Tailored apoptotic vesicles promote bone regeneration by releasing the osteoinductive brake

Abstract

Accumulating evidence has demonstrated that apoptotic vesicles (apoVs) derived from mesenchymal stem cells (MSCs; MSC-apoVs) are vital for bone regeneration, and possess superior capabilities compared to MSCs and other extracellular vesicles derived from MSCs (such as exosomes). The osteoinductive effect of MSC-apoVs is attributed to their diverse contents, especially enriched proteins or microRNAs (miRNAs). To optimize their osteoinduction activity, it is necessary to determine the unique cargo profiles of MSC-apoVs. We previously established the protein landscape and identified proteins specific to MSC-apoVs. However, the features and functions of miRNAs enriched in MSC-apoVs are unclear. In this study, we compared MSCs, MSC-apoVs, and MSC-exosomes from two types of MSC. We generated a map of miRNAs specific to MSC-apoVs and identified seven miRNAs specifically enriched in MSC-apoVs compared to MSCs and MSC-exosomes, which we classified as apoV-specific miRNAs. Among these seven specific miRNAs, hsa-miR-4485-3p was the most abundant and stable. Next, we explored its function in apoV-mediated osteoinduction. Unexpectedly, hsa-miR-4485-3p enriched in MSC-apoVs inhibited osteogenesis and promoted adipogenesis by targeting the AKT pathway. Tailored apoVs with downregulated hsa-miR-4485-3p exhibited a greater effect on bone regeneration than control apoVs. Like releasing the brake, we acquired more powerful osteoinductive apoVs. In summary, we identified the miRNA cargos, including miRNAs specific to MSC-apoVs, and generated tailored apoVs with high osteoinduction activity, which is promising in apoV-based therapies for bone regeneration.

References

1. Green D. R.The Coming Decade of Cell Death Research: Five Riddles.Cell 177, 1094-1107 (2019).
2. Atkin-Smith, G. K. & Poon, I. K. H. Disassembly of the Dying: Mechanisms and Functions.Trends Cell Biol. 27, 151-162 (2017).
3. Argüelles S.,Guerrero-Castilla, A., Cano, M., Muñoz, M. F. & Ayala, A. Advantages and disadvantages of apoptosis in the aging process.Ann. N.Y. Acad. Sci. 1443, 20-33 (2019).
4. Arandjelovic S.& Ravichandran, K. S. Phagocytosis of apoptotic cells in homeostasis.Nat. Immunol. 16, 907-917 (2015).
5. Nagata S.Apoptosis and Clearance of Apoptotic Cells.Annu. Rev. Immunol. 36, 489-517 (2018).
6. Caruso S.& Poon, I. K. H. Apoptotic Cell-Derived Extracellular Vesicles: More Than Just Debris.Front. Immunol. 9, 1486(2018).
7. Tixeira, R.et al.Defining the morphologic features and products of cell disassembly during apoptosis.Apoptosis 22, 475-477 (2017).
8. Kakarla R., Hur J., Kim Y. J., Kim J.& Chwae, Y. J. Apoptotic cell-derived exosomes: messages from dying cells.Exp. Mol. Med. 52, 1-6 (2020).
9. Dou, G.et al.Chimeric apoptotic bodies functionalized with natural membrane and modular delivery system for inflammation modulation.Sci. Adv. 6, eaba2987 (2020).
10. Chen, H.et al.Extracellular Vesicles from Apoptotic Cells Promote TGFβ Production in Macrophages and Suppress Experimental Colitis.Sci. Rep. 9, 5875(2019).
11. Li, M., Liao, L.& Tian, W. Extracellular Vesicles Derived From Apoptotic Cells: An Essential Link Between Death and Regeneration.Front. Cell Dev. Biol. 8, 573511(2020).
12. Bunpetch, V.et al.Strategies for MSC expansion and MSC-based microtissue for bone regeneration.Biomaterials 196, 67-79 (2019).
13. Galipeau J.& Sensébé, L. Mesenchymal Stromal Cells: Clinical Challenges and Therapeutic Opportunities.Cell Stem Cell 22, 824-833 (2018).
14. Fu, J.et al.Systemic therapy of MSCs in bone regeneration: a systematic review and meta-analysis.Stem Cell Res. Ther. 12, 377(2021).
15. Weiss, D. J.et al.The Necrobiology of Mesenchymal Stromal Cells Affects Therapeutic Efficacy.Front. Immunol. 10, 1228(2019).
16. Liu, D.et al.Circulating apoptotic bodies maintain mesenchymal stem cell homeostasis and ameliorate osteopenia via transferring multiple cellular factors.Cell Res. 28, 918-933 (2018).
17. Liu, H.et al.Donor MSCs release apoptotic bodies to improve myocardial infarction via autophagy regulation in recipient cells.Autophagy 16, 2140-2155 (2020).
18. Pavlyukov, M. S.et al. Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors. Cancer Cell 34, 119-135.e110 (2018).
19. Qu, Y.et al.Apoptotic vesicles inherit SOX2 from pluripotent stem cells to accelerate wound healing by energizing mesenchymal stem cells.Acta. Biomater. 149, 258-272 (2022).
20. Dong, J., Wu, B.& Tian, W. Preparation of Apoptotic Extracellular Vesicles from Adipose Tissue and Their Efficacy in Promoting High-Quality Skin Wound Healing.Int. J. Nanomed. 18, 2923-2938 (2023).
21. Ye, Q.et al.MSCs-derived apoptotic extracellular vesicles promote muscle regeneration by inducing Pannexin 1 channel-dependent creatine release by myoblasts.Int. J. Oral Sci. 15, 7(2023).
22. Wang, Y. et al. Delivering Antisense Oligonucleotides across the Blood-Brain Barrier by Tumor Cell-Derived Small Apoptotic Bodies. Adv. Sci. (Weinh) 8, 2004929 (2021).
23. Huang, X.et al.Apoptotic vesicles resist oxidative damage in noise-induced hearing loss through activation of FOXO3a-SOD2 pathway.Stem Cell Res. Ther. 14, 88(2023).
24. You, Y.et al.Tailored Apoptotic Vesicle Delivery Platform for Inflammatory Regulation and Tissue Repair to Ameliorate Ischemic Stroke.ACS Nano. 17, 8646-8662 (2023).
25. Shao, Y.et al.Apoptotic vesicles derived from human red blood cells promote bone regeneration via carbonic anhydrase 1.Cell Prolif. 57, e13547(2023).
26. Zhu, Y.et al.Apoptotic Vesicles Regulate Bone Metabolism via the miR1324/SNX14/SMAD1/5 Signaling Axis.Small 19, e2205813(2023).
27. Ma, L.et al.Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair.Bioact. Mater. 19, 626-641 (2023).
28. Zhang, X.et al.Proteomic analysis of MSC-derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions.J. Extracell. Vesicles 11, e12240(2022).
29. Zheng, C.et al.Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes.J. Extracell. Vesicles 10, e12109(2021).
30. Wang, J.et al.Apoptotic Extracellular Vesicles Ameliorate Multiple Myeloma by Restoring Fas-Mediated Apoptosis.ACS Nano. 15, 14360-14372 (2021).
31. Jiang, Y.et al.Lyophilized apoptotic vesicle-encapsulated adhesive hydrogel sponge as a rapid hemostat for traumatic hemorrhage in coagulopathy.J. Nanobiotechnol. 21, 407(2023).
32. Wang, R.et al.Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling.Bioact. Mater. 25, 472-484 (2023).
33. Zhu, Y.et al.Macrophage-derived apoptotic vesicles regulate fate commitment of mesenchymal stem cells via miR155.Stem Cell Res. Ther. 13, 323(2022).
34. Poon I. K., Lucas C. D., Rossi A. G.& Ravichandran, K. S. Apoptotic cell clearance: basic biology and therapeutic potential.Nat. Rev. Immunol. 14, 166-180 (2014).
35. Poon I. K.H. et al. Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell-derived extracellular vesicles in vitro.J. Extracell. Vesicles 8, 1608786(2019).
36. Shao, H.et al.New Technologies for Analysis of Extracellular Vesicles.Chem. Rev. 118, 1917-1950 (2018).
37. Wiklander, O. P.et al.Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting.J. Extracell. Vesicles 4, 26316(2015).
38. Krützfeldt, J.et al.Silencing of microRNAs in vivo with ‘antagomirs.Nature 438, 685-689 (2005).
39. Lin Z., He H., Wang M.& Liang, J. MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate.Cell Prolif. 52, e12688(2019).
40. Fröhlich L. F.Micrornas at the Interface between Osteogenesis and Angiogenesis as Targets for Bone Regeneration.Cells 8, 121(2019).
41. Dixson A. C., Dawson T. R., Di Vizio, D. & Weaver, A. M. Context-specific regulation of extracellular vesicle biogenesis and cargo selection.Nat. Rev. Mol. Cell Biol. 24, 454-476 (2023).
42. Garcia-Martin, R. et al. MicroRNA sequence codes for small extracellular vesicle release and cellular retention.Nature 601, 446-451 (2022).
43. Zheng, W.et al.Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles.Nat. Commun. 14, 4734(2023).
44. Lei, F.et al.Apoptotic vesicles rejuvenate mesenchymal stem cells via Rab7-mediated autolysosome formation and alleviate bone loss in aging mice.Nano. Res. 16, 822-833 (2023).
45. Sripada, L. et al. hsa-miR-4485 regulates mitochondrial functions and inhibits the tumorigenicity of breast cancer cells. J. Mol. Med. (Berl) 95, 641-651 (2017).
46. Guo, L., Wang, Q.& Zhang, D. MicroRNA-4485 ameliorates severe influenza pneumonia via inhibition of the STAT3/PI3K/AKT signaling pathway.Oncol. Lett. 20, 215(2020).
47. Chen, J.et al.Circulating microRNAs as potential biomarkers of HBV infection persistence.Infect. Genet. Evol. 54, 152-157 (2017).
48. Mi, B.et al.SARS-CoV-2-induced Overexpression of miR-4485 Suppresses Osteogenic Differentiation and Impairs Fracture Healing.Int. J. Biol. Sci. 17, 1277-1288 (2021).
49. O’Brien, J., Hayder, H., Zayed, Y. & Peng, C. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front. Endocrinol. (Lausanne) 9, 402 (2018).
50. Wu, Y.et al.Mettl3-mediated m(6)A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis.Nat. Commun. 9, 4772(2018).
51. Li, L.et al.Tailored Extracellular Vesicles: Novel Tool for Tissue Regeneration.Stem Cells Int. 2022, 7695078(2022).
52. Cao, Z.et al.Encapsulation of Nano-Bortezomib in Apoptotic Stem Cell-Derived Vesicles for the Treatment of Multiple Myeloma.Small 19, e2301748(2023).
53. Jo, H.et al.Applications of Mesenchymal Stem Cells in Skin Regeneration and Rejuvenation.Int. J. Mol. Sci. 22, 2410(2021).
54. Liu, S.et al.Mesenchymal stem cells prevent hypertrophic scar formation via inflammatory regulation when undergoing apoptosis.J. Invest. Dermatol. 134, 2648-2657 (2014).
55. Deng, P.et al.Loss of KDM4B impairs osteogenic differentiation of OMSCs and promotes oral bone aging.Int. J. Oral Sci. 14, 24(2022).
56. Yu, B.et al. PGC-1α Controls Skeletal Stem Cell Fate and Bone-Fat Balance in Osteoporosis and Skeletal Aging by Inducing TAZ. Cell Stem Cell 23, 193-209.e195 (2018).
57. Langmead B., Trapnell C., Pop M.& Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol. 10, R25(2009).
58. Nawrocki E. P.& Eddy, S. R. Infernal 1.1: 100-fold faster RNA homology searches.Bioinformatics 29, 2933-2935 (2013).
59. Kivioja, T.et al.Counting absolute numbers of molecules using unique molecular identifiers.Nat. Methods 9, 72-74 (2011).
60. Wang L., Feng Z., Wang X., Wang X.& Zhang, X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data.Bioinformatics 26, 136-138 (2010).