Center for Research in Tissue Engineering and Cellular Therapies, Maimónides University, Buenos Aires, Argentina.
Ophthalmic Res. 2012;48 Suppl 1:1-5. doi: 10.1159/000339839. Epub 2012 Aug 21.
It has been previously shown that adult mesenchymal stem cells (MSCs) differentiate into neural progenitor cells (NPCs) and that the differentiation process was completed in 24-48 h. In this previous study, MSCs from a bone marrow or fat source were co-incubated with homologous autoaggressive cells (ECs) against nerve tissue, and these NPCs were successfully used in human regenerative therapeutic approaches. The present study was conducted to investigate whether a similar differentiation method could be used to obtain autologous retinal progenitor cells (RPCs).
Human Th1 cells against retinal tissue were obtained by challenging human blood mononuclear cells with an eye lysate of bovine origin; negative selection was performed using a specific immunomagnetic bead cocktail. Fat MSCs were obtained from a human donor through mechanical and enzymatic dissociation of a surgical sample. The ECs and MSCs were co-cultured in a serum-free medium without the addition of cytokines for 0, 24, 48 and 72 h. The plastic adherent cells were morphologically examined using inverted-phase microscopy and characterized by immunofluorescent staining using antibodies against Pax 6, TUBB3, GFAP, Bestrophin 2, RPE 65, OPN1 SW, and rhodopsin antigens.
The early signs of MSC differentiation into RPCs were observed at 24 h of co-culture, and the early differentiated retinal linage cells appeared at 72 h (neurons, rods, Müller cells, retinal ganglion cells and retinal pigmented epithelial cells). These changes increased during further culture.
The results reported here support the development of a method to obtain a large number of autologous adult RPCs, which could be used to treat different retinopathies.
此前已经证明，成体间充质干细胞（MSCs）可分化为神经祖细胞（NPCs），且分化过程在 24-48 小时内完成。在之前的研究中，骨髓或脂肪来源的 MSCs 与针对神经组织的同源自身反应性细胞（ECs）共同孵育，这些 NPC 成功地用于人类再生治疗方法中。本研究旨在探讨是否可以使用类似的分化方法获得自体视网膜祖细胞（RPCs）。
用人血单核细胞与牛眼匀浆孵育获得针对视网膜组织的人 Th1 细胞，采用特异性免疫磁珠鸡尾酒进行阴性选择。脂肪 MSCs 从人类供体中通过机械和酶消化手术样本获得。将 ECs 和 MSCs 在无血清培养基中共同培养，不添加细胞因子，分别在 0、24、48 和 72 小时后进行检测。采用倒置相差显微镜观察贴壁细胞的形态，并通过针对 Pax6、TUBB3、GFAP、Bestrophin 2、RPE 65、OPN1SW 和视紫红质抗原的免疫荧光染色进行鉴定。
共培养 24 小时后观察到 MSC 向 RPC 分化的早期迹象，72 小时时出现早期分化的视网膜谱系细胞（神经元、视杆细胞、Müller 细胞、视网膜神经节细胞和视网膜色素上皮细胞）。这些变化在进一步培养过程中增加。
本研究结果支持开发一种获得大量自体成年 RPC 的方法，该方法可用于治疗不同的视网膜病变。
