Glaucoma is a complex, multivariate, irreversible blinding eye disease. The blockade of retinal ganglion cell apoptosis, the reduction of intraocular pressure, and nourishment of the optic nerve are known to be somewhat effective in prolonging the life of ganglion cells and retarding disease progression for patients with early glaucoma or progressing glaucoma. However, such treatment strategies are ineffective for patients with advanced glaucoma. This discrepancy lies in the fact that most or all retinal ganglion cells have undergone apoptosis in patients with advanced glaucoma, and the number of surviving ganglion cells is too few to reverse pathological changes resulting from glaucoma. Therefore, there is an urgent need to develop novel strategies to regenerate retinal ganglion cells to reverse disease progression or even restore the vision.
Stem cell engineering has emerged as a promising approach for retinal regeneration therapy [23, 24]. By incorporating stem cells into the retina and inducing their proliferation and differentiation into target cells, it is possible to replenish retinal neurons and restore retinal function. Retinal Müller cells, the glial cells in the retina, retain proliferation potential and offer an abundant source for cell engineering [9, 10]. In addition, Müller cells span the entire width of the retina and are widely distributed among ganglion cells. This increases the chance to better integrate the cells converted from Müller cells into the ganglion cell layer. All these features suggest that retinal Müller cells are the most promising source of stem cells in the are features suggest that retinal Müller cells are the most promising source of stem cells in the treatment of glaucoma. Although there is substantial evidence that retinal Müller cells can dedifferentiate into retinal stem cells in certain conditions, the potential of their differentiation into ganglion cells remains unclear. Therefore, in the present study, we selected stem cells dedifferentiated from rat retinal Müller cells as the target cells. We successfully transfected these stem cells with lentivirus pGC-FU-Atoh7-GFP, and demonstrated that Atoh7 could promote the differentiation of Müller cells derived stem cells into ganglion cells.
Glutamine synthetase (GS) is a key enzyme that transfers glutamic acid into glutamine and is only expressed in Müller cells . Therefore, we chose GS as the specific marker to identify Müller cells. The results showed that the cells isolated from rat retina displayed the general morphology of Müller cells and more than 98.10 ± 2.18% of the cells were immunopositive for GS. FACS showed that 98.01% of the purified cells were immunoreactive for GS. RT-PCR revealed that the cells expressed a battery of transcripts characteristic of Müller cells, such as GS, vimentin, CRALBP, clusterin and carbonic anhydrase. In contrast, transcripts corresponding to rod photoreceptors, bipolar cells, amacrine cells, retinal ganglion cells, endothelial cells and RPE/pigmented ciliary epithelium were not detected. Taken together, these results demonstrate that our method of purifying Müller cells is effective.
Currently, two methods are commonly used to induce the dedifferentiation of retinal Müller cells: one is to use ouabain, N-methyl-D-aspartate (NMDA) or other agents to induce the effects of injury, and the other is to add various cell growth factors [11–13]. In the present study, we chose a serum-free DMEM/F12 medium supplemented with EGF, bFGF and other cytokines to induce the dedifferentiation of retinal Müller cells. Characterization of the cell spheres dedifferentiated from retinal Müller cells showed that the cells within the neurospheres highly expressed retinal stem cell-specific markers Nestin and Pax6, which, however, are not expressed in normal Müller cells. Furthermore, positive Edu staining, an important indicator of proliferation, was observed in 82.80 ± 6.65% of the cells, proving that the cells derived from dedifferentiation have the proliferative ability of stem cells. These data suggest that upon cytokine stimulation, Müller cells acquire the property of neural stem cells.
The serum is a natural inducer of stem cell differentiation, but previous study showed that stem cells dedifferentiated from retinal Müller cells did not undergo neuronal cell differentiation when cultured in medium only containing serum . BDNF and RA have been shown to induce neural stem cells to differentiate into neurons and promote their maturation [26, 27]. Thus, we chose to induce re-differentiation of stem cells by using a culture medium supplemented with BDNF and RA. We found that the stem cells had the ability to re-differentiate. Cells of various shapes were obtained as early as at days 7 to 10 of culture. Even without transfection with Atoh7 expression vector, ganglion cells induced by our medium could account for 9.10 ± 3.21% of the total differentiated cells.
The directed differentiation of retinal stem cells is mainly co-regulated by the extracellular microenvironment factors and endogenous cytokines. The bHLH (basic helix-loop-helix) family plays an important role in regulating retinal cell differentiation . Atoh7 is a member of the bHLH family and its expression pattern is consistent with the spatiotemporal pattern of retinal ganglion cell differentiation . Atoh7 is a key regulatory factor essential for the development of retinal ganglion cells in vertebrates . Ectopic expression of Atoh7 has been shown to increase the number of retinal ganglion cells differentiated from stem cells . Therefore, in the present study, we transfected Müller cells-derived stem cells with the Atoh7 expression vector to promote the differentiation into ganglion cells. The results showed that transfection with lentivirus PGC-FU-Atoh7-GFP led to the differentiation into ganglion cells at a frequency nearly five times that of transfection with empty vector, confirming the crucial role of Atoh7 in promoting the directed differentiation of stem cells into ganglion cells.
To explore the signaling mechanisms by which Atoh7 promotes the differentiation of Müller cells-derived stem cells into ganglion cells, we employed the loss of function approach to intervene with the expression of Brn-3b, Isl-1 and Notch1. Our results showed that knockdown of Brn-3b or Isl-1 inhibited the differentiation of Müller cells-derived stem cells into retinal ganglion cells, while Notch signal pathway inhibitor GSI promoted the differentiation into retinal ganglion cells. These results suggest that Brn-3b and Isl-1 promote while Notch signaling inhibits the differentiation of Müller cells-derived stem cells into retinal ganglion cells. In particular, we found that overexpression of Atoh7 and inhibition of Notch signaling by GSI exhibited synergistic effects to promote the differentiation into retinal ganglion cells. Considering our results that Atoh7 inhibited the expression of Notch1, we propose that Atoh7 promotes the differentiation of retinal stem cells derived from Müller cells into retinal ganglion cells by inhibiting Notch signaling.