experimental Ophthalmology.²³ The manuscript suggests that the neuronal loss induced by RD and PVR might stimulate, in the ciliary epithelium (CE) of the human eye in vivo, the proliferation of retinal progenitor cells (RPCs) with neuronal and photoreceptor differentiation and their migration to the anterior vitreous base.

This morphological study was carried out on three human eyes eviscerated for RDs and PVR, and a normal eye exenterated for an orbital tumor served as a control. It showed an intense proliferation of the CE, which migrates and colonizes the anterior vitreous base and forms aggregates, which resemble the neurospheres produced in stem cell cultures (Figure 1).

Figure 1. Case C55 HES x200. Ciliary epithelium: RD with PVR. Proliferation and migration of CE cells (arrows). Colonization of the adjacent vitreous base (VB) (colored in yellow), with a floating, clustered, “NeuroSphere-like” (NS) appearance, resembling stem cells cultured in vitro.

This active proliferation was unquestionably demonstrated by immunohistochemistry testing for Ki67, a nuclear antigen marker present in all of the active phases of the cell cycle. CD133, a mature marker expressed by neural and hematopoietic SCs, was absent from the CE and from the controls. Neuronal differentiation was demonstrated by testing for neuron-specific endolase (NSE), a mature neuronal cytoplasmic cell marker: NSE+ cell proliferation involved the entire thickness of the CE (Figure 2); in contact with the pigmentary CE, some cells were positive for antirhodopsin antibodies, which are cytoplasmic rod photoreceptor retinal cell markers, demonstrating photoreceptor differentiation (Figure 3). Intense overexpression of the epithelial growth factor receptor (EGFR) was also observed in the CE cells (Figure 4).

Figure 2. Immunohistochemistry study: Neuron-specific enolase (NSE) (marker for mature neuronal differentiation) and ciliary epithelium (CE). a. Case 04B1864 NSE x100. Normal eye: The inner, nonpigmented CE is negative (arrows). b. Case C55 NSE x100. RD and PVR : The retracted detached retina (R) is disorganized and shows neuronal loss (arrow). A few optically empty vacuoles of silicone oil are visible (S). The inner hyperplasic nonpigmented CE staining is very positive (asterisk).

Figure 3. Immunohistochemistry study: case C55. Rhodopsin (photoreceptor differentiation) x200. An intense cytoplasmic staining was observed in some cells at the vicinity of the pigmented CE (arrow).

Figure 4. Immunohistochemistry study: case C55. Epithelial growth factor receptor (EGFR) x200. Ciliary epithelium (CE). Overexpression (in red) of EGFR in hyperplasic NPCE (arrow).

Simultaneously, the detached neuroepithelial retinal cells showed neuronal loss with a considerable decrease in NSE+ cells and reactional gliosis with intense expression of glial fibrillary astrocytic protein (GFAP), a marker of mature astrocytes (Figure 5). Additionally, proliferation of retinal pigment epithelium cells, which colonized the retinal surface, was also present, as commonly seen in PVR. Notably, GFAP testing in the CE was negative, indicating that this ciliary proliferation did not present any glial differentiation (Figure 5).

Figure 5. Immunohistochemistry study: case C55. Glial fibrillary astrocytic protein (GFAP) (marker for astrocytes and gliosis) x200. a. GFAP is negative in the hyperplasic ciliary epithelium: no glial differentiation (arrow). b. The retracted retina is strongly stained by GFAP (arrow), demonstrating intense gliosis, in contrast with the adjacent hyperplasic ciliary epithelium (asterisk), not stained in red.

We have studied two additional human eyes since this work was published, and the more recent results are in agreement with our original published manuscript.

Our studies in the human eye in vivo have added pathoclinical information to numerous previous works in animals in vitro and in vivo, as well as in human cadaver eyes. To our knowledge, this is the first time that a process of neuronal regeneration contemporaneous with, and thus, possibly as a reaction to, a retinal injury has been observed in the adult human eye in vivo.

It is important to emphazise that the normal CE in the adult human eye (Figure 6) does not express neuronal or photoreceptor differentiation. CE proliferation seems to reproduce retinal organogenesis.¹³ Although the existence of a quiescent population of stem or transdifferentiated cells in the ciliary body has been verified, both in vivo and by cellular cultures in vitro, first in animals and then in the human cadaver eye, they have never been reproduced in experiments with injury to the ciliary body itself.

Figure 6. Morphological study: hematoxylin and eosin Safran (HES) x25. Normal ciliary body (CB) and pars plana (PP): The ciliary epithelium (CE) consists of two layers of cells, the outer, pigmented CE (arrow); and the inner, nonpigmented CE (NPCE) (asterisk).

It seems that our observations in the human eye in vivo have confirmed what had been shown previously in mammalian eyes, ie, the cells leave the quiescent state in the event of retinalneuronal loss/injury.^14-16

In vivo and in vitro studies in animals and in human cadaver eyes suggest that CE-derived RPCs are rare.¹¹ In our study, CDD133 was absent from CE proliferation. This finding might correspond with transitory expression of this neural SC marker (an acknowledged phenomenon), or it might occur as a consequence of a transdifferentiation process.

Indeed, the proliferation marker Ki67 was positive in a few cells only, but one must consider that Ki67, while highly present in tissue cultures, is extremely difficult to demonstrate in tissues in vivo that are not tumor- or cancer-related. Therefore, although the significance of this finding in vivo is very great, the frequency of duplication was low.

The nature of this regenerative process seems to be at first purely neuronal, because GFAP, which characterizes the cicatricial tissue of the retina, is not expressed in CE proliferation. Müller cells, the retinal candidate cells for progenitor cell properties, respond to retinal injury by inducing reactive gliosis, which forms scar tissue; however, the rescue potential of this process is still unclear.

In our in vivo study, the CE RPC population was naturally preprogrammed toward a mature functional fate. This finding is in contrast with, and might reduce the potential for, the simultaneous glial cicatricial orientations that had been previously observed in cultures in vitro from the ciliary margin/body.

Indeed, early experiments suggested that the extrinsic microenvironment (“niche”) and/or cellular intrinsic signals play a significant role in ciliary RP cell fate determination.¹⁷ As of today, the concept of niches is well established. As with any epithelial tissue, EGFR is normally expressed in the ciliary body and in the retina in a subliminal state only. Its overexpression in cases of retinal injury might constitute the natural niche necessary for this proliferation and differentiation.

Based on observations in the adult brain in vivo,^18,19 as well as after central nervous system injury, one role of neurotrophic factors is the promotion of axonal regeneration without scar tissue formation.^18,20 Furthermore, there is the ability of multipotent progenitor cells harbored by the human brain in vivo to develop into neurons, as demonstrated on cerebral resections in cases of epilepsy.²¹

Further investigations are necessary, and we are currently conducting experimental studies to investigate the mechanism of proliferation and differentiation of CE cells.

POTENTIAL FUTURE PERSPECTIVE

The ciliary epithelium in embryogenesis is represented by the edge of the optic cup, which does not differentiate into retinal tissue. Our speculative hypothesis, since our original presentation at the 2008 annual meeting of the Association for Research in Vision and Ophthalmology, is that the CE has the capacity, in the presence of retinal injury, to start a process of retinal regeneration induced by the growth factor EGF (and possibly by other factors) (Figure 7). With this perspective, the CE might be a potential donor source of cells for human subretinal autologous and homologous transplantation.

Figure 7. Diagram of the simultaneous observations in the retinal neuroepithelium and the ciliary epithelium in five human eyes with RD and PVR in vivo. Top: The normal retinal neuroepithelium is NSE-positive (marker for mature neuronal differentiation). The ciliary epithelium (CE) is NSE-negative. Retinal detachment induces neuronal loss illustrated by a decrease in NSE+ cells in the retina. Bottom, from left to right: Retinal neuronal loss might induce epithelial growth factor (EGF), illustrated by an intense reactivity to EGFR in the CE; retinal neuronal loss might induce proliferation demonstrated by Ki67 (nuclear antigen marker) positivity in the CE (Ki67+ cells are usually extremely difficult to observe among tissues in vivo); retinal neuronal loss might induce neuronal differentiation (NSE+ cells) with photoreceptor differentiation (rhodopsin + cells) in the CE.

Our speculative hypothesis is supported by new insights. Experimental studies in mice by Derek Van der Kooy's team have already shown that adult RPCs derived from the CE can generate all retinal cell types with functional recovery.¹¹ Aruta et al. demonstrated that RPE cells that had been derived from adult ciliary RPCs matured into functional RPE cells in murine cultures.²²

At ARVO 2012, Van der Kooy's team (Ballios et al.) showed that an injectable and biodegradable material could overcome the glial distribution barrier to survive. Furthermore, they demonstrated that ciliary body-derived RPCs integrate into neural murine retina.²⁴

Also, Dong Feng Chen, MD, from the Schepens Research Institute, identified the protein eprin 3, a tyrosine kinase receptor, as a matrix signal suppressive of the progenicity of ciliary body RPCs in mice. This signal, previously established as a negative regulator of adult neurogenesis in the central nervous system, might be responsible for the dormant state of the CE population under normal conditions.

CONCLUSION

We have observed in three human adult ciliary bodies in vivo a population of proliferating neuronal cells. These cells strongly express the growth factor EGF, and some cells also express the photoreceptor marker rhodopsin. This transformation might be induced by neuronal loss in the pathological condition of RD with PVR, which might promote signals to the RPCs to quit the dormant state and generate the niche required for proliferation. Other retinal injuries might also generate similar reactional processes.

We can speculate that these suppressed cells could be removed by surgical tissue biopsy and activated through manipulation of signaling pathways (or other unidentified techniques). This potential source of autotransplantation seems to present an exclusively neuronal and nonglial orientation and could have the following advantages:

1. It comes from adult donors.
2. Because it is autologous, it would eliminate the need for immunosuppressive treatment.
3. Its proliferation index is low; thus, it would decrease the risk of potential tumor growth.
4. Because it comes from the RPCs and is nonglial, it would eliminate the need to be reprogrammed or genetically modified.

Retinal neuronal progenitor cells seem to exist in the ciliary body in adult human eyes with RD and PVR. These RPCs seem to be suppressed under normal conditions but might be an autologous source for retinal repair in pathologic conditions. RP

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