The inducing effect of rat nucleus pulposus cells derived exosomes on the differentiation of mesenchymal stem cells into nucleus pulposus-like cells

English Keywords:Exosomes  Bone marrow mesenchymal stem cell  Nucleus pulposus cell  Differentiation

English Abstract:

  【Abstract】 Objectives: To investigate the roles of exosomes derived from rat nucleus pulposus cells(NPCs) in the differentiation of rat bone marrow mesenchymal stem cells(BMSCs) into nucleus pulposus-like cells. Methods: SD rats were sacrificed to obtain NPCs and BMSCs. BMSCs were identified by flow cytometry, and its multipotent differentiation ability was proved by the induction of osteogenic, adipogenic and chondrogenic differentiation. NPC exosomes were extracted by differential centrifugation and identified by transmission electron microscope, and western blot analyses of exosomes surface makers, such as CD81, Tsg101. NPC exosomes and MSCs were labeled by fluorescent dye CM-Dil and CM-Dio respectively and incubated for 24h. Fluorescence microscopy was used to detect the uptake of NPC exosomes by MSCs. The third generation BMSCs were divided into three groups: exosomes group(treated with NPCs exosomes, 50μg/ml), coculture group(indirect cocultured with NPCs) and control group(no treatment). Quantitative RT-PCR(qRT-PCR) was performed to detect the expressions of Collagen Ⅱ, Aggrecan and SOX9 in exosomes group and control group in 7, 14, 21 days. QRT-PCR was performed to detect the expressions of Collagen Ⅱ, Aggrecan and SOX9 in three groups at 14d. Results: The third generation NPCs were polygonal. The third generation BMSCs were spindle and regular. BMSCs were positive for CD29(99.77%), CD44(93.97%), CD90(99.67%) and negative for CD34(0.82%), CD45(0.68%). BMSCs showed the ability to differentiate into osteoblasts, adipocytes and chondrocytes. The NPCs exosomes were revealed as round-shaped vesicles with double-layer membrane structure and <100nm in diameter under the transmission electron microscope. It was positive for CD81, Tsg101 and negative for calnexin. CM-DIL labeled NPCs exosomes could be internalized by CM-DIO labeled BMSCs under fluorescence microscopy. The mRNA expressions of Collagen Ⅱ, Aggrecan and SOX9 in exosomes group were higher than those in control group in 7, 14, 21 days(P<0.05). The mRNA expressions of Collagen Ⅱ, Aggrecan and SOX9 in coculture group were higher than those in control group, lower than those in exosomes group(P<0.05). Conclusions: Rat NPC exosomes can induce MSCs differentiate into NP-like cells in vitro, and its effects are better than indirect coculture system. It can provide an effective source of seed cells for intervertebral disc tissue engineering.