Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR-23a and miR-374.

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR-23a and miR-374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR-23a during E10-19, PN1-7, and adult retina revealed an inverse relation with Brn3b expression. Though miR-374 did not show such a pattern, its co-expression with miR-23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR-23a and miR-374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co-ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors.

Umbilical cord blood-derived mesenchymal stem cells consist of a unique population of progenitors co-expressing mesenchymal stem cell and neuronal markers capable of instantaneous neuronal differentiation.

INTRODUCTION: Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are self-renewing multipotent progenitors with the potential to differentiate into multiple lineages of mesoderm, in addition to generating ectodermal and endodermal lineages by crossing the germline barrier. In the present study we have investigated the ability of UCB-MSCs to generate neurons, since we were able to observe varying degrees of neuronal differentiation from a few batches of UCB-MSCs with very simple neuronal induction protocols whereas other batches required extensive exposure to combination of growth factors in a stepwise protocol. Our hypothesis was therefore that the human UCB-MSCs would contain multiple types of progenitors with varying neurogenic potential and that the ratio of the progenitors with high and low neurogenic potentials varies in different batches of UCB. METHODS: In total we collected 45 UCB samples, nine of which generated MSCs that were further expanded and characterized using immunofluorescence, fluorescence-activated cell sorting and RT-PCR analysis. The neuronal differentiation potential of the UCB-MSCs was analyzed with exposure to combination of growth factors. RESULTS: We could identify two different populations of progenitors within the UCB-MSCs. One population represented progenitors with innate neurogenic potential that initially express pluripotent stem cell markers such as Oct4, Nanog, Sox2, ABCG2 and neuro-ectodermal marker nestin and are capable of expanding and differentiating into neurons with exposure to simple neuronal induction conditions. The remaining population of cells, typically expressing MSC markers, requires extensive exposure to a combination of growth factors to transdifferentiate into neurons. Interesting to note was that both of these cell populations were positive for CD29 and CD105, indicating their MSC lineage, but showed prominent difference in their neurogenic potential. CONCLUSION: Our results suggest that the expanded UCB-derived MSCs harbor a small unique population of cells that express pluripotent stem cell markers along with MSC markers and possess an inherent neurogenic potential. These pluripotent progenitors later generate cells expressing neural progenitor markers and are responsible for the instantaneous neuronal differentiation; the ratio of these pluripotent marker expressing cells in a batch determines the innate neurogenic potential.

In vitro differentiation of retinal ganglion-like cells from embryonic stem cell derived neural progenitors.

ES cells have been reported to serve as an excellent source for obtaining various specialized cell types and could be used in cell replacement therapy. Here, we demonstrate the potential of ES cells to differentiate along retinal ganglion cell (RGC) lineage. FGF2-induced ES cell derived neural progenitors (ES-NPs) were able to generate RGC-like cells in vitro upon differentiation. These cells expressed RGC regulators and markers such as, Ath5, Brn3b, RPF-1, Thy-1 and Islet-1, confirming their potential to differentiate into RGCs. The generation of RGCs from ES-NPs was enhanced with the exposure of FGF2 and Sonic hedgehog (Shh), although Shh treatment alone did not affect RGC differentiation significantly. ES-NPs, after exposure to FGF2, were capable of integrating and differentiating into RGCs in vivo upon transplantation. Thus, our study suggests that ES cells can serve an excellent renewable source for generating RGCs that can be used to treat neurodegenerative diseases like glaucoma.