State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Cytotherapy. 2011 Mar;13(3):294-303. doi: 10.3109/14653249.2010.523075. Epub 2010 Oct 27.
Retinal progenitor cells (RPC) are an excellent resource for retinal replacement therapy but usually unavailable. We attempted to induce bone marrow mesenchymal stromal cells (BMSC) into RPC.
BMSC and embryonic day 13.5 (E13.5) RPC derived from wild-type or enhanced green fluorescence protein (EGFP) transgenic (Egfp(+/+)) mice were co-cultured in a transwell or re-aggregation system. Gene and protein expressions were investigated by reverse transcription-polymerase chain reaction (PCR) and immunofluorescence, respectively. Spontaneous cell fusion was evaluated by Chloromethylbenzamido derivative of 1,1'- dioctadecyl-3,3,3',3' - tetramethylindocarbocyanine perchlorate (CM-DiI) labeling together with EGFP tracing.
BMSC from both wild-type and Egfp(+/+) mice displayed similar spindle shapes. The undifferentiated BMSC already expressed immature neural markers but did not express Nfl, Gfap or the retina-related genes Pax6, Math5 and Brn3b. When co-cultured with E13.5 RPC in the transwell system, BMSC displayed transient expression of early retinal development genes, including Pax6, Math5 and Brn3b at 3 days, as well as long-term expression of Nfl (up to 21 days). No expression of the late photoreceptor gene rhodopsin could be detected at any time. In re-aggregation co-culture, E13.5 RPC induced EGFP-positive BMSC to express not only the early retinal development genes but also the late gene rhodopsin. Furthermore, a small fraction of BMSC could be induced to express the synaptophysin protein. Re-aggregation co-culture of CM-DiI-labeled BMSC and EGFP-positive E13.5 RPC displayed minimal co-localization of the two fluorescence signals.
E13.5 RPC are capable of inducing BMSC towards an RPC fate. The differentiation is independent of cell fusion. Cytokines and cell-cell interactions exert this induction effect, but they have different functions.
视网膜祖细胞（RPC）是视网膜替代治疗的极好资源，但通常无法获得。我们试图诱导骨髓间充质基质细胞（BMSC）成为 RPC。
将 BMSC 和源自野生型或增强型绿色荧光蛋白（EGFP）转基因（Egfp（+/+））小鼠的胚胎第 13.5 天（E13.5）RPC 在 Transwell 或重新聚集系统中共培养。通过逆转录聚合酶链反应（PCR）和免疫荧光分别检测基因和蛋白表达。通过氯甲基苯甲酰胺衍生物 1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（CM-DiI）标记与 EGFP 示踪一起评估自发细胞融合。
来自野生型和 Egfp（+/+）小鼠的 BMSC 均显示出相似的纺锤形。未分化的 BMSC 已经表达了不成熟的神经标记物，但不表达 Nfl、Gfap 或与视网膜相关的基因 Pax6、Math5 和 Brn3b。当在 Transwell 系统中与 E13.5 RPC 共培养时，BMSC 在第 3 天短暂表达早期视网膜发育基因，包括 Pax6、Math5 和 Brn3b，以及长期表达 Nfl（长达 21 天）。在任何时间都无法检测到晚期光感受器基因视紫红质的表达。在重新聚集共培养中，E13.5 RPC 诱导 EGFP 阳性 BMSC 不仅表达早期视网膜发育基因，还表达晚期基因视紫红质。此外，一小部分 BMSC 可被诱导表达突触小泡蛋白。CM-DiI 标记的 BMSC 和 EGFP 阳性 E13.5 RPC 的重新聚集共培养显示两种荧光信号的最小共定位。
E13.5 RPC 能够诱导 BMSC 向 RPC 命运分化。这种分化独立于细胞融合。细胞因子和细胞-细胞相互作用发挥这种诱导作用，但它们具有不同的功能。
