Shweta Singhal

MIO‐M1 Cells and Similar Müller Glial Cell Lines Derived from Adult Human Retina Exhibit Neural Stem Cell Characteristics

Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor‐like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO‐M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor‐2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including βIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S‐opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell‐based therapies to restore retinal function.

Chondroitin Sulfate Proteoglycans and Microglia Prevent Migration and Integration of Grafted Müller Stem Cells into Degenerating Retina

At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68‐positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5‐week‐old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N‐terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5‐week‐old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell‐based therapies to treat human retinal disease.

Distribution of Müller stem cells within the neural retina: evidence for the existence of a ciliary margin-like zone in the adult human eye.

Much interest has been generated by the identification of neural stem cells in the human neural retina and ciliary body. However, it is not clear whether stem cells identified in these ocular compartments are of the same origin or whether they ontogenically derive from different cell populations. This study examined the in situ anatomical distribution of these cells within the neural retina and ciliary body, as well as their ability to proliferate in response to EGF. Human retinae and ciliary body were examined for co-expression of Nestin, cellular retinaldehyde binding (CRALBP) or Vimentin, and the stem cell markers SOX2, CHX10, NOTCH1 and SHH. Retinal explants were cultured with epidermal growth factor (EGF) to assess retinal cell proliferation. Intense Nestin and CRALBP staining was observed in the neural retinal margin, where cells formed bundles of spindle cells (resembling glial cells) that lacked lamination and co-stained for SOX2, CHX10 and SHH. This staining differentiated the neural retina from the ciliary epithelium, which expressed SOX2, CHX10 and NOTCH1 but not Nestin or CRALBP. Nestin and CRALBP expression decreased towards the posterior retina, where it anatomically identified a population of Müller glia. All Vimentin positive Müller glia co-stained for SOX2, but only few Vimentin positive cells expressed Nestin and SOX2. Cells of the retinal margin and the inner nuclear layer (INL), where the soma of Müller glia predominate, re-entered the cell cycle upon retinal explant culture with EGF. Lack of lamination and abundance of Müller glia expressing stem cell markers in the marginal region of the adult human retina resemble the ciliary marginal zone (CMZ) of fish and amphibians. The findings that cells in this CM-like zone, as well in the inner nuclear layer proliferate in response to EGF suggest that the adult human retina has regenerative potential. Identification of factors that may promote retinal regeneration in the adult human eye would provide efficient treatments for retinal degenerative conditions for which treatments are not yet available.

Human Müller Glia with Stem Cell Characteristics Differentiate into Retinal Ganglion Cell (RGC) Precursors In Vitro and Partially Restore RGC Function In Vivo Following Transplantation

Müller glia with stem cell characteristics have been identified in the adult human eye, and although there is no evidence that they regenerate retina in vivo, they can be induced to grow and differentiate into retinal neurons in vitro. We differentiated human Müller stem cells into retinal ganglion cell (RGC) precursors by stimulation with fibroblast growth factor 2 together with NOTCH inhibition using the γ‐secretase inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT). Differentiation into RGC precursors was confirmed by gene and protein expression analysis, changes in cytosolic [Ca2+] in response to neurotransmitters, and green fluorescent protein (GFP) expression by cells transduced with a transcriptional BRN3b‐GFP reporter vector. RGC precursors transplanted onto the inner retinal surface of Lister hooded rats depleted of RGCs by N‐methyl‐d‐aspartate aligned onto the host RGC layer at the site of transplantation but did not extend long processes toward the optic nerve. Cells were observed extending processes into the RGC layer and expressing RGC markers in vivo. This migration was observed only when adjuvant anti‐inflammatory and matrix degradation therapy was used for transplantation. RGC precursors induced a significant recovery of RGC function in the transplanted eyes as determined by improvement of the negative scotopic threshold response of the electroretinogram (indicative of RGC function). The results suggest that transplanted RGC precursors may be capable of establishing local interneuron synapses and possibly release neurotrophic factors that facilitate recovery of RGC function. These cells constitute a promising source of cells for cell‐based therapies to treat retinal degenerative disease caused by RGC dysfunction.

Adult Retinal Stem Cells Revisited

Recent advances in retinal stem cell research have raised the possibility that these cells have the potential to be used to repair or regenerate diseased retina. Various cell sources for replacement of retinal neurons have been identified, including embryonic stem cells, the adult ciliary epithelium, adult Müller stem cells and induced pluripotent stem cells (iPS). However, the true stem cell nature of the ciliary epithelium and its possible application in cell therapies has now been questioned, leaving other cell sources to be carefully examined as potential candidates for such therapies. The need for identification of the ontogenetic state of grafted stem cells in order to achieve their successful integration into the murine retina has been recognized. However, it is not known whether the same requirements may apply to achieve transplant cell integration into the adult human eye. In addition, the existence of natural barriers for stem cell transplantation, including microglial accumulation and abnormal extracellular matrix deposition have been demonstrated, suggesting that several obstacles need to be overcome before such therapies may be implemented. This review addresses recent scientific developments in the field and discusses various strategies that may be potentially used to design cell based therapies to treat human retinal disease.

SOX2 is required for adult human muller stem cell survival and maintenance of progenicity in vitro.

PURPOSE SOX2, a high-mobility group transcription factor, is expressed by retinal progenitors during development. It has been associated with the ability of progenitor cells to differentiate into retinal neurons and is highly expressed by human Müller stem cells (hMSCs) in culture. The authors investigated the role of this factor in the maintenance of progenicity and neural differentiation of hMSCs in vitro. METHODS SOX2 silencing was induced by transfection of hMSCs in culture with two pGSU6-GFP SOX2 silencing constructs and a scrambled control vector. Silencing was confirmed by examination of gene and protein expression coding for SOX2. Effects of SOX2 downregulation were investigated by expression of proliferation (Ki67) and apoptotic (TUNEL, caspase) cell markers and by the expression of markers of retinal neurons (HuD, βIII tubulin, rhodopsin, BRN3B, ISL1), glia (vimentin), and the progenitor marker PAX6. RESULTS SOX2 silencing caused hMSCs to rapidly adopt a neural-like morphology and was accompanied by the upregulation of specific markers of retinal neurons, including βIII tubulin, rhodopsin, BRN3B, and ISL1, and by the downregulation of the neural progenitor marker PAX6 and the glial cell marker vimentin. Interestingly, SOX2 silencing induced apoptosis, suggesting a crucial role of this factor on hMSC survival in vitro. CONCLUSIONS These in vitro results parallel that seen when Sox2 is silenced in neural stem cells of lower species during development, and they suggest that Sox2 may have an important role in adult hMSC differentiation into retinal neurons in vitro.