Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (13): 2056-2061.doi: 10.12307/2022.333

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Isolation and identification of human urine-derived stem cell-derived exosomes

Wang Shuai1, Li Chaoran2, Zhang Xiru3, Huang Guilin4   

  1. 1School of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China; 2Dongfang Hospital Affiliated to Tongji University, Shanghai 310000, China; 3First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550002, Guizhou Province, China; 4Hospital of Stomatology, Zunyi Medical University, Zunyi 563003, Guizhou Province, China
  • Received:2020-12-14 Revised:2020-12-18 Accepted:2021-01-23 Online:2022-05-08 Published:2021-12-20
  • Contact: Wang Shuai, PhD, Associate professor, School of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China Huang Guilin, MD, Professor, Hospital of Stomatology, Zunyi Medical University, Zunyi 563003, Guizhou Province, China
  • About author:Wang Shuai, PhD, Associate professor, School of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81860193 (to WS); a grant from Department of Science and Technology of Guizhou Province, No. [2016]1170 (to WS), No. [2019]1340 (to WS); the Youth Science and Technology Talents Growth Project of Guizhou Provincial Education Department, No. [2016]198 (to WS) 

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Abstract: BACKGROUND: Recent studies have found that the tissue repair and regeneration function of stem cells are largely realized by paracrine function. Exosomes, as one of important paracrine factors, have been proven to play an important role in the repair and regeneration of heart, nerve, bone and retina injury. Researchers are looking for a better source of stem cells-derived exosomes.
OBJECTIVE: To isolate and identify exosomes secreted by human urine-derived stem cells. 
METHODS: Human urine-derived stem cells were isolated from urine of healthy male adults and then cultured and passaged. The surface markers were identified by flow cytometry. Human urine-derived stem cells were induced to differentiate into osteoblasts and adipocytes, and identified by alizarin red, Von Kossa and oil red O staining, respectively. Modified ultra-high speed centrifugation was used to isolate exosomes secreted by human urine-derived stem cells from human urine-derived stem cells conditioned medium. Transmission electron microscopy was used to analyze their morphology. Nanoparticle Tracking Analysis was used to analyze the size and concentration. Western blot assay was used to detect the expression of specific protein CD9, CD63 and HSP70. 
RESULTS AND CONCLUSION: (1) Adherent cell growth was identified in human urine-derived stem cells after 3-5 days of culturing. They resembled “slabstone” in the primary passage, and then resembled “rice” in the first and second passages, and in the third passage, they gradually turned into a “spindle-like shape” or “shuttle-like shape”. Flow cytometry results showed the positive expression of human urine-derived stem cells with mesenchymal stem cell specific surface markers (CD44, CD73, CD90 and CD105). After osteogenic and adipogenic induction in vitro, human urine-derived stem cells successfully differentiated into osteoblasts and adipocytes. (2) After low speed, high speed, ultra-high speed centrifugation, exosomes secreted by human urine-derived stem cells were extracted from conditioned medium of human urine-derived stem cells. Observation by transmission electron microscope showed round or oval membranous vesicles, resembling “saucer”, mainly concentrated with the size of 64-141 nm. Western blot assay showed the protein expression of CD9, CD63 and HSP70 in the exosomes secreted by human urine-derived stem cells. (3) This study confirmed the feasibility of the improved ultra-high speed centrifugation method to extract the exosomes secreted by human urine-derived stem cells.

Key words: stem cells, human urine-derived stem cells, exosomes, ultrahigh speed centrifugation, osteogenic differentiation, particle size, nanoparticle

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