Xing Zhao

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.

Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.

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.

Stem Cell Therapy for Retinal Degeneration: Retinal Neurons from Heterologous Sources

Over the past few years a great deal of interest has been generated in using stem cells/progenitors to treat degenerative diseases that afflict different tissues, including retina. This interest is due to the defining properties of stem cells/progenitors, the ability of these cells to self-renew and generate all the basic cell types of the particular tissue to which they belong. In addition, the recent reports of plasticity of the adult tissue-specific stem cells/progenitors and directed differentiation of the embryonic cells (ES) has fueled the hope for cell and gene therapy using stem cells from heterologous sources. Will this approach work for treating retinal degeneration? Here, we review the current state of knowledge about obtaining retinal cells from heterologous sources, including ES cells.

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.

Differentiation of Embryonic Stem Cells to Retinal Cells In Vitro

Currently, there is no effective treatment for photoreceptor degeneration, the most common cause of blindness caused by diseases like retinitis pigmentosa, age-related macular degeneration, and diabetic retinopathy. Two promising approaches include cell therapy to replace degenerating cells and neuroprotection to rescue affected cells from premature death. Determination of the potential of embryonic stem (ES) cells to differentiate into photoreceptors will provide reagents for both approaches. First, neural progenitors with retinal potential will be available in unlimited supply to test the efficacy of cell therapy; second, the controlled differentiation of ES cells into photoreceptors, in addition to providing cells to replace degenerating photoreceptors, will offer a robust in vitro model of photoreceptor differentiation for better understanding of degenerative processes and screening of neuroprotective drugs/reagents. In addition, it will allow the identification of genes (gene discovery) that play critical roles in photoreceptor differentiation and degeneration. Here, we describe the protocol to promote differentiation of the mouse ES cell-derived neural progenitors into retinal cells, specifically the rod photoreceptors.

Direct Differentiation of Adult Ocular Progenitors into Striatal Dopaminergic Neurons

Parkinson’s disease, characterized by motor dysfunction due to the loss of nigrostriatal dopaminergic neurons, is one of the most prevalent age-related neurodegenerative disorders. Given there is no current cure, the stem cell approach has emerged as a viable therapeutic option to replace the dopaminergic neurons that are progressively lost to the disease. The success of the approach is likely to depend upon accessible, renewable, immune compatible, and non-tumorigenic sources of neural progenitors from which stable dopaminergic neurons can be generated efficaciously. Here, we demonstrate that neural progenitors derived from limbus, a regenerative and accessible ocular tissue, represent a safe source of dopaminergic neurons. When the limbus-derived neural progenitors were subjected to a well-established protocol of directed differentiation under the influence of Shh and FGF8, they acquired the biochemical and functional phenotype of dopaminergic neurons that included the ability to synthesize dopamine. Their intrastriatal transplantation in the rat model of hemi-Parkinsonism was associated with a reduction in the amphetamine-induced rotation. No tumor formation was observed 6 weeks post-transplantation. Together, these observations posit limbus-derived neural progenitors as an accessible and safe source of dopaminergic neurons for a potential autologous ex-vivo stem cell approach to Parkinson’s disease.