Michael Young

Bioengineering and Stem Cells to Treat Optic Neuropathy

We investigated a treatment for glaucoma using scaffolds seeded with stem cells derived from the brain and retina to investigate the repair of the optic nerve. Since humans do not have the ability to regenerate the optic nerve following injury, permanent loss of sight results. However, we now understand a great deal about the complex organization of the visual system, why neural regeneration fails, and how this problem might be solved.

The optic nerve is the largest sensory tract of the human central nervous system, and it connects the eye with the visual centers of the brain by way of approx. 1.2 million separate axons from each retina. The organization of these fibers is critical for maintaining an accurate topographic map of the visual world.

We, together with our collaborators at MIT (Drs. Langer and Lavic), have begun to investigate a means of repairing the optic nerve, drawing upon a wide range of emerging technologies including tissue engineering, stem cell biology, genetic engineering, and recombinant growth factors. The combined application of these technologies now provides a potential means for restoring useful visual communication between the eye and the brain. We are developing animal models including mice and pigs, so that our most promising findings may become ready for human clinical studies as soon as is possible.

The best approach to the problem of retinal cell loss is to attack it from many directions. Our paradigm is to replace lost cells via stem cell/biodegradable polymer composite grafts. The polymer scaffolds used to support the stem cells also provide a vehicle for drug delivery. Our expertise in drug delivery allows us to pursue new means of preserving the life of these vital cells through the delivery of survival factors to the eye. We are incorporating growth factors and other drugs in the scaffolds to augment the incorporation of stem cells and promote repair. In addition, we are also examining the architecture of the scaffold along with the release of growth factors in the hopes of not only reconnecting the eye and brain but reconnecting it in a topographic manner.

Differentiation of Retinal Progenitor Cells into Specific Cell Types

We began to develop strategies to differentiate retinal progenitor cells (RPC's) into specific cell types (bipolar and photoreceptor cells) using exogenous factors, to enhance survival and functional integration of transplanted cells. The RPC's that were used in the differentiation studies were isolated from GFP transgenic mice. These cells were characterized by immunostaining with Nestin (a marker for neuronal progenitor cells) and Ki67 (a marker for mitotically active cells). They were then treated with different reagents including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), potassium chloride (KCl), retinoic acid (RA), retinal pigmented epithelial (RPE) cell conditioned medium, sodium butyrate, succinylated concanavalin A (SCA) and taurine for up to two weeks. In vitro studies showed that CNTF (20ng/ml) treatment resulted in changes in cellular morphology and was able to increase the number of cells expressing bipolar markers (PKC and MGluR6). Retinoic acid (500 nM) and sodium butyrate (4 mM), in combination, induced changes in cellular morphology and the differentiated cells stained positive for photoreceptor marker (Recoverin). Next, RPC's were seeded onto biodegradable poly (lactic-co-glycolic acid) polymers and treated with CNTF for up to 14 days. The aim was to use these polymers not only as substrates for transplanting cells but also for controlled release of incorporated molecules (such as CNTF) over time resulting in differentiation of RPC's in vivo. These cells were then transplanted to retinal explants from C3H (retinal degeneration) mice to determine if the cells would preferentially integrate into the inner nuclear layer and express the appropriate bipolar cell markers. RPC's differentiated into bipolar (>90%) and photoreceptor (>10%) cell types upon treatment with CNTF and retinoic acid and sodium butyrate, in combination. In addition, the RPC's treated with CNTF prior to transplantation, integrated into the inner nuclear layer and expressed bipolar cell markers.

We have shown that RPCs can differentiate and integrate into the inner nuclear layer in retinal explants. The next step is to transplant the CNTF treated RPCs in vivo. RPCs will be transplanted by intravitreal injections in rhodopsin knockout mice. In addition, RPCs will be seeded onto polymer substrates into which CNTF has been incorporated. This will allow controlled release of CNTF in vivo, which in turn will induce differentiation and integration of RPCs.