Mike O. Karl

Efficient generation of retinal progenitor cells from human embryonic stem cells.

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cell-derived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.

Stimulation of neural regeneration in the mouse retina.

Müller glia can serve as a source of new neurons after retinal damage in both fish and birds. Investigations of regeneration in the mammalian retina in vitro have provided some evidence that Müller glia can proliferate after retinal damage and generate new rods; however, the evidence that this occurs in vivo is not conclusive. We have investigated whether Müller glia have the potential to generate neurons in the mouse retina in vivo by eliminating ganglion and amacrine cells with intraocular NMDA injections and stimulating Müller glial to re-enter the mitotic cycle by treatment with specific growth factors. The proliferating Müller glia dedifferentiate and a subset of these cells differentiated into amacrine cells, as defined by the expression of amacrine cell-specific markers Calretinin, NeuN, Prox1, and GAD67-GFP. These results show for the first time that the mammalian retina has the potential to regenerate inner retinal neurons in vivo.

Neural Regeneration and Cell Replacement: A View from the Eye

Neuronal degenerations in the retina are leading causes of blindness. Like most other areas of the CNS, the neurons of the mammalian retina are not replaced following degeneration. However, in nonmammalian vertebrates, endogenous repair processes restore neurons very efficiently, even after complete loss of the retina. We describe the phenomenon of retinal regeneration in nonmammalian vertebrates and attempts made in recent years to stimulate similar regenerative processes in the mammalian retina. In addition, we review the various strategies employed to replace lost neurons in the retina and the recent use of stem cell technologies to address problems of retinal repair.

Regenerative medicine for retinal diseases: activating endogenous repair mechanisms

The retina is subject to degenerative diseases that often lead to significant visual impairment. Non-mammalian vertebrates have the remarkable ability to replace neurons lost through damage. Fish, and to a limited extent birds, replace lost neurons by the dedifferentiation of Müller glia to a progenitor state followed by the replication of these neuronal progenitor cells. Over the past five years, studies have investigated whether regeneration can be stimulated in the mouse and rat retina. Several groups have reported that at least some types of neurons can be regenerated in the mammalian retina in vivo or in vitro, and that the regeneration of neurons can be stimulated using growth factors, transcription factors or subtoxic levels of excitatory amino acids. These recent results suggest that some part of the regenerative program that occurs in non-mammalian vertebrates remains in the mammalian retina, and could provide a basis to develop new strategies for retinal repair in patients with retinal degenerations.

Strategies for retinal repair: cell replacement and regeneration.

The retina, like most other regions of the central nervous system, is subject to degeneration from both genetic and acquired causes. Once the photoreceptors or inner retinal neurons have degenerated, they are not spontaneously replaced in mammals. In this review, we provide an overview of retinal development and regeneration with emphasis on endogenous repair and replacement seen in lower vertebrates and recent work on induced mammalian retinal regeneration from Müller glia. Additionally, recent studies demonstrating the potential for cellular replacement using postmitotic photoreceptors and embryonic stem cells are also reviewed.

Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Genome-wide analysis of Müller glial differentiation reveals a requirement for Notch signaling in postmitotic cells to maintain the glial fate.

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.

Retinal Organoids from Pluripotent Stem Cells Efficiently Recapitulate Retinogenesis

Summary The plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine.

P53 is required for the developmental restriction in Müller glial proliferation in mouse retina

Müller glia are normally mitotically quiescent cells, but in certain pathological states they can re‐enter the mitotic cell cycle. While several cell cycle regulators have been shown to be important in this process, a role for the tumor suppressor, p53, has not been demonstrated. Here, we investigated a role for p53 in limiting the ability of Müller glia to proliferate in the mature mouse retina. Our data demonstrate that Müller glia undergo a developmental restriction in their potential to proliferate. Retinal explants or dissociated cultures treated with EGF become mitotically quiescent by the end of the second postnatal week. In contrast, Müller glia from adult trp53−/+ or trp53−/− mice displayed a greater ability to proliferate in response to EGF stimulation in vitro. The enhanced proliferative ability of trp53 deficient mice correlates with a decreased expression of the mitotic inhibitor Cdkn1a/p21cip and an increase in c‐myc, a transcription factor that promotes cell cycle progression. These data show that p53 plays an essential role in limiting the potential of Müller glia to re‐enter the mitotic cycle as the retina matures during postnatal development.

BAF60c is a component of the neural progenitor specific BAF complex in developing retina

Remodeling of the chromatin network plays an important role regulating embryonic development as well as differentiation. The SWI/SNF complex is an ATP‐dependent chromatin‐remodeling complex. It consists of several proteins, including an ATPase subunit, either Brg1 or Brm. Two subunits of this complex, Baf53a and Baf45, have been previously identified as being neural progenitor‐specific. In this study, we show that Baf60c, another important part of this large complex, acts in a similar neural progenitor–specific manner. We show that during development Baf60c is expressed in neural progenitors in human retinas as well as mouse retina, cortex and spinal cord. Baf60c expression is lost during neural differentiation and its overexpression keeps the progenitors in a proliferative state through its interaction with the Notch pathway. Finally, we show that Baf60c is re‐expressed in the Müller glial cells that re‐enter the cell cycle after neurotoxic damage. Developmental Dynamics 237:3016–3023, 2008.