Méndez-Flores Orquidia Guadalupe, Costa Anna Carolina Rego, de Aguiar Andrey Fabiano Lourenço, Paes-Colli Yolanda, Batista Cláudia Maria, Ribeiro-Resende Victor T, Ortega Arturo, de Melo Reis Ricardo A
Laboratory of Neurochemistry, Institute of Biophysics Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, 21941-902, Brazil.
DACS-Universidad Juárez Autónoma de Tabasco, Av. Gregorio Méndez No. 2838-A. Col. Tamulté, 86150, Villahermosa, Tabasco, Mexico.
Neurochem Res. 2025 Jul 21;50(4):242. doi: 10.1007/s11064-025-04500-0.
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 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.
哺乳动物视网膜祖细胞（RPC）通过内在和外在因子的信号传导退出细胞周期，并按照有组织的时空模式产生几种类型的神经元和穆勒胶质细胞。细胞外基质（ECM）在视网膜发育中起重要作用，影响RPC增殖以及向促胶质细胞生成和/或神经细胞生成表型的分化。在此，我们研究了添加在先前用10μg/mL聚-L-赖氨酸处理过的盖玻片上的四种不同ECM成分，即纤连蛋白、玻连蛋白、IV型胶原和层粘连蛋白-1（α1β1γ1），如何影响用20ng/mL表皮生长因子（EGF）生成并培养四天的视网膜神经球的分化。祖细胞（由GABA激动剂蝇蕈醇激活）、神经元（由KCl和/或谷氨酸能激动剂AMPA激活）和穆勒胶质细胞（由ATP激活）由于发育过程中表达的选择性受体和通道模式，在钙成像方面表现出不同的功能反应。当神经球在不同的ECM分子中培养时，产生了高度异质性的细胞群体，表明存在高反应性、中等反应性和低反应性细胞。如图所示，在DMEM F12中用IV型胶原或层粘连蛋白-1处理6天有相似的反应，表明近55%的细胞对KCl有反应，28 - 39%对AMPA有反应，18 - 28%对ATP有反应，几乎没有细胞对蝇蕈醇有反应（小于0.5%）。另一方面，在纤连蛋白存在的情况下，56%的视网膜神经球被诱导对KCl有反应，32%对AMPA有反应，33%对ATP有反应，2.8%对蝇蕈醇有反应。最后，在玻连蛋白中培养的神经球约67%的细胞对KCl有反应，41%对AMPA有反应，对ATP有反应的细胞少于20%，对蝇蕈醇有反应的细胞为3%。正如预期的那样，与其他底物相比，在纤连蛋白存在下分化的细胞进行免疫标记并表达更高水平的胶质纤维酸性蛋白（GFAP），而在玻连蛋白存在下制备的培养物中神经元特异性III类β-微管蛋白（TUJ-1，一种神经元标志物）的表达增加。总之，我们的数据表明，与标准底物层粘连蛋白相比，胶原和玻连蛋白增加了功能性神经元的数量，而纤连蛋白使小鼠视网膜发育细胞中的胶质细胞数量增加了两倍。
