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RESULTS
Differentiation of BMSC In this assay, we detected the differentiation characteristics of BMSC cocultured with inactivated RPE cells. In order to distinguish BMSC from RPE cells, BMSC were labeled with CFSE before coculture. After 3 days, we observed that some of cytoplasm of cultured cells retracted toward the nucleus, presenting a more spherical shape and extending processes; the changed cells were CFSEpositive demonstrating the BMSC origin(Figure 1). Furthermore, the cocultured BMSC aggregated into neurospherelike body and cells with a spindleshaped morphology were observed 10 days later (Figure 2). Immunocytochemistry was performed to investigate the expression of neuronal markers, including Nestin (neural precursor marker), NSE (neuronal marker) , and GFAP (astroglial maker). These analysis revealed that BMSC expressed the neural makers when cocultured with RPE. On the contrary, no neural marker was found in untreated fetal BMSC (Figure 3).Figure 1The morphological changes of fetal BMSC after induced for 3 days A and D, BMSC cocultured with RPE cells without addition of BRE; B and E, BMSC cocultured with RPE cells with addition of BRE; C, untreated BMSC. A, B and C, phasecontrast photomicrographs; D and E, confocal photomicrographs presenting the CFSEstaining cells, ×100 (略)Figure 2The morphological changes of fetal BMSC after induced for 10 days A and D, BMSC cocultured with RPE cells without addition of BRE; B and E, BMSC cocultured with RPE cells addition of BRE; C, untreated BMSC. A, B and C, phasecontrast photomicrographs; D and E, confocal photomicrographs presenting the CFSEstaining cells, ×100 (略)Figure 3Nestin, NSE and GFAP expression of BMSC upregulated in BRE treated group after induced for 14 days(Bar=100μm)A, D and G, +RPE cells, BRE; B, E and H, +RPE cells +BRE; C, F and I, untreated(略)
Effects of BRE In some assay, 10μg BRE was added to the induction system. The neuronlike morphology of BMSC treated with BRE in the medium was induced much more markedly at the early induction stage (Figure 1). CFSE is used to fluorescently label live cells and is equally partitioned to daughter cells during division and can be used to measure cell proliferation. After 10 days, the CFSE intensity of the cells was markedly decreased by addition of BRE in culture medium(Figure 2), suggesting that BRE may promote the proliferation of the cells. The immunoreactivity for specific neural markers was increased obviously (Figure 3). Especially, the expression of NSE and GFAP was significantly upregulated as compared with the group without BRE.
DISCUSSION
In this study, we detected the effects of RPE cells and BRE on the differentiation of BMSC to determine the potential induction function of retinaderived components on BMSC. Before induction, fetal BMSC had a flat, elongated, spindlelike structure, similar to that of fibroblasts. After induction, the CFSE positive BMSC showed the morphological characteristics of neuronal cells such as long multipolar extensions and branching ends after 3 days; and then aggregated into spheroid 10 days later. And the neural lineage differentiation of BMSC was demonstrated by the expression of some specific neural markers detected by immunocytochemistry assay. In addition, BRE could promote the neurallike cells differentiation of BMSC in this coculture system. We didnt get the similar results of differentiation BMSC into retinal lineage cells as Chiou et al by coculture of BMSC with RPE cells in this experiment. This may be attributed to the existence of some differences between our induction methods. Chiou et al[8] first induced the BMSC to a spheroid body using neurogenic selection medium for 2 weeks, and then followed a further differentiation in the medium containing RPE cells as a feeder layer for another 23 weeks. However, we directly combined the undifferentiated BMSC and RPE cells together and induced differentiation for 2 weeks. Considering the neural ectoderm developing origin of RPE, RPE cells may play a role in inducing BMSC into neurallineage cells.
Previous reports seemed that the protein of BRE has the function of maintenance the proliferation and differentiation of RPE cells[9]. In order to detect whether BRE could have some function in BMSC differentiation, 10μg protein/mL of BRE was used. The results revealed that BRE could promote a higher expression of nestin, an intermediate filament protein that is predominantly expressed during neural development to some degree[10]. Especially, BRE also enhanced the expression of NSE, a unique form of the glycolytic enzyme enolase found in neurons and in virtually all of the neuroendocrine, paraneuronal cell types, and GFAP, a glial protein that is found in glial cells such as astrocytes. These results indicated that BRE could promote the neural differentiation of BMSC. Given that the retina is developed from neural ectoderm and considering that BRE contains prominent portion of intracellular proteins which do not release and contact cell during retinal development; our results suggested that some of the retinal component must have functions in inducing the differentiation of neural lineage cells. Interesting, these neurallike cells differentiated from BMSC in this assay are both NSE and GFAP positive, we suppose that these neurallike cells are neuron/astrocyte precursor cells which may further differentiated into neuron or astrocyte cells. In conclusion, BMSC from fetal bone marrow were differentiated into neurallike cells expressing the special markers of neural cells by coculture with RPE cells, and the component from retinal may promote BMSC changing into neural lineage cells.
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