Extracellular Matrix Proteins Differentiate Postnatal Mouse Retina Neurospheres into Neurons or Glia Profiles

The mammalian retinal progenitor cells (RPC) exit the cell cycle through signaling of intrinsic and extrinsic factors and give rise to several types of neurons and Müller glia, following an organized spatial-temporal pattern. Extracellular matrix (ECM) plays an important role in retinal development, influencing RPC proliferation and differentiation into pro-gliogenic and/or neurogenic phenotypes. Here, we investigated how four different ECM constituents, fibronectin, vitronectin, collagen type IV and laminin-1 (α1β1γ1), added on coverslips previously treated with 10 µg/mL poly-L-lysine, could impact differentiation of retinal neurospheres generated with epidermal growth factor (EGF) 20 ng/mL and cultivated for four days. Progenitors (activated by muscimol, a GABA_A agonist), neurons (by KCl and/or AMPA, a glutamatergic agonist) and Müller glia (by ATP) show distinct functional responses in terms of calcium imaging due to the pattern of selective receptors and channels expressed during development. A highly heterogeneous cell population was generated when neurospheres were cultivated in different ECM molecules, suggesting the presence of high, medium, and low-responsive cells. As shown, collagen type IV or laminin-1 for 6 days in DMEM F12 had similar responses, revealing that nearly 55% of cells were responsive to KCl, 28-39% to AMPA, 18-28% to ATP and almost none to muscimol (less than 0.5%). On the other hand, in the presence of fibronectin, 56% of retinal neurospheres were induced to respond to KCl, 32% to AMPA, 33% to ATP and 2.8% to muscimol. Finally, neurospheres raised in vitronectin had around 67% of cells responsive to KCl, 41% to AMPA, less than 20% to ATP and 3% to muscimol. As expected, differentiated cells in the presence of fibronectin were immuno-labelled and expressed higher levels of glial fibrillary acidic protein (GFAP), compared to other substrates, while cultures prepared in the presence of vitronectin had increased expression of neuron-specific class III β-tubulin (TUJ-1), a neuronal marker. Altogether, our data suggest that, compared to laminin, a standard substrate, collagen and vitronectin increased the number of functional neurons, while fibronectin induced a two-fold increase in the number of glial cells in the developing cells of the mice retina.