Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

BMT

Biomaterials Translational

TBA2096-112X

Biomaterials Translational

10.12336/biomatertransl.2023.04.006

Research Article

Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoidshttps://artdesignp.com/journal/BMT/4/4/10.12336/biomatertransl.2023.04.006DengShunshu,ZhuFuwei,DaiKai,WangJing,LiuChangsheng

2023

2023-12-28

Bone marrow-derived mesenchymal stem cells (BM-MSCs) play a crucial role in stem cell therapy and are extensively used in regenerative medicine research. However, current methods for harvesting BM-MSCs present challenges, including a low yield of primary cells, long time of in vitro expansion, and diminished differentiation capability after passaging. Meanwhile mesenchymal stem cells (MSCs) recovered from cell banks also face issues like toxic effects of cryopreservation media. In this study, we provide a detailed protocol for the isolation and evaluation of MSCs derived from in vivo osteo-organoids, presenting an alternative to autologous MSCs. We used recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds to construct in vivo osteo-organoids, which were stable sources of MSCs with large quantity, high purity, and strong stemness. Compared with protocols using bone marrow, our protocol can obtain large numbers of high-purity MSCs in a shorter time (6 days vs. 12 days for obtaining passage 1 MSCs) while maintaining higher stemness. Notably, we found that the in vivo osteo-organoid-derived MSCs exhibited stronger anti-replicative senescence capacity during passage and amplification, compared to BM-MSCs. The use of osteo-organoid-derived MSCs addresses the conflict between the limitations of autologous cells and the risks associated with allogeneic sources in stem cell transplantation. Consequently, our protocol emerges as a superior alternative for both stem cell research and tissue engineering.anti-replicative senescence ; in vivo osteo-organoid ; mesenchymal stem cell ; recombinant human bone morphogenetic protein 2 ; stem cell therapy

1. Zhu, H.; Guo, Z. K.; Jiang, X. X.; Li, H.; Wang, X. Y.; Yao, H. Y.; Zhang, Y.; Mao, N. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 2010, 5, 550-560. 2. Li, W. Y.; Choi, Y. J.; Lee, P. H.; Huh, K.; Kang, Y. M.; Kim, H. S.; Ahn, Y. H.; Lee, G.; Bang, O. Y. Mesenchymal stem cells for ischemic stroke: changes in effects after ex vivo culturing. Cell Transplant. 2008, 17, 1045-1059. 3. Kurth, T. B.; Dell’accio, F.; Crouch, V.; Augello, A.; Sharpe, P. T.; De Bari, C. Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo. Arthritis Rheum. 2011, 63, 1289-1300. 4. Huang, S.; Xu, L.; Sun, Y.; Wu, T.; Wang, K.; Li, G. An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. J Orthop Translat. 2015, 3, 26-33. 5. Rossi, C. A.; Flaibani, M.; Blaauw, B.; Pozzobon, M.; Figallo, E.; Reggiani, C.; Vitiello, L.; Elvassore, N.; De Coppi, P. In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel. FASEB J. 2011, 25, 2296-2304. 6. Zhai, W.; Yong, D.; El-Jawhari, J. J.; Cuthbert, R.; McGonagle, D.; Win Naing, M.; Jones, E. Identification of senescent cells in multipotent mesenchymal stromal cell cultures: current methods and future directions. Cytotherapy. 2019, 21, 803-819. 7. Karnoub, A. E.; Dash, A. B.; Vo, A. P.; Sullivan, A.; Brooks, M. W.; Bell, G. W.; Richardson, A. L.; Polyak, K.; Tubo, R.; Weinberg, R. A. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007, 449, 557-563. 8. Salazar-Noratto, G. E.; Luo, G.; Denoeud, C.; Padrona, M.; Moya, A.; Bensidhoum, M.; Bizios, R.; Potier, E.; Logeart-Avramoglou, D.; Petite, H. Understanding and leveraging cell metabolism to enhance mesenchymal stem cell transplantation survival in tissue engineering and regenerative medicine applications. Stem Cells. 2020, 38, 22-33. 9. Wang, Y.; Zhang, W.; Yao, Q. Copper-based biomaterials for bone and cartilage tissue engineering. J Orthop Translat. 2021, 29, 60-71. 10. Uezumi, A.; Fukada, S.; Yamamoto, N.; Takeda, S.; Tsuchida, K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol. 2010, 12, 143-152. 11. Tidu, F.; De Zuani, M.; Jose, S. S.; Bendíčková, K.; Kubala, L.; Caruso, F.; Cavalieri, F.; Forte, G.; Frič, J. NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses. iScience. 2021, 24, 102683. 12. Wang, G.; Cao, K.; Liu, K.; Xue, Y.; Roberts, A. I.; Li, F.; Han, Y.; Rabson, A. B.; Wang, Y.; Shi, Y. Kynurenic acid, an IDO metabolite, controls TSG-6-mediated immunosuppression of human mesenchymal stem cells. Cell Death Differ. 2018, 25, 1209-1223. 13. Kfoury, Y.; Scadden, D. T. Mesenchymal cell contributions to the stem cell niche. Cell Stem Cell. 2015, 16, 239-253. 14. McCullen, S. D.; Chow, A. G.; Stevens, M. M. In vivo tissue engineering of musculoskeletal tissues. Curr Opin Biotechnol. 2011, 22, 715-720. 15. Li, Z.; Niu, S.; Guo, B.; Gao, T.; Wang, L.; Wang, Y.; Wang, L.; Tan, Y.; Wu, J.; Hao, J. Stem cell therapy for COVID-19, ARDS and pulmonary fibrosis. Cell Prolif. 2020, 53, e12939. 16. Hejcl, A.; Sedý, J.; Kapcalová, M.; Toro, D. A.; Amemori, T.; Lesný, P.; Likavcanová-Ma&scaron;ínová, K.; Krumbholcová, E.; Prádný, M.; Michálek, J.; Burian, M.; Hájek, M.; Jendelová, P.; Syková, E. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury. Stem Cells Dev. 2010, 19, 1535-1546. 17. Maruyama, M.; Pan, C. C.; Moeinzadeh, S.; Storaci, H. W.; Guzman, R. A.; Lui, E.; Ueno, M.; Utsunomiya, T.; Zhang, N.; Rhee, C.; Yao, Z.; Takagi, M.; Goodman, S. B.; Yang, Y. P. Effect of porosity of a functionally-graded scaffold for the treatment of corticosteroid-associated osteonecrosis of the femoral head in rabbits. J Orthop Translat. 2021, 28, 90-99. 18. Sun, L.; Akiyama, K.; Zhang, H.; Yamaza, T.; Hou, Y.; Zhao, S.; Xu, T.; Le, A.; Shi, S. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells. 2009, 27, 1421-1432. 19. Cao, X.; Duan, L.; Hou, H.; Liu, Y.; Chen, S.; Zhang, S.; Liu, Y.; Wang, C.; Qi, X.; Liu, N.; Han, Z.; Zhang, D.; Han, Z. C.; Guo, Z.; Zhao, Q.; Li, Z. IGF-1C hydrogel improves the therapeutic effects of MSCs on colitis in mice through PGE(2)-mediated M2 macrophage polarization. Theranostics. 2020, 10, 7697-7709. 20. Soleimani, M.; Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc. 2009, 4, 102-106. 21. Houlihan, D. D.; Mabuchi, Y.; Morikawa, S.; Niibe, K.; Araki, D.; Suzuki, S.; Okano, H.; Matsuzaki, Y. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α. Nat Protoc. 2012, 7, 2103-2111. 22. Lin, W.; Xu, L.; Lin, S.; Shi, L.; Wang, B.; Pan, Q.; Lee, W. Y. W.; Li, G. Characterisation of multipotent stem cells from human peripheral blood using an improved protocol. J Orthop Translat. 2019, 19, 18-28. 23. Matsuda, K.; Falkenberg, K. J.; Woods, A. A.; Choi, Y. S.; Morrison, W. A.; Dilley, R. J. Adipose-derived stem cells promote angiogenesis and tissue formation for in vivo tissue engineering. Tissue Eng Part A. 2013, 19, 1327-1335. 24. Lin, W.; Xu, L.; Li, G. A novel protocol for isolation and culture of multipotent progenitor cells from human urine. J Orthop Translat. 2019, 19, 12-17. 25. Xu, Y.; Zhang, T.; Chen, Y.; Shi, Q.; Li, M.; Qin, T.; Hu, J.; Lu, H.; Liu, J.; Chen, C. Isolation and characterization of multipotent canine urine-derived stem cells. Stem Cells Int. 2020, 2020, 8894449. 26. Huang, R. L.; Kobayashi, E.; Liu, K.; Li, Q. Bone graft prefabrication following the in vivo bioreactor principle. EBioMedicine. 2016, 12, 43-54. 27. Yin, J. Q.; Zhu, J.; Ankrum, J. A. Manufacturing of primed mesenchymal stromal cells for therapy. Nat Biomed Eng. 2019, 3, 90-104. 28. Mauney, J. R.; Nguyen, T.; Gillen, K.; Kirker-Head, C.; Gimble, J. M.; Kaplan, D. L. Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cells with silk fibroin 3D scaffolds. Biomaterials. 2007, 28, 5280-5290. 29. Cui, L.; Xiang, S.; Chen, D.; Fu, R.; Zhang, X.; Chen, J.; Wang, X. A novel tissue-engineered bone graft composed of silicon-substituted calcium phosphate, autogenous fine particulate bone powder and BMSCs promotes posterolateral spinal fusion in rabbits. J Orthop Translat. 2021, 26, 151-161. 30. Dai, K.; Deng, S.; Yu, Y.; Zhu, F.; Wang, J.; Liu, C. Construction of developmentally inspired periosteum-like tissue for bone regeneration. Bone Res. 2022, 10, 1. 31. Dai, K.; Shen, T.; Yu, Y.; Deng, S.; Mao, L.; Wang, J.; Liu, C. Generation of rhBMP-2-induced juvenile ossicles in aged mice. Biomaterials. 2020, 258, 120284. 32. Dai, K.; Zhang, Q.; Deng, S.; Yu, Y.; Zhu, F.; Zhang, S.; Pan, Y.; Long, D.; Wang, J.; Liu, C. A BMP-2-triggered in vivo osteo-organoid for cell therapy. Sci Adv. 2023, 9, eadd1541. 33. Dey, D.; Bagarova, J.; Hatsell, S. J.; Armstrong, K. A.; Huang, L.; Ermann, J.; Vonner, A. J.; Shen, Y.; Mohedas, A. H.; Lee, A.; Eekhoff, E. M.; van Schie, A.; Demay, M. B.; Keller, C.; Wagers, A. J.; Economides, A. N.; Yu, P. B. Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic ossification. Sci Transl Med. 2016, 8, 366ra163. 34. Smith, E.; Yang, J.; McGann, L.; Sebald, W.; Uludag, H. RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells. Biomaterials. 2005, 26, 7329-7338. 35. Zhou, B. O.; Yue, R.; Murphy, M. M.; Peyer, J. G.; Morrison, S. J. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell. 2014, 15, 154-168. 36. Leong, D. J.; Sun, H. B. Mesenchymal stem cells in tendon repair and regeneration: basic understanding and translational challenges. Ann N Y Acad Sci. 2016, 1383, 88-96. 37. Wagner, W.; Horn, P.; Castoldi, M.; Diehlmann, A.; Bork, S.; Saffrich, R.; Benes, V.; Blake, J.; Pfister, S.; Eckstein, V.; Ho, A. D. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One. 2008, 3, e2213. 38. Dimri, G. P.; Lee, X.; Basile, G.; Acosta, M.; Scott, G.; Roskelley, C.; Medrano, E. E.; Linskens, M.; Rubelj, I.; Pereira-Smith, O.; et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995, 92, 9363-9367. 39. Julien, A.; Kanagalingam, A.; Martínez-Sarrà, E.; Megret, J.; Luka, M.; Ménager, M.; Relaix, F.; Colnot, C. Direct contribution of skeletal muscle mesenchymal progenitors to bone repair. Nat Commun. 2021, 12, 2860. 40. Wang, X.; Matthews, B. G.; Yu, J.; Novak, S.; Grcevic, D.; Sanjay, A.; Kalajzic, I. PDGF modulates BMP2-induced osteogenesis in periosteal progenitor cells. JBMR Plus. 2019, 3, e10127. 41. Lees-Shepard, J. B.; Yamamoto, M.; Biswas, A. A.; Stoessel, S. J.; Nicholas, S. E.; Cogswell, C. A.; Devarakonda, P. M.; Schneider, M. J., Jr.; Cummins, S. M.; Legendre, N. P.; Yamamoto, S.; Kaartinen, V.; Hunter, J. W.; Goldhamer, D. J. Activin-dependent signaling in fibro/adipogenic progenitors causes fibrodysplasia ossificans progressiva. Nat Commun. 2018, 9, 471. 42. Zhang, Q.; Liu, Y.; Li, J.; Wang, J.; Liu, C. Recapitulation of growth factor-enriched microenvironment via BMP receptor activating hydrogel. Bioact Mater. 2023, 20, 638-650. 43. Fernández-Santos, M. E.; Garcia-Arranz, M.; Andreu, E. J.; García-Hernández, A. M.; López-Parra, M.; Villarón, E.; Sepúlveda, P.; Fernández-Avilés, F.; García-Olmo, D.; Prosper, F.; Sánchez-Guijo, F.; Moraleda, J. M.; Zapata, A. G. Optimization of mesenchymal stromal cell (MSC) manufacturing processes for a better therapeutic outcome. Front Immunol. 2022, 13, 918565. 44. Saha, D.; Hofmann, N.; Mueller, T.; Niemann, H.; Glasmacher, B. C-2014: Investigation of genetic and epigenetic changes of cryopreserved mesenchymal stem cells. Cryobiology. 2014, 69, 519. 45. Shu, Z.; Heimfeld, S.; Gao, D. Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplant. 2014, 49, 469-476. 46. Lagonda, C. A.; Tjahjadi, F. B.; Fauza, D.; Kusnadi, Y. Hypoxia increases VEGF secretion in multiple sources of mesenchymal stem cells. Cytotherapy. 2018, 20, S44-S45. 47. Wang, X.; Li, F.; Xie, L.; Crane, J.; Zhen, G.; Mishina, Y.; Deng, R.; Gao, B.; Chen, H.; Liu, S.; Yang, P.; Gao, M.; Tu, M.; Wang, Y.; Wan, M.; Fan, C.; Cao, X. Inhibition of overactive TGF-β attenuates progression of heterotopic ossification in mice. Nat Commun. 2018, 9, 551.