Ani V. Das

Transplantation of ocular stem cells: the role of injury in incorporation and differentiation of grafted cells in the retina.

The incorporation of transplanted cells into the host retina is one of the prerequisites for successful cell replacement therapy to treat retinal degeneration. To test the hypothesis that injury promotes cell incorporation, stem cells/progenitors were isolated from the retina, ciliary epithelium or limbal epithelium and transplanted into the eyes of rats with retinal injury. Different stem cell/progenitor populations incorporated into traumatized or diseased retina but not into the normal retina. The proportion of cells incorporated into the inner retina was consistently higher than in the outer retina. The transplanted cells expressed markers specific to cells of the lamina into which they were incorporated suggesting that cues for specific differentiation are localized within the inner and outer retina. These findings demonstrate that injury-induced cues play a significant role in promoting the incorporation of ocular stem cells/progenitors regardless of their origin or their differentiation along specific retinal sublineage.

In vitro differentiation of retinal ganglion-like cells from embryonic stem cell derived neural progenitors

ES cells have been reported to serve as an excellent source for obtaining various specialized cell types and could be used in cell replacement therapy. Here, we demonstrate the potential of ES cells to differentiate along retinal ganglion cell (RGC) lineage. FGF2-induced ES cell derived neural progenitors (ES-NPs) were able to generate RGC-like cells in vitro upon differentiation. These cells expressed RGC regulators and markers such as, Ath5, Brn3b, RPF-1, Thy-1 and Islet-1, confirming their potential to differentiate into RGCs. The generation of RGCs from ES-NPs was enhanced with the exposure of FGF2 and Sonic hedgehog (Shh), although Shh treatment alone did not affect RGC differentiation significantly. ES-NPs, after exposure to FGF2, were capable of integrating and differentiating into RGCs in vivo upon transplantation. Thus, our study suggests that ES cells can serve an excellent renewable source for generating RGCs that can be used to treat neurodegenerative diseases like glaucoma.

Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling

ABCG2 belongs to the ATP-binding cassette superfamily of transmembrane proteins and is ubiquitously expressed in stem cells including those in the developing nervous system. The ability of ABCG2 to preferentially exclude DNA-intercalating dyes is regarded to be the basis for the enrichment of stem cells or progenitors as dyelow side population (SP) cells. However, the role of ABCG2 in neural stem cells remains speculative and poorly understood. Here, we demonstrate using retinal stem cells, that ABCG2 is the molecular determinant of SP cell phenotype of neural stem cells and plays an important role in their maintenance. Overexpression of ABCG2 prevents the SP cell phenotype and adversely affects the lineage commitment of retinal stem cells. By contrast, targeted attenuation of ABCG2 depletes retinal SP cells and promotes their differentiation along pan neural and retinal lineages. In addition, we demonstrate for the first time that ABCG2 is a target of Notch signaling, and as such, constitutes one of the genes in the regulatory network of Notch signaling, involved in the maintenance of stem cells.

Retinal properties and potential of the adult mammalian ciliary epithelium stem cells

The ciliary epithelium (CE) in the adult mammalian eye harbors a mitotic quiescent population of neural stem cells. Here we have compared the cellular and molecular properties of CE stem cells and populations of retinal progenitors that define the early and late stages of histogenesis. The CE stem cells and retinal progenitors proliferate in the presence of mitogens and share the expression of universal neural and retinal progenitor markers. However, the expression of the majority of retinal progenitor markers (e.g., Chx10) is transient in the former when compared to the latter, in vitro. They are similar to early than late retinal progenitors in their proliferative response to FGF2 and/or EGF. Analysis of the differentiation potential of CE stem cells shows that they are capable of generating both early (e.g., retinal ganglion cells) and late (e.g., rod photoreceptors) born retinal neurons. However, under identical differentiation conditions, i.e., in the presence of 1% FBS, they generate more early-born retinal neurons than late-born retinal neurons showing a preference for generating early retinal neurons. Transcription profiling of these cells and retinal progenitors demonstrate that they share approximately 80% of the expressed genes. The CE stem cells have more unique genes in common with early retinal progenitors than late retinal progenitors. Both proliferative/differential potential and transcription profiles suggest that CE stem cells may be a residual population of stem cells of optic neuroepithelium, representing a stage antecedent to retinal progenitors.

Growth factor-responsive progenitors in the postnatal mammalian retina.

It is thought that the adult mammalian retina lacks the regenerative capacity of fish and amphibians retina because it does not harbor a progenitor population. However, recent observations suggest that another derivative of the optic neuroepithelium, the ciliary body, contains a mitotically quiescent population of neural progenitors that proliferate in the presence of growth factors and demonstrate properties of neural stem cells. Examination of the hypothesis that similar mitotically quiescent and growth factor‐responsive progenitors may exist in the postnatal retina revealed a population of cells located in the periphery of the retina that displayed proliferative responsiveness to growth factors and possessed potential to support neurogenesis. Given their marginal position and neural properties and potential, these cells may represent a residual population of retinal progenitors, analogous to those found in the ciliary marginal zone of fish and amphibians. Their progressive decrease in proliferative potential and number in postnatal stages suggests a temporal decline in regulatory signaling that supports their maintenance during retinal neurogenesis. Developmental Dynamics 232:349–358, 2005.

Derivation of Neurons with Functional Properties from Adult Limbal Epithelium: Implications in Autologous Cell Therapy for Photoreceptor Degeneration

The limbal epithelium (LE), a circular and narrow epithelium that separates cornea from conjunctiva, harbors stem cells/progenitors in its basal layer that regenerate cornea. We have previously demonstrated that cells in the basal LE, when removed from their niche and cultured in reduced bond morphogenetic protein signaling, acquire properties of neural progenitors. Here, we demonstrate that LE‐derived neural progenitors generate neurons with functional properties and can be directly differentiated along rod photoreceptor lineage in vitro and in vivo. These observations posit the LE as a potential source of neural progenitors for autologous cell therapy to treat photoreceptor degeneration in age‐related macular degeneration and retinitis pigmentosa.

Müller Glia: A Promising Target for Therapeutic Regeneration

In the past 10 years, there has been a paradigm shift in our understanding of brain development and approaches to treat degenerative diseases, including those that affect the retina. The latest knowledge includes (1) the discovery that the adult brain harbors proliferating progenitors and that neurons are born throughout life, particularly in the subventricular zone (SVZ) of the lateral ventricle and the subgranular layer (SGL) of the dentate gyrus of the hippocampus 1 and (2) the observation that glia perform dual functions, providing homeostatic support and serving as the source of stem cells in the embryonic brain and the adult SVZ and SGL. 2 In contrast to the SVZ and SGL, active neurogenesis has not been detected in adult mammalian retina. However, neurogenic changes have been observed in injured retina, and the source of injury-induced neu

Ciliary neurotrophic factor-mediated signaling regulates neuronal versus glial differentiation of retinal stem cells/progenitors by concentration-dependent recruitment of mitogen-activated protein kinase and Janus kinase-signal transducer and activator of transcription pathways in conjunction with Notch signaling.

In the retina, as elsewhere in the central nervous system, neurogenesis precedes gliogenesis; that is, the only glia in the retina, Müller cells, are born when the majority of neurons have already been generated. However, our understanding of how the multipotent retinal stem cells/progenitors choose to differentiate along neuronal and glial lineages is unclear. This information is important in promoting directed differentiation of retinal stem cells/progenitors in an ex vivo or in vivo stem cell approach to treating degenerative retinal diseases. Here, using the neurosphere assay, we demonstrate that ciliary neurotrophic factor (CNTF), acting in a concentration‐dependent manner, influences the simultaneous differentiation of retinal stem cells/progenitors into neurons or glia. At low CNTF concentrations differentiation of bipolar cells is promoted, whereas high CNTF concentrations facilitate Müller cell differentiation. The two concentrations of CNTF lead to differential activation of mitogen‐activated protein kinase and Janus kinase‐signal transducer and activator of transcription (Jak‐STAT) pathways, with recruitment of the former and the latter for the differentiation of bipolar and Müller cells, respectively. The concentration‐dependent recruitment of two disparate pathways toward neurogenesis and gliogenesis occurs in concert with Notch signaling. Furthermore, we demonstrate that the attenuation of Jak‐STAT signaling along with Notch signaling facilitates the differentiation of retinal stem cells/progenitors along the rod photoreceptor lineage in vivo. Our observations posit CNTF‐mediated signaling as a molecular switch for neuronal versus glial differentiation of retinal stem cells/progenitors and a molecular target for directed neuronal differentiation of retinal stem cells/progenitors as an approach to addressing degenerative changes in the retina.

The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with notch signaling

The canonical Wnt pathway is known to influence multiple developmental events such as patterning, cell proliferation and cell specification. Recent studies have provided evidence of the involvement of the canonical Wnt pathway in the emergence and development of the optic neuroepithelium and its derivatives, particularly the retina. However, the mechanism of its action during retinal development remains rather obscure. Here, we demonstrate that (in agreement with observations in the blood, intestine, and skin) the canonical Wnt pathway influences retinal development by maintaining stem cells/progenitors. For example, the activation of this pathway keeps the early retinal stem cells/progenitors proliferating and uncommitted, while its attenuation facilitates their differentiation into retinal ganglion cells in vitro and in vivo. In addition, we demonstrate that Wnt signaling acts in concert with Notch signaling during retinal histogenesis, where the latter calibrates the influence of the former on the differentiation status of retinal stem cells/progenitors by regulating Lef1 and sFRP2.

SWI/SNF chromatin remodeling ATPase Brm regulates the differentiation of early retinal stem cells/progenitors by influencing Brn3b expression and Notch signaling.

Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation.