Mammalian retinal development follows a well-conserved progression of events beginning with the formation of the optic vesicle from the presumptive eye field. The optic vesicle invaginates to form the bilayered optic cup, which produces the retinal pigment epithelium (RPE) and retinal neuroblastic layer from the outer and inner levels, respectively. The neuroblastic layer is composed of undifferentiated, highly proliferative retinal progenitor cells (RPCs) whose apical surface lies in direct apposition with that of the RPE. Differentiation of RPCs and formation of the normal laminar structure of the retina occur in a precise spatiotemporal order, with long projection neurons (ganglion cells) appearing early, followed by photoreceptors and retinal interneurons and, ultimately, Müller glia cells. Lineage tracing studies have shown that these cells are derived from a common progenitor whose competency to generate particular retinal cell-types changes with time. Therefore, the expansion and targeted differentiation of human stem and progenitor cells in vitro could provide an essential source of biological material for modeling retinal development and producing cell-based treatments for degenerative disease. The work of our laboratory is focused on the derivation, characterization, differentiation and transplantation of human cells to 1) investigate cellular and molecular events that occur during retinogenesis and 2) provide cells for use in rescue or replacement therapies for retinal degenerative diseases. In order to meet these aims, we study a repertoire of cell types including prenatal cortical and retinal progenitors, retinal pigmented epithelial cells, human embryonic stem cells (hES) and human induced pluripotent stem cells (iPS).
Although RPCs in lower species have been well-studied in vitro, progenitors harvested from pre- and postnatal human neural retina have proven difficult to maintain and expand for extended periods in culture. Application of successful techniques for long-term prenatal cortical progenitor growth supported human RPC (hRPC) growth for only approximately one month. Moreover, in both retinal and cortical culture, the competency of progenitor cells to produce neurons declined over time while the propensity to yield glia increased. Within the embryonic retina, progenitor cells are located adjacent to and supported by the RPE, which has the capacity to secrete a multitude of growth factors. In an effort to improve the growth potential of hRPC derived from primary prenatal retina, we developed a serum-free monolayer method for the culture of primary prenatal RPE. Conditioned media (CM) was collected from these cultures and used to treat hRPC cultures. Compared to untreated retinal cultures, those treated with RPE CM displayed a greatly enhanced growth period lasting up to one year in culture. Examination of the cell fate potential of differentiated, RPE CM-treated cultures confirmed a time-dependent reduction in neurogenic potential. However, following transduction with a lentiviral vector expressing the neurogenic basic helix-loop-helix (bHLH) protein Mash1, the capacity of late-passage retinal neurosphere cultures to produce neurons was restored. Overexpression of Mash-1 succeeded in directing these cells to adopt phenotypic characteristics of multiple inner retinal cell types, including ganglion, bipolar and amacrine neurons. Thus, a novel combination of extrinsic and intrinsic factors was required to promote both proliferation and neuronal multipotency in hRPCs. These studies suggest strategies for overcoming the limitation of hRPC cell fate restriction in vitro and are the focus of ongoing research in our laboratory.
Unlike hRPCs, hES cells can be used to study the full extent of human retinal development in vitro, including stages that occur during embryogenesis and organogenesis. We use hES cells to delineate the genetic “checkpoints” necessary to efficiently generate cells with the capacity to produce eye field, optic vesicle, retinal progenitor and RPE cells. By understanding the steps involved in this process, we hope to establish hESC as a model system for studying human retinal development. Our laboratory has successively directed undifferentiated hESC through the primitive neuroepithelial (Pax6+), presumptive eye field (Pax6+/Rx+) and optic vesicle (Pax6+/Mitf+) stages to become either definitive Chx10+ retinal progenitor cells or Mitf+ RPE cells. Interestingly, the appearance of these cell types follows a temporal sequence nearly identical to that observed in vivo. Knowledge obtained from these experiments has allowed us to examine the roles of exogenous factors and the interactions between specific transcription factors during human retinogenesis. We are currently extending this culture system toward the efficient production of photoreceptor precursors and opsin-expressing cells. By understanding the early steps involved in the production of retinal cells from hES and having the capacity to produce a particular cell type in a stepwise manner that capitulates normal development, the efficacy of various cell populations at multiple stages of differentiation to reconstitute a diseased retina can be determined. Our efforts to generate human retinal progenitors from hES will provide enriched sources of several cell types amenable for transplant studies in retinal degenerative animal models and hopefully lead to clinical treatments.
Another potential source for human retinal cell replacement is the induced pluripotent stem (iPS) cell. iPS cells represent a recent breakthrough in stem cell biology whereby mature cells (e.g., fibroblasts) harvested non-invasively are converted to hESC-like cells by forced expression of key stem cell genes. In collaboration with Dr. James Thomson, our laboratory has initiated a study designed to direct wild-type iPS cells towards a retinal lineage in a manner similar to hES cells. This research may allow for the creation of disease models from individual patients with retinal degenerative diseases. Alternatively, using gene repair techniques, customized cell replacement therapies can be envisioned.
In addition to potential applications for retinal cell repopulation, stem and progenitor cells may have a role in preserving at-risk host photoreceptors in retinal degenerative diseases. In collaborative studies using various neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) uniformly protected dying host neurons within both the brain and spinal cord. Based on these reports, the participating laboratories explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons (RCS) rat. When implanted into the subretinal space prior to the onset of photoreceptor loss, human neural progenitor cells (hNPCs) isolated from prenatal cortex promoted rescue of photoreceptors and vision in the RCS rat. The donor hNPCs migrated extensively within the retina and subretinal space where they had the capacity to secrete neuroprotective factors. Furthermore, the rescue effect was augmented by genetically modifying hNPCs to release glial cell line-derived neurotrophic factor, a strategy that currently holds promise for the treatment of numerous neurodegenerative diseases. The long term effects of subretinal hNPC transplantation into the RCS model was also examined, which revealed prolonged protection of retinal anatomy and visual function to an extent previously unattained by other donor cell types.
In summary, stem and progenitor cell technology holds the potential to enhance our knowledge of human retinal development and supply cell populations for use in retinal rescue and repopulation therapies. Once such information and tools are available, the scientific community will be faced with the task of coaxing these cells to integrate into host retina, survive an inhospitable environment and partially restore visual function. While these are challenging goals, recent advances suggest they are achievable, offering hope for patients afflicted with certain types of blinding disorders.
- Customized iPS cell therapy for recessive monogenic retinal degenerative disease
- Human stem cell-based therapies for age-related macular degeneration
- Walsh retinal stem cell research
- Macular Degeneration Stem Cell Research
- Research To Prevent Blindness
- Retina Research Foundation
- Culture And Transplantation Of Human Retinal Spheres
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