Exosomes derived from nucleus pulposus cells induced bone marrow mesenchymal stem cells into nucleus pulposus like cells

English Keywords:Intervetebral disc degeneration  Exosomes  Mesenchymal stem cells  Nucleus pulposus cells

English Abstract:

  【Abstract】 Objectives: To investigate the role of exosomes derived from nucleus pulposus cells(NPCs) in inducing bone marrow mesenchymal stem cells(BMSCs) differentiation into NP-like cells. Methods: The nucleus pulposus was obtained from the surgical specimen of the patient diagnosed as lumbar disc herniation. The NPCs were separated and cultured in vitro, and then the NPC-exosomes were isolated and purified by differential centrifugation. NPC-exosomes were identified by transmission electron microscope(TEM) and immuneblot analysis. To test the uptake of NPC-exosomes by BMSCs, PKH67 labeled NPC-exosomes were co-incubated with BMSCs for 0.5, 2, 4 hours. Fluorescence confocal microscope(FCFM) was used to examine the uptake of NPC-exosomes. After 3, 7, 10, 14 days stimulated by NPC-exosomes, RT-PCR analysis was performed to detect the mRNA expressions of ACAN, SOX9, COL2A1, KRT19 and HIF-1α in BMSCs. Results: Exosomes derived from NPCs were observed on TEM and the size of them was in the range of 30-100nm. Additionally, NPC-exosomes expressed the exosomal markers CD63, TSG101 and no endoplasmic reticulum marker Calnexin. FCM confirmed that NPCs derived exosomes could be transferred into BMSCs. The RT-PCR results showed that with the duration of interaction, ACAN, SOX-9, COL2A1, HIF-1 α and KRT19 mRNA expres?鄄sions in BM-MSCs had significant increases compared with those of the control group(P<0.05). Conclusions: NPCs can secrete exosomes and then interact with BMSCs to induce BMSCs differentiation into NP-like cells. This study provides a more efficient and simple source of NPCs for tissue engineering repair of degenerative intervertebral disc.