Isolation, identification and ovarian uptake of exosomes derived from human bone marrow mesenchymal stem cells

doi:10.11958/20221305

Abstract

Abstract:

Objective To extract and identify exosomes derived from human bone marrow mesenchymal stem cells (hBMSC), and to investigate the exosome uptake by granulosa cells and the ovaries of autoimmune premature ovarian insufficiency (POI) mouse. Methods hBMSC of generation P3 to P9 were cultured, and the cell supernatant was collected. The morphology of hBMSC at P4 generation was observed under an inverted microscope. The morphology of the exosomes was observed by transmission electron microscopy, and the particle size of the exosomes was measured by nanoflow cytometry. Western blot assay was used to detect the expression of exosome surface-associated specific proteins CD63, CD9 and TSG101. DiR was used for fluorescence labeling of exosomes. The exosome uptake by human ovarian granulosa cells (KGN) was observed by confocal microscopy after 24 h and 48 h co-incubation. The autoimmune POI mouse model was constructed with ZP3 protein polypeptide, and 150 μg DiR-labeled exosomes were intraperitoneally injected on the 1st and 7th days after successful modeling. Exosome uptake of mouse ovaries were monitored by an in vivo imager on day 14 after the second injection of exosomes. Results hBMSC cells were spindle-shaped and uniform in size. Transmission electron microscopy showed that exosomes were double-layered membranes with a diameter of (81.12 ± 17.23) nm. Exosome expression marker proteins CD63, CD9 and TSG101 were positive. Under confocal microscopy, it was observed that KGN cells could absorb exosomes, and the volume of exosomes taken up by KGN cells at 48 h was more than that of co-incubation for 24 h. In the autoimmune POI mouse model, luminescent signals were observed in mouse ovaries on the 14th day after the second injection of exosomes. Conclusion In this study, exosomes derived from hBMSC are successfully extracted, and the uptake of exosomes by KGN cells and ovarian tissue of autoimmune POI mice is observed.

Key words: mesenchymal stem cells, exosomes, autoimmunity, ultracentrifugation, premature ovarian insufficiency, granulosa cells

CLC Number:

R711.75

Figures/Tables 6

Fig.1 The morphology of hBMSC (×400)

Fig.2 The particle size of hBMSC-derived Exo

Fig.3 The morphology of hBMSC-derived Exo (The scale is 100 nm)

Fig.4 Expression of CD63, CD9 and TSG101 membrane proteins in Exo

Fig.5 The uptake of hBMSC-derived Exo by KGN cells observed by confocal microscopy

Fig.6 Exo uptake in ovarian tissue observed by fluorescence imaging in vivo

References 23

[1]	FU X, LIU G, HALIM A, et al. Mesenchymal stem cell migration and tissue repair[J]. Cells, 2019, 8(8):784. doi:10.3390/cells8080784.
[2]	BATSALI A K, GEORGOPOULOU A, MAVROUDI I, et al. The role of bone marrow mesenchymal stem cell derived extracellular vesicles(MSC-EVs)in normal and abnormal hematopoiesis and their therapeutic potential[J]. J Clin Med, 2020, 9(3):856. doi:10.3390/jcm9030856.
[3]	KALLURI R, LEBLEU V S. The biology,function,and biomedical applications of exosomes[J]. Science, 2020, 367(6478):eaau6977. doi:10.1126/science.aau6977.
[4]	ZHAO J, LI X, HU J, et al. Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization[J]. Cardiovasc Res, 2019, 115(7):1205-1216. doi:10.1093/cvr/cvz040.
[5]	MIANEHSAZ E, MIRZAEI H R, MAHJOUBIN-TEHRAN M, et al. Mesenchymal stem cell-derived exosomes:A new therapeutic approach to osteoarthritis?[J]. Stem Cell Res Ther, 2019, 10(1):340. doi:10.1186/s13287-019-1445-0.
[6]	KESHTKAR S, AZARPIRA N, GHAHREMANI M H. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine[J]. Stem Cell Res Ther, 2018, 9(1):63. doi:10.1186/s13287-018-0791-7.
[7]	NELSON L R, BULUN S E. Estrogen production and action[J]. J Am Acad Dermatol, 2001, 45(3 Suppl):S116-S124. doi:10.1067/mjd.2001.117432.
[8]	TAKAHASHI A, YOUSIF A, HONG L, et al. Premature ovarian insufficiency:Pathogenesis and therapeutic potential of mesenchymal stem cell[J]. J Mol Med(Berl), 2021, 99(5):637-650. doi:10.1007/s00109-021-02055-5.
[9]	LIU W, CHEN M, LIU C, et al. Epg5 deficiency leads to primary ovarian insufficiency due to WT1 accumulation in mouse granulosa cells[J]. Autophagy, 2023, 19(2):644-659. doi:10.1080/15548627.2022.2094671.
[10]	HUANG Y, ZHANG X, ZHAN J, et al. Bone marrow mesenchymal stem cell-derived exosomal miR-206 promotes osteoblast proliferation and differentiation in osteoarthritis by reducing Elf3[J]. J Cell Mol Med, 2021, 25(16):7734-7745. doi:10.1111/jcmm.16654.
[11]	SUN Z, XIE Y, LEE R J, et al. Erratum:Myocardium-targeted transplantation of PHD2 shRNA-modified bone mesenchymal stem cells through ultrasound-targeted microbubble destruction protects the heart from acute myocardial infarction: Erratum[J]. Theranostics, 2022, 12(6):2637-2638. doi:10.7150/thno.72333.
[12]	HUANG Q Y, CHEN S R, CHEN J M, et al. Therapeutic options for premature ovarian insufficiency:An updated review[J]. Reprod Biol Endocrinol, 2022, 20(1):28. doi:10.1186/s12958-022-00892-8.
[13]	RHIM S H, MILLAR S E, ROBEY F, et al. Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida[J]. J Clin Invest, 1992, 89(1):28-35. doi:10.1172/JCI115572.
[14]	COUMANS F, BRISSON A R, BUZAS E I, et al. Methodological guidelines to study extracellular vesicles[J]. Circ Res, 2017, 120(10):1632-1648. doi:10.1161/CIRCRESAHA.117.309417.
[15]	LI P, KASLAN M, LEE S H, et al. Progress in exosome isolation techniques[J]. Theranostics, 2017, 7(3):789-804. doi:10.7150/thno.18133.
[16]	JEPPESEN D K, HVAM M L, PRIMDAHL-BENGTSON B, et al. Comparative analysis of discrete exosome fractions obtained by differential centrifugation[J]. J Extracell Vesicles, 2014, 3:25011. doi:10.3402/jev.v3.25011.
[17]	TANG Y, ZHOU Y, LI H J. Advances in mesenchymal stem cell exosomes:A review[J]. Stem Cell Res Ther, 2021, 12(1):71. doi:10.1186/s13287-021-02138-7.
[18]	SUN B, MA Y, WANG F, et al. miR-644-5p carried by bone mesenchymal stem cell-derived exosomes targets regulation of p53 to inhibit ovarian granulosa cell apoptosis[J]. Stem Cell Res Ther, 2019, 10(1):360. doi:10.1186/s13287-019-1442-3.
[19]	ZHOU X, LI Z, SUN W, et al. Delivery efficacy differences of intravenous and intraperitoneal injection of exosomes:Perspectives from tracking dye labeled and miRNA encapsulated exosomes[J]. Curr Drug Deliv, 2020, 17(3):186-194. doi:10.2174/1567201817666200122163251.
[20]	DE JONG B, BARROS E R, HOENDEROP J, et al. Recent advances in extracellular vesicles as drug delivery systems and their potential in precision medicine[J]. Pharmaceutics, 2020, 12(11):1006. doi:10.3390/pharmaceutics12111006.
[21]	GENG Z, CHEN H, ZOU G, et al. Human amniotic fluid mesenchymal stem cell-derived exosomes inhibit apoptosis in ovarian granulosa cell via miR-369-3p/YAF2/PDCD5/p53 pathway[J]. Oxid Med Cell Longev, 2022, 2022:3695848. doi:10.1155/2022/3695848.
[22]	XIAO G Y, CHENG C C, CHIANG Y S, et al. Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy[J]. Sci Rep, 2016, 6:23120. doi:10.1038/srep23120.
[23]	YANG M, LIN L, SHA C, et al. Bone marrow mesenchymal stem cell-derived exosomal miR-144-5p improves rat ovarian function after chemotherapy-induced ovarian failure by targeting PTEN[J]. Lab Invest, 2020, 100(3):342-352. doi:10.1038/s41374-019-0321-y.