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Journal of Peking University(Health Sciences) ›› 2020, Vol. 52 ›› Issue (1): 43-50. doi: 10.19723/j.issn.1671-167X.2020.01.007

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Mediated pathways of exosomes uptake by stem cells of apical papilla

Xiao-min GAO,Xiao-ying ZOU(),Lin YUE()   

  1. Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology & National Clinical research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2019-10-10 Online:2020-02-18 Published:2020-02-20
  • Contact: Xiao-ying ZOU,Lin YUE E-mail:zouxiaoying1125@163.com;kqlinyue@bjmu.edu.cn
  • Supported by:
    Support by the National Natural Science Foundation of China(81650005);Support by the National Natural Science Foundation of China(81200773)

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Abstract:

Objective: To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP. Methods: (1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 ℃ as positive control group, and co-cultured with SCAP at 4 ℃ as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluore-scence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluore-scence. Results: (1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 ℃ for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 ℃ for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%,and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively. Conclusion: SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.

Key words: Dental pulp stem cells, Stem cells from apical papilla, Exosome, Endocytosis

CLC Number: 

  • R329.2

Figure 1

Cell morphology of human DPSCs and SCAP A, primary DPSCs cultured for 5 days; B, primary DPSCs cultured for 14 days; C, Primary SCAP cultured for 5 days; D, Primary SCAP cultured for 14 days."

Figure 2

Isolation and identification results of DPSCs derived exosomes A and B, morphologic analysis of DPSCs derived exosomes by transmission electron microscopy; C, nanoparticle tracking analysis measurement of particle size of DPSCs derived exosomes; D, detection of exosomal marker expression in DPSCs derived exosomes by Western blot."

Figure 3

Uptake of DPSCs derived exosomes by SCAP DAPI, 4’,6-diamidino-2-phenylindole."

Figure 4

Uptake of DPSCs derived exosomes by SCAP with the treatment of different endocytosis inhibitors DMSO, dimethyl sulfoxide; CPZ, chlorpromazine; DAPI, 4’,6-diamidino-2-phenylindole."

Figure 5

Role of different endocytosis inhibitors in the uptake of DPSCs derived exosomes by SCAP in flow cytometry DMSO, dimethyl sulfoxide; CPZ, chlorpromazine."

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