Extracellular Vesicles from Hypoxic Pretreated Urine-Derived Stem Cells Enhance the Proliferation and Migration of Chondrocytes by Delivering miR-26a-5p

Abstract

Objective: Stem-cell therapy is a promising treatment for cartilage defects. The newly identified urine-derived stem cells (USCs), which have multipotency and sufficient proliferative ability, are promising candidates for several tissue engineering therapies. In this study, we investigated the role of USC extracellular vehicles (EVs) in promoting the proliferation and migration of chondrocytes.

Design: USCs were characterized by measuring induced multipotent differentiation and flow cytometry analysis of surface marker expression. The EVs were isolated from USCs under normoxic conditions (nor-EVs) and hypoxic conditions (hypo-EVs). Transmission electron microscopy and western blot analysis characterized the EVs. The chondrocytes were cultured in the USC-EVs. CCK-8 assay and EdU staining detected the proliferation of chondrocytes, and transwell assay detected their migration. miR-26a-5p expression in EVs was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The target relationship of miR-26a-5p and phosphatase and tensin homolog (PTEN) was predicted and confirmed. The roles of EVs-miR-26a-5p and PTEN on the proliferation and migration of chondrocytes were also investigated.

Results: Hypo-EVs showed a superior effect in promoting the proliferation and migration of chondrocytes than nor-EVs. Mechanistically, USC-EVs delivered miR-26a-5p into chondrocytes to overexpress miR-26a-5p. PTEN was identified as an miR-26a-5p target in chondrocytes. The effects of EVs-miR-26a-5p on promoting the proliferation and migration of chondrocytes were mediated by its regulation of PTEN.

Conclusion: Our study suggested that hypoxic USC-EVs may represent a promising strategy for osteoarthritis by promoting the proliferation and migration of chondrocytes via miR-26a-5p transfer.

Keywords: extracellular vesicles; hypoxia; miR-26a-5p; osteoarthritis; urine-derived stem cells.

Figure 1.

Characterization of USCs. (A) The morphology of USCs under optical microscopy. (B) USC was characterized by flow cytometry analysis using the surface markers CD44, CD73, CD90, CD105, CD31, CD34, and CD45. (C) Verification of the multidirectional differentiation potential of USCs. Osteogenic differentiation ability was accessed by Alizarin red staining, adipogenic differentiation ability was accessed by Oil red O staining, Alcian blue staining accessed the chondrogenic differentiation ability. USCs = urine-derived stem cells.

Figure 2.

USC-EVs promoted the chondrocytes proliferation and migration. (A) TEM scanning of the USC-EVs. (B) EV surface biosignatures CD63, TSG101, Syntenin, and Calnexin, were detected by western blot. (C) The Chondrocytes uptake USC-EVs. (D) CCK-8 assay detection of the cell viability of chondrocytes. (E) EdU staining detection of the proliferation of chondrocytes. (F) Transwell assay was applied to detect the migration ability of chondrocytes. USCs = urine-derived stem cells; EVs = extracellular vehicles; TEM = transmission electron microscopy; CCK-8 = cell counting kit-8; EdU = 5-Ethynyl-2’-Deoxyuridine; DAPI = 4’,-6-diamidino-2-phenylindole. *P < 0.05, ** P < 0.01, compared with the PBS group.

Figure 3.

Hypo-EVs enhanced the promoting effect of USC-EVs on chondrocytes. (A) BCA method to determine the protein concentration of the nor-EVs and hypo-EVs. (B) Western blot detection of EV surface biosignatures CD63, Syntenin, and TSG101. (C-D) The concentration (C) and mean size (D) of nor-EVs and hypo-EVs. (E) Dil-labeled EVs in chondrocytes. (F) CCK-8 assay detection of the cell viability of chondrocytes. (G) EdU staining of the cell proliferation of chondrocytes. (H) Transwell assay was applied to detect the migration ability of chondrocytes. EVs = extracellular vehicles; USCs = urine-derived stem cells; BCA = bicinchoninic acid; EdU = 5-Ethynyl-2’-Deoxyuridine; DAPI = 4’,-6-diamidino-2-phenylindole; PBS = phosphate-buffered saline; CCK-8 = cell counting kit-8. *P < 0.05, **P < 0.01, compared with the PBS group; #P < 0.05, ##P < 0.01, compared with the Nor-EVs group.

Figure 4.

miR-26a-5p is transferred to chondrocytes through USC-EVs. (A) Expression of miRNAs in nor-EVs and hypo-EVs was detected by qRT-PCR. (B) The expression of miR-26a-5p after transfection was assessed by qRT-PCR in USCs. (C) The expression of miR-26a-5p in EVs. (D) The expression of miR-26a-5p in chondrocytes after treatment. (E-F) Detection of cell proliferation using CCK-8 and EdU staining assays. (G) Transwell assay detection of cell migration. USCs = urine-derived stem cells; EVs = extracellular vehicles; NC = negative control; qRT-PCR = quantitative real-time polymerase chain reaction; CCK-8 = cell counting kit-8;  EdU = 5-Ethynyl-2’-Deoxyuridine; DAPI = 4’,-6-diamidino-2-phenylindole; PBS = phosphate-buffered saline. *P < 0.05, **P < 0.01, compared with the PBS group; ##P < 0.01, compared with the hypo-EVs (inhibitor-NC) group.

Figure 5.

miR-26a-5p targeted PTEN in chondrocytes. (A) Prediction of miR-26-5p targets using TargetScan, miRDB, DIANA, and miRanda. (B) PTEN binding sites with miR-26-5p. (C) Dual-luciferase reporter assay confirmed the target relationship between miR-26a-5p and PTEN. (D) miR-26-5p expression in chondrocytes detected by qRT-PCR. (E-F) PTEN expression in chondrocytes after transfection. miRDB = MicroRNA Target Prediction Database (mirdb.org); DIANA = DIANA-microT-CDS (www.microrna.gr/microT-CDS), TargetScan = TargetScan Database (targetscan.org); PTEN = phosphatase and tensin homolog; WT = wild-type; MUT = mutant type; NC = negative control; qRT-PCR = quantitative real-time polymerase chain reaction. *P < 0.05, **P < 0.01.

Figure 6.

EV miR-26a-5p promoted chondrocyte proliferation and migration through mediating PTEN. (A-B) PTEN expression in chondrocytes after treatment with inhibitor-NC-Hypo-EVs or miR-inhibitor-Hypo-EVs. (C-D) PTEN expression in chondrocytes after sh-NC or sh-PTEN transfection. (E-F) After transfection with sh-NC or sh-PTEN and administration of inhibitor-NC-Hypo-EVs or miR-inhibitor-Hypo-EVs, the expression level of PTEN was measured in chondrocytes. (G-I) CCK-8, EdU, and transwell assays were applied to evaluate cell proliferation and migration. EVs = extracellular vehicles; PTEN = phosphatase and tensin homolog; NC = negative control; CCK-8 = cell counting kit-8; EdU = 5-Ethynyl-2’-Deoxyuridine; DAPI = 4’,-6-diamidino-2-phenylindole. *P < 0.05, **P < 0.01.