【摘要】 目的：检测视网膜色素上皮细胞对骨髓间充质干细胞分化的调节作用。方法：体外培养人骨髓间充质干细胞（BMSC）和视网膜色素上皮（RPE）细胞。RPE细胞采用紫外线照射处理，而后与CFSE标记的BMSC共培养14d。并在共培养体系中加入牛眼视网膜提取物（BRE）以研究视网膜成分对此分化过程的影响。采用NSE, Nestin和GFAP抗体标记检测BMSC分化前后的表达特征。结果：BMSC在与RPE细胞共培养后，能够分化成为神经样细胞，并表达神经性细胞的特异性标记NSE，Nestin和GFAP。BRE能够显著促进共培养体系中BMSC向神经样细胞的分化。结论：RPE和BRE能够诱导BMSC分化为神经样细胞。
【关键词】 髓间充质干细胞；视网膜色素上皮细胞；神经样细胞；牛眼视网膜提取物
Differentiation of human bone marrowderived mesenchymal stem cells into neurallike cells by coculture with retinal pigment epithelial cells
LingLing Yang, QingJun Zhou, Yao Wang, YiQiang Wang
Foundation items: Natural Science Foundation of China (No.30471645); Taishan Scholar Program QDUEYE, Qingdao University, Shandong Province, China
State Key Laboratory Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao 266071, Shandong Province, China
AbstractAIM: To detect the differentiation effects of retinal cells or extracts on bone marrowderived mesenchymal stem cells (BMSC).METHODS: Human fetal BMSC were previously labeled by carboxyfluorescein succinimidyl ester (CFSE), and cocultured with retinal pigment epithelial （RPE） cells which were pretreated with ultraviolet irradiation at a ratio of 1∶1 to induce the differentiation of BMSC for up to 14 days. In some assays, a retinal extract of bovine retinal extract (BRE) was added to detect the potential effects of retinal component on the differentiation of BMSC. In addition, neuronspecific enolase (NSE), Nestin and Glial fibrillary acidic protein (GFAP) immunostaining were performed to determine the characteristics of BMSC.RESULTS: The results indicated that by cocultured with RPE cells, fetal BMSC were differentiated into neurallike cells expressing special neuronal markers Nestin, GFAP and NSE. And the expression of these markers was obviously increased by BRE. CONCLUSION: Retina derived cells and extracts can induce the differentiation of BMSC into neurallike cells.
KEYWORDS: bone marrowderived mesenchymal stem cells; retinal pigment epithelial cells; neurallike cells; bovine retinal extract
INTRODUCTION
According to the origin and potential of differentiation, two types of stem cells can be distinguished: embryonic stem cells (ESC) and somatic stem cells (SSC). SSC are isolated from fetal (after gastrulation) or adult tissues, but classically the differentiation fate of these cells are believed to be limited to the cell types that belong to the tissue from which they originate. However, previous studies have suggested that these tissuespecific stem cells might be able to differentiate into cell types of other lineages[1]. Bone marrowderived mesenchymal stem cells (BMSC) are one of the major subpopulations of SSC, which is extensively studied with respect to transdifferentiation potential[2]. Recent studies have described that BMSC can be differentiated into neurallike cells in vitro under specific induced culture conditions[3,4] or developed spontaneously differentiation under noninduced standard culture conditions[57]. Usually the differentiation is induced just by addition of growth factors and/or chemicals in culture medium. The agents include βmercaptoethanol and dimethylsulfoxide, epidermal growth factor (EGF) and brainderived neurotrophic factor (BDNF), isobutylmethylxanthine/dibutyryl cAMP, or 5AzaC/growth factors. Moreover, a small proportion of BMSCderived cells differentiated into neuronlike cells expressing NeuN and glial cells expressing Glial fibrillary acidic protein (GFAP) when cocultured with rat fetal mesencephalic or striatal cells. The retinal pigment epithelium(RPE) is a hexagonally packed monolayer cell in ocular retina that performs critical functions in the maintenance of the physiology of photoreceptors which is developed from the outer layer of the optic cup. The research of Chiou et al[8] shows that coculture of human BMSC with human retinal pigment epithelium (HRPE) cells facilitates the retina and photoreceptorlineage differentiation of adult human BMSC. This system indicates the retinal differentiation potential of BMSC. In this study, we established a coculture fetal BMSC/RPE system by directly mixing them and showed that under such condition, fetal BMSC was differentiated into neural phenotypelike cells and expressed neuronal markers such as Nestin, GFAP and neural specific enolase[5]. Besides, our results demonstrated that bovine retinal extract (BRE) could markedly promoted the neurallineage differentiation of fetal BMSC in vitro.
MATERIALS AND METHODS
Materials Human fetal BMSC were cultured in this assay. The use of human tissues in this study was approved by the Shandong Eye Institute Medical Ethics Committee and was in compliance with the Declaration of Helsinki. In order to eliminate unwanted types of cells presented in the marrow aspiration, mononuclear cells were obtained by Ficoll Hypaque density gradient centrifugation (lymphoprep, 1073g/L; TBD, China) and BMSC were selected by plastic adhesion. Briefly, a small percentage of cells isolated from the density interface of 1073g/L were seeded in 6 well plates using the medium of LG DMEM supplemented with 100mL/L FBS (Gibco, USA). After 3 days undisturbed to promote cell attachment, the nonadherent cells were removed by changing the medium. At nearconfluence, about two weeks after initial plating, cells were subcultured after trypsin digestion. Fetal BMSC were isolated by Ficoll Hypaque density gradient centrifugation and the physical property of adherence to plastic culture dishes were confirmed. BMSC cultured in our experiment grew as fibroblastic cells with scant cytoplasm and contain granules around nuclei, which were similar to the cells described in previous reports[8]. Fetal RPE cells were isolated and cultured as described previously[9] and these cells showed typical polygonal shape. Briefly, sheets of RPE were dissected from the choroids of fetal eye cups and cultured in lowcalcium (0.05mmol/L Ca2+) MEM (Sigma, St. Louis, MO; catalog number M8028) medium containing proposed supplements according to the reports of Hu and Bok [9]. Floating RPE cells were collected and passaged with normalcalcium medium (Sigma, St. Louis, MO; catalog number M2279) containing the same supplements.
BMSC/RPE coculture Firstly, fetal BMSC at 35 passage were labeled with CFSE (PKCA705C375, Probior GmbH) in order to be distinguished from RPE cells. A total of 106 to 107 cells were washed twice with phosphate buffered saline (PBS) and then incubated with 2.5μmol/L CFSE in PBS for 10 minutes in dark at room temperature. Then cells were washed twice with media containing 100mL/L FBS. Secondly, the second passage of fetal RPE cells were pretreated under a UV lamp for 15 minutes to abolish its selfroliferation as the method of Chiou et al[8] and washed three times with DHanks. Then the inactivated cells were digested with 2.5g/L trypsin0.2g/L EDTA and resuspended in medium containing 100mL/L FBS. Lastly, 2×105 of fetal BMSC and RPE cells prepared as above were mixed together and plated on gelatincoated 24wells plates for up to 14 days. In some assays, 10μg BRE was added in the medium. BRE was prepared by homogenizing 12 fresh bovine retinas per 100mL CMFBSS and stirring overnight in the dark at 4℃. The mixture was cleared by centrifugation (12000g for 20 minutes) and the supernatants were collected[9]. The noninduced fetal BMSC were cultured as the control group. In each group four duplicate wells were set up, and the assay was repeated for 3 times independently.
Immunocytochemistry Cells were fixed at 20℃ with cold methanol for 5 minutes. For staining, samples were rehydrated in PBS/2mL/L Triton X100. Nonspecific binding was blocked with 100mL/L normal serum in PBS/2mL/L Triton X100 for 30 minutes at room temperature. Cells were then incubated overnight at 4℃ with the following primary antibodies diluted in blocking solution, polyclonal rabbit antinestin (1∶100, Boster, China); polyclonal rabbit antiNSE (1∶100, Boster, China); polyclonal rabbit antiGFAP (ready to use, Beijing Zhongshan Golden Bridge Biotechnology CO, LTD, China). Rhodamine conjugated Goat anti rabbit IgG (1∶100; Beijing Zhongshan Golden Bridge Biotechnology CO, LTD, China) was used as secondary antibody. After washing with PBS for 3 times, samples were counterstained with DAPI (Santa Cruz, USA). Negative controls were performed by omitting primary antibodies.
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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