Research progress and clinical prospect of three-dimensional spheroid culture of mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHU Yuanzheng ,  YI Yangyan

Department of Plastic Surgery, Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China;

Corresponding author：

YI Yangyan, Email: yyy0218@126.com

Keywords：

Three-dimensional culture; spheroid culture; mesenchymal stem cells; stem cell therapy; repair and reconstruction

DOI：

10.7507/1002-1892.201612056

Video：

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Objective To review the research progress and clinical prospect of three-dimensional spheroid culture of mesenchymal stem cells (MSCs). Methods Recent literature about three-dimensional spheroid culture of MSCs was summarized, mainly on the formation of MSCs spheroids collected by three-dimensional culture, differences between MSCs spheroids and MSCs collected by traditional two-dimensional culture, and the mechanism underlying these differences. Last, its clinical prospect was discussed. Results Compared with MSCs collected by traditional two-dimensional culture, MSCs spheroids collected by three-dimensional culture get a salient up-regulation in anti-apoptosis, multiple differentiation potential, paracrine, and anti-inflammatory effect, which may be related to the morphology and cytoskeleton organization, cell-to-cell contact and gap junctions, and the hypoxia microenvironment. The animal experiments show obvious effects in repair of refractory wounds, repair of ischemic injury, and tissue remodeling, so MSCs spheroid has broad clinical prospect. Conclusion MSCs spheroids collected by three-dimensional culture have stronger biological potential and treatment effect than MSCs collected by traditional two-dimensional culture, MSCs spheroids can be used to optimize stem cell therapy and improve its treatment effect.

Citation： ZHU Yuanzheng, YI Yangyan. Research progress and clinical prospect of three-dimensional spheroid culture of mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(4): 497-503. doi: 10.7507/1002-1892.201612056 Copy

1.	Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968, 6(2): 230-247.
2.	Sart S, Schneider YJ, Agathos SN. Ear mesenchymal stem cells: an efficient adult multipotent cell population fit for rapid and scalable expansion. J Biotechnol, 2009, 139(4): 291-299.
3.	Murphy KC, Fang SY, Leach JK. Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell Tissue Res, 2014, 357(1): 91-99.
4.	Galipeau J. The mesenchymal stromal cells dilemma——does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy, 2013, 15(1): 2-8.
5.	Mitchell JB, Mcintosh K, Zvonic S, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromalassociated and stem cell-associated markers. Stem Cells, 2006, 24(2): 376-385.
6.	Bartosh TJ, Ylostalo JH, Mohammadipoor A, et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A, 2010, 107(31): 13724-13729.
7.	Steinberg MS. Cell-cell recognition in multicellular assembly: levels of specificity. Symp Soc Exp Biol, 1978, 32: 25-49.
8.	Foty R. A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp, 2011, (51). pii: 2720.
9.	Huang GS, Dai LG, Yen BL, et al. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials, 2011, 32(29): 6929-6945.
10.	Schmidt S, Friedl P. Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res, 2010, 339(1): 83-92.
11.	Li Y, Guo G, Li L, et al. Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res, 2015, 360(2): 297-307.
12.	Yeh HY, Liu BH, Sieber M, et al. Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes. BMC Genomics, 2014, 15: 10.
13.	Shoham N, Gefen A. Mechanotransduction in adipocytes. J Biomech, 2012, 45(1): 1-8.
14.	Shoham N, Gefen A. The influence of mechanical stretching on mitosis, growth, and adipose conversion in adipocyte cultures. Biomech Model Mechanobiol, 2012, 11(7): 1029-1045.
15.	Young DA, Choi YS, Engler AJ, et al. Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue. Biomaterials, 2013, 34(34): 8581-8588.
16.	Tran TD, Yao S, Hsu WH, et al. Arginine vasopressin inhibits adipogenesis in human adipose-derived stem cells. Mol Cell Endocrinol, 2015, 406: 1-9.
17.	Cheng NC, Wang S, Young TH. The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials, 2012, 33(6): 1748-1758.
18.	Cheng NC, Chen SY, Li JR, et al. Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med, 2013, 2(8): 584-594.
19.	Lee JH, Han YS, Lee SH. Long-duration three-dimensional spheroid culture promotes angiogenic activities of adipose-derived mesenchymal stem cells. Biomol Ther (Seoul), 2016, 24(3): 260-267.
20.	Bartosh TJ, Ylöstalo JH, Bazhanov N, et al. Dynamic compaction of human mesenchymal stem/precursor cells (MSC) into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6 and STC1). Stem Cells, 2013, 31(11): 2443-2456.
21.	Tamama K, Kawasaki H, Kerpedjieva SS, et al. Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition. J Cell Biochem, 2011, 112(3): 804-817.
22.	Ylöstalo JH, Bartosh TJ, Coble K, et al. Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype. Stem Cells, 2012, 30(10): 2283-2296.
23.	Frith JE, Thomson B, Genever PG. Dynamic threedimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods, 2010, 16(4): 735-749.
24.	Lee EJ, Park SJ, Kang SK, et al. Spherical bullet formation via E-cadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction. Mol Ther, 2012, 20(7): 1424-1433.
25.	Jackson WM, Nesti LJ, Tuan RS. Mesenchymal stem cell therapy for attenuation of scar formation during wound healing. Stem Cell Res Ther, 2012, 3(3): 20.
26.	Peng R, Yao X, Cao B, et al. The effect of culture conditions on the adipogenic and osteogenic inductions of mesenchymal stem cells on micropatterned surfaces. Biomaterials, 2012, 33(26): 6008-6019.
27.	Zhong Z, Williams BO. Integration of cellular adhesion and Wnt signaling: Interactions between N-cadherin and LRP5 and their role in regulating bone mass. J Bone Miner Res, 2012, 27(9): 1849-1851.
28.	Alan T, Tufan AC. C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions. J Cell Biochem, 2008, 105(1): 227-235.
29.	Hay E, Buczkowski T, Marty C, et al. Peptide-based mediated disruption of N-cadherin-LRP5/6 interaction promotes Wnt signaling and bone formation. J Bone Miner Res, 2012, 27(9): 1852-1863.
30.	Hsu SH, Huang GS. Substrate-dependent Wnt signaling in MSC differentiation within biomaterial-derived 3D spheroids. Biomaterials, 2013, 34(20): 4725-4738.
31.	Yeganeh A, Stelmack GL, Fandrich RR, et al. Connexin 43 phosphorylation and degradation are required for adipogenesis. Biochim Biophys Acta, 2012, 1823(10): 1731-1744.
32.	Van Winkle AP, Gates ID, Kallos MS. Mass transfer limitations in embryoid bodies during human embryonic stem cell differentiation. Cells Tissues Organs, 2012, 196(1): 34-47.
33.	Kaufman G, Nunes L, Eftimiades A, et al. Enhancing the Three-Dimensional Structure of Adherent Gingival Fibroblasts and Spheroids via a Fibrous Protein-Based Hydrogel Cover. Cells Tissues Organs, 2016, 202(5-6): 343-354.
34.	Becavin T, Kuchler-Bopp S, Kokten T, et al. Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development. Cell Tissue Res, 2016, 366(3): 601-615.
35.	Charles-de-Sá L, Gontijo-de-Amorim NF, Maeda Takiya C, et al. Antiaging treatment of the facial skin by fat graft and adipose-derived stem cells. Plast Reconstr Surg, 2015, 135(4): 999-1009.
36.	Xie L, Mao M, Zhou L, et al. Spheroid Mesenchymal Stem Cells and Mesenchymal Stem Cell-Derived Microvesicles: Two Potential Therapeutic Strategies. Stem Cells Dev, 2016, 25(3): 203-213.
37.	Palomäki S, Pietilä M, Laitinen S, et al. HIF-1α is upregulated in human mesenchymal stem cells. Stem Cells, 2013, 31(9): 1902-1909.
38.	Barbeau DJ, La KT, Kim DS, et al. Early growth response-2 signaling mediates immunomodulatory effects of human multipotential stromal cells. Stem Cells Dev, 2014, 23(2): 155-166.
39.	Zhang Q, Nguyen AL, Shi S, et al. Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis. Stem Cells Dev, 2012, 21(6): 937-947.
40.	Mineda K, Feng J, Ishimine H, et al. Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel. Stem Cells Transl Med, 2015, 4(12): 1511-1522.
41.	Guo L, Ge J, Zhou Y, et al. Three-dimensional spheroid-cultured mesenchymal stem cells devoid of embolism attenuate brain stroke injury after intra-arterial injection. Stem Cells Dev, 2014, 23(9): 978-989.
42.	Cesarz Z, Tamama K. Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int, 2016, 2016: 9176357.
43.	Ho SS, Murphy KC, Binder BY, et al. Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels. Stem Cells Transl Med, 2016, 5(6): 773-781.
44.	Kuk M, Kim Y, Lee SH, et al. Osteogenic Ability of Canine Adipose-Derived Mesenchymal Stromal Cell Sheets in Relation to Culture Time. Cell Transplant, 2016, 25(7): 1415-1422.
45.	Obara C, Tomiyama K, Takizawa K, et al. Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates. Cell Tissue Res, 2016, 366(1): 113-127.

1. Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968, 6(2): 230-247.
2. Sart S, Schneider YJ, Agathos SN. Ear mesenchymal stem cells: an efficient adult multipotent cell population fit for rapid and scalable expansion. J Biotechnol, 2009, 139(4): 291-299.
3. Murphy KC, Fang SY, Leach JK. Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell Tissue Res, 2014, 357(1): 91-99.
4. Galipeau J. The mesenchymal stromal cells dilemma——does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy, 2013, 15(1): 2-8.
5. Mitchell JB, Mcintosh K, Zvonic S, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromalassociated and stem cell-associated markers. Stem Cells, 2006, 24(2): 376-385.
6. Bartosh TJ, Ylostalo JH, Mohammadipoor A, et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A, 2010, 107(31): 13724-13729.
7. Steinberg MS. Cell-cell recognition in multicellular assembly: levels of specificity. Symp Soc Exp Biol, 1978, 32: 25-49.
8. Foty R. A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp, 2011, (51). pii: 2720.
9. Huang GS, Dai LG, Yen BL, et al. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials, 2011, 32(29): 6929-6945.
10. Schmidt S, Friedl P. Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res, 2010, 339(1): 83-92.
11. Li Y, Guo G, Li L, et al. Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res, 2015, 360(2): 297-307.
12. Yeh HY, Liu BH, Sieber M, et al. Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes. BMC Genomics, 2014, 15: 10.
13. Shoham N, Gefen A. Mechanotransduction in adipocytes. J Biomech, 2012, 45(1): 1-8.
14. Shoham N, Gefen A. The influence of mechanical stretching on mitosis, growth, and adipose conversion in adipocyte cultures. Biomech Model Mechanobiol, 2012, 11(7): 1029-1045.
15. Young DA, Choi YS, Engler AJ, et al. Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue. Biomaterials, 2013, 34(34): 8581-8588.
16. Tran TD, Yao S, Hsu WH, et al. Arginine vasopressin inhibits adipogenesis in human adipose-derived stem cells. Mol Cell Endocrinol, 2015, 406: 1-9.
17. Cheng NC, Wang S, Young TH. The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials, 2012, 33(6): 1748-1758.
18. Cheng NC, Chen SY, Li JR, et al. Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med, 2013, 2(8): 584-594.
19. Lee JH, Han YS, Lee SH. Long-duration three-dimensional spheroid culture promotes angiogenic activities of adipose-derived mesenchymal stem cells. Biomol Ther (Seoul), 2016, 24(3): 260-267.
20. Bartosh TJ, Ylöstalo JH, Bazhanov N, et al. Dynamic compaction of human mesenchymal stem/precursor cells (MSC) into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6 and STC1). Stem Cells, 2013, 31(11): 2443-2456.
21. Tamama K, Kawasaki H, Kerpedjieva SS, et al. Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition. J Cell Biochem, 2011, 112(3): 804-817.
22. Ylöstalo JH, Bartosh TJ, Coble K, et al. Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype. Stem Cells, 2012, 30(10): 2283-2296.
23. Frith JE, Thomson B, Genever PG. Dynamic threedimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods, 2010, 16(4): 735-749.
24. Lee EJ, Park SJ, Kang SK, et al. Spherical bullet formation via E-cadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction. Mol Ther, 2012, 20(7): 1424-1433.
25. Jackson WM, Nesti LJ, Tuan RS. Mesenchymal stem cell therapy for attenuation of scar formation during wound healing. Stem Cell Res Ther, 2012, 3(3): 20.
26. Peng R, Yao X, Cao B, et al. The effect of culture conditions on the adipogenic and osteogenic inductions of mesenchymal stem cells on micropatterned surfaces. Biomaterials, 2012, 33(26): 6008-6019.
27. Zhong Z, Williams BO. Integration of cellular adhesion and Wnt signaling: Interactions between N-cadherin and LRP5 and their role in regulating bone mass. J Bone Miner Res, 2012, 27(9): 1849-1851.
28. Alan T, Tufan AC. C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions. J Cell Biochem, 2008, 105(1): 227-235.
29. Hay E, Buczkowski T, Marty C, et al. Peptide-based mediated disruption of N-cadherin-LRP5/6 interaction promotes Wnt signaling and bone formation. J Bone Miner Res, 2012, 27(9): 1852-1863.
30. Hsu SH, Huang GS. Substrate-dependent Wnt signaling in MSC differentiation within biomaterial-derived 3D spheroids. Biomaterials, 2013, 34(20): 4725-4738.
31. Yeganeh A, Stelmack GL, Fandrich RR, et al. Connexin 43 phosphorylation and degradation are required for adipogenesis. Biochim Biophys Acta, 2012, 1823(10): 1731-1744.
32. Van Winkle AP, Gates ID, Kallos MS. Mass transfer limitations in embryoid bodies during human embryonic stem cell differentiation. Cells Tissues Organs, 2012, 196(1): 34-47.
33. Kaufman G, Nunes L, Eftimiades A, et al. Enhancing the Three-Dimensional Structure of Adherent Gingival Fibroblasts and Spheroids via a Fibrous Protein-Based Hydrogel Cover. Cells Tissues Organs, 2016, 202(5-6): 343-354.
34. Becavin T, Kuchler-Bopp S, Kokten T, et al. Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development. Cell Tissue Res, 2016, 366(3): 601-615.
35. Charles-de-Sá L, Gontijo-de-Amorim NF, Maeda Takiya C, et al. Antiaging treatment of the facial skin by fat graft and adipose-derived stem cells. Plast Reconstr Surg, 2015, 135(4): 999-1009.
36. Xie L, Mao M, Zhou L, et al. Spheroid Mesenchymal Stem Cells and Mesenchymal Stem Cell-Derived Microvesicles: Two Potential Therapeutic Strategies. Stem Cells Dev, 2016, 25(3): 203-213.
37. Palomäki S, Pietilä M, Laitinen S, et al. HIF-1α is upregulated in human mesenchymal stem cells. Stem Cells, 2013, 31(9): 1902-1909.
38. Barbeau DJ, La KT, Kim DS, et al. Early growth response-2 signaling mediates immunomodulatory effects of human multipotential stromal cells. Stem Cells Dev, 2014, 23(2): 155-166.
39. Zhang Q, Nguyen AL, Shi S, et al. Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis. Stem Cells Dev, 2012, 21(6): 937-947.
40. Mineda K, Feng J, Ishimine H, et al. Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel. Stem Cells Transl Med, 2015, 4(12): 1511-1522.
41. Guo L, Ge J, Zhou Y, et al. Three-dimensional spheroid-cultured mesenchymal stem cells devoid of embolism attenuate brain stroke injury after intra-arterial injection. Stem Cells Dev, 2014, 23(9): 978-989.
42. Cesarz Z, Tamama K. Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int, 2016, 2016: 9176357.
43. Ho SS, Murphy KC, Binder BY, et al. Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels. Stem Cells Transl Med, 2016, 5(6): 773-781.
44. Kuk M, Kim Y, Lee SH, et al. Osteogenic Ability of Canine Adipose-Derived Mesenchymal Stromal Cell Sheets in Relation to Culture Time. Cell Transplant, 2016, 25(7): 1415-1422.
45. Obara C, Tomiyama K, Takizawa K, et al. Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates. Cell Tissue Res, 2016, 366(1): 113-127.

Chinese Journal of Reparative and Reconstructive Surgery

Research progress and clinical prospect of three-dimensional spheroid culture of mesenchymal stem cells

Abstract Full text Figures/Tables Video References Cited by

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