The loss of retinal ganglion cells (RGC) and their axons is one of the leading causes of blindness and includes traumatic (optic neuropathy) and degenerative (glaucoma) eye diseases. Although no clinical therapies are in use, mesenchymal stem cells (MSC) have demonstrated significant neuroprotective and axogenic effects on RGC in both of the aforementioned models. Recent evidence has shown that MSC secrete exosomes, membrane enclosed vesicles (30–100nm) containing proteins, mRNA and miRNA which can be delivered to nearby cells. The present study aimed to isolate exosomes from bone marrow-derived MSC (BMSC) and test them in a rat optic nerve crush (ONC) model. Treatment of primary retinal cultures with BMSC-exosomes demonstrated significant neuroprotective and neuritogenic effects. Twenty-one days after ONC and weekly intravitreal exosome injections; optical coherence tomography, electroretinography, and immunohistochemistry was performed. BMSC-derived exosomes promoted statistically significant survival of RGC and regeneration of their axons while partially preventing RGC axonal loss and RGC dysfunction. Exosomes successfully delivered their cargo into inner retinal layers and the effects were reliant on miRNA, demonstrated by the diminished therapeutic effects of exosomes derived from BMSC after knockdown of Argonaute-2, a key miRNA effector molecule. This study supports the use of BMSC-derived exosomes as a cell-free therapy for traumatic and degenerative ocular disease.

We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2).Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6. [Key Words: Dach2; Eya2; Six1; Pax3; myogenesis; dachshund; somite development] Received August 20, 1999; revised version accepted November 1, 1999. Somites are segmentally organized mesodermal structures that are the embryonic precursors of the axial skeleton and of all skeletal muscle (for review, see Christ and Ordahl 1995). Somites form by budding off from the anterior end of the presegmental mesoderm (PSM) to form epithelial balls of tissue. Patterning signals from surrounding tissues induce different regions of the somite to acquire distinct fates: The dorsal somite develops into the dermamyotome, the precursor to the dermis and to the muscles; and the ventral somite gives rise to the sclerotome, the precursor of the axial skeleton and ribs (Christ and Ordahl 1995). Subsequent inductive signaling leads to further subdivision of cell fates within the somite.The best studied aspect of this patterning and differentiation process is the specification of the myogenic cells. The establishment of muscle cell fate requires inductive signals both from axial tissues and from the dorsal ectoderm that overlays the somite (Christ and Ordahl 1995;Cossu et al. 1996). The progress of myogenic induction can be observed by following the expression of the paired-type transcription factor Pax3. In the chick embryo, Pax3 is initially expressed throughout the PSM (Williams and Ordahl 1994). However, early inductive infl...

Purpose. Retrograde neurotrophic factor transport blockade has been implicated in the pathophysiology of glaucoma. Stem cell transplantation appears to ameliorate some neurodegenerative conditions in the brain and spinal cord, in part by neurotrophic factor secretion. The present study was conducted to determine whether local or systemic bone marrow-derived mesenchymal stem cell (MSC) transplantation can confer neuroprotection in a rat model of laser-induced ocular hypertensive glaucoma. Methods. MSCs were isolated from the bone marrow of adult wild-type and transgenic rats that ubiquitously express green fluorescent protein. MSCs were transplanted intravitreally 1 week before, or intravenously on the day of, ocular hypertension induction by laser photocoagulation of the trabecular meshwork. Ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was quantified by counting axons within optic nerve cross-sections 4 weeks after laser treatment. Results. After intravitreal transplantation, MSCs survived for at least 5 weeks. Cells were found mainly in the vitreous cavity, though a small proportion of discrete cells migrated into the host retina. Intravitreal MSC transplantation resulted in a statistically significant increase in overall RGC axon survival and a significant decrease in the rate of RGC axon loss normalized to cumulative intraocular pressure exposure. After intravenous transplantation, MSCs did not migrate to the injured eye. Intravenous transplantation had no effect on optic nerve damage. Conclusions. Local, but not systemic, transplantation of MSCs was neuroprotective in a rat glaucoma model. Autologous intravitreal transplantation of MSCs should be investigated further as a potential neuroprotective therapy for glaucoma.