缺血性腦中風為臨床上最常見的中風類別，其主要原因為腦血管栓塞所導致。目前最常用來治療腦中風的方式為使用血栓溶解藥物，但腦中風區域的神經細胞在中風後即開始壞死，且需要長期的修復時間，僅用血栓溶解藥物無法對於病患神經功能給予太大的改善。為了提升神經功能的新生及修復，細胞療法已被認為是具展望性的治療方式，但因細胞植入後缺乏有效的結構支持以及腦內發炎的環境限制了細胞療法的療效；先前研究已證實三維幹細胞球體可透過保存細胞間及細胞外基質的連結而提高其臨床應用潛力，並可有效治療下肢缺血的小鼠，但目前針對缺血性腦中風尚未有任何研究。在本篇研究中，我們建立了以間葉幹細胞與內皮細胞共培養的三維幹細胞球體系統，並與傳統二維培養幹細胞的療法進行比較，分析其治療效益。在體外實驗中，我們以免疫螢光染色證明三維幹細胞球體可藉由分泌更多的促神經生長、促存活及血管新生因子以達成神經保護的作用；除此之外，我們發現三維幹細胞球體的條件培養液可有效提升神經細胞的存活、分化及促進血管新生。在動物實驗中，我們在小鼠腦中風後將三維幹細胞球體施打到小鼠腦受損區域，並分析植入細胞的留存情形。實驗結果顯示，相較於二維培養的幹細胞，三維幹細胞球體可完整滯留於損傷區域。透過動物行為及腦受損區域染色，可觀察到小鼠的運動行為明顯獲得改善，且腦受損區域有明顯縮小。由組織切片免疫染色結果可知，三維幹細胞球體可有效抑制腦受損區域星狀膠質細胞及微膠細胞之活化，並能夠促進腦內源性神經幹細胞的聚集而加速神經再生，以及促進腦損傷區域周圍的血管新生。綜合以上結果，三維幹細胞球體可促進神經保護作用並促進神經再生及血管新生，期望未來可將三維幹細胞球體應用於缺血性腦中風的細胞療法，提升臨床治療效果。

Ischemic stroke is one of the most frequent causes of death and disability globally. The cause of ischemic stroke is due to the plaque rupture of atherosclerotic arteries which supply the brain. To date, no treatment is available that would grant functional recovery when administered in the post-ischemic phase. Therefore, the novel therapeutic strategies for enhancing the repair of neural structures and the recovery of functions are urgently warranted. In this work, we aim to employ a stem cell-based approach for treating ischemic stroke. Three-dimensional (3D) spheroids of human mesenchymal stem cells and human umbilical vein endothelial cells, which are capable of secreting neuroprotective agents and inducing therapeutic angiogenesis, are constructed using a methylcellulose hydrogel system. Our in vitro study reveals that the developed 3D stem cell spheroids can secrete several neurotrophic and pro-survival, pro-angiogenic factors, thus holding great neuroprotective and neovascularization potential. In animal study, a surgically established mouse model of ischemic stroke is employed for evaluating the therapeutic efficacy of the fabricated 3D stem cell spheroids. Our in vivo study indicates that transplantation of 3D stem cell spheroids into mice with ischemic stroke significantly promotes functional recovery, with the potential of reducing lesion area. Moreover, 3D stem cell spheroids may induce a conducive environment for neural repair through the downregulation of macrophage recruitment and astrocyte activity into peri-infarct tissue, facilitating neurogenesis and angiogenesis. These experimental data highlight the fabrication of 3D stem cell spheroids may represent a novel therapeutic approach of stem cell based therapy for ischemic stroke.

1.Johnson, C.O., et al., Global, regional, and national burden of stroke, 1990-2013;2016: a systematic analysis for the Global Burden of Disease Study 2016. The Lancet Neurology, 2019. 18(5): p. 439-458.
2.Feigin, V.L., et al., Global, Regional, and Country-Specific Lifetime Risks of Stroke, 1990 and 2016. N Engl J Med, 2018. 379(25): p. 2429-2437.
3.Zhang, D., et al., Wnt-3a alleviates neuroinflammation after ischemic stroke by modulating the responses of microglia/macrophages and astrocytes. Int Immunopharmacol, 2019. 75: p. 105760.
4.Shaaban, A.M. and A.J. Duerinckx, Wall shear stress and early atherosclerosis: a review. AJR Am J Roentgenol, 2000. 174(6): p. 1657-65.
5.Campbell, B.C.V., et al., Ischaemic stroke. Nat Rev Dis Primers, 2019. 5(1): p. 70.
6.Mozaffarian, D., et al., Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation, 2016. 133(4): p. e38-360.
7.Bernhardt, J., et al., Agreed definitions and a shared vision for new standards in stroke recovery research: The Stroke Recovery and Rehabilitation Roundtable taskforce. Int J Stroke, 2017. 12(5): p. 444-450.
8.Howells, D.W., et al., Different strokes for different folks: the rich diversity of animal models of focal cerebral ischemia. J Cereb Blood Flow Metab, 2010. 30(8): p. 1412-31.
9.Anrather, J. and C. Iadecola, Inflammation and Stroke: An Overview. Neurotherapeutics, 2016. 13(4): p. 661-670.
10.Li, Q. and B.A. Barres, Microglia and macrophages in brain homeostasis and disease. Nat Rev Immunol, 2018. 18(4): p. 225-242.
11.Otxoa-de-Amezaga, A., et al., Microglial cell loss after ischemic stroke favors brain neutrophil accumulation. Acta Neuropathol, 2019. 137(2): p. 321-341.
12.Patel, A.R., et al., Microglia and ischemic stroke: a double-edged sword. Int J Physiol Pathophysiol Pharmacol, 2013. 5(2): p. 73-90.
13.Tam, W.Y. and C.H.E. Ma, Bipolar/rod-shaped microglia are proliferating microglia with distinct M1/M2 phenotypes. Scientific reports, 2014. 4: p. 7279-7279.
14.Liddelow, S.A., et al., Neurotoxic reactive astrocytes are induced by activated microglia. Nature, 2017. 541(7638): p. 481-487.
15.Tarassishin, L., H.S. Suh, and S.C. Lee, LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14. Glia, 2014. 62(6): p. 999-1013.
16.Choudhury, G.R. and S. Ding, Reactive astrocytes and therapeutic potential in focal ischemic stroke. Neurobiol Dis, 2016. 85: p. 234-244.
17.Machado-Pereira, M., et al., Anti-Inflammatory Strategy for M2 Microglial Polarization Using Retinoic Acid-Loaded Nanoparticles. Mediators of inflammation, 2017. 2017: p. 6742427-6742427.
18.Lu, J., A. Manaenko, and Q. Hu, Targeting Adult Neurogenesis for Poststroke Therapy. Stem Cells Int, 2017. 2017: p. 5868632.
19.Koh, S.H. and H.H. Park, Neurogenesis in Stroke Recovery. Transl Stroke Res, 2017. 8(1): p. 3-13.
20.Yu, Q.-J., et al., Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke. Neural regeneration research, 2015. 10(11): p. 1882-1891.
21.Navaratna, D., et al., Mechanisms and targets for angiogenic therapy after stroke. Cell adhesion & migration, 2009. 3(2): p. 216-223.
22.Wechsler, L.R., et al., Cell Therapy for Chronic Stroke. Stroke, 2018. 49(5): p. 1066-1074.
23.Rajkovic, O., G. Potjewyd, and E. Pinteaux, Regenerative Medicine Therapies for Targeting Neuroinflammation After Stroke. Front Neurol, 2018. 9: p. 734.
24.Birkenmeier, R.L., E.M. Prager, and C.E. Lang, Translating animal doses of task-specific training to people with chronic stroke in 1-hour therapy sessions: a proof-of-concept study. Neurorehabilitation and neural repair, 2010. 24(7): p. 620-635.
25.Richards, C.L., F. Malouin, and S. Nadeau, Stroke rehabilitation: clinical picture, assessment, and therapeutic challenge. Prog Brain Res, 2015. 218: p. 253-80.
26.Michinaga, S. and Y. Koyama, Pathogenesis of brain edema and investigation into anti-edema drugs. Int J Mol Sci, 2015. 16(5): p. 9949-75.
27.Simard, J.M., et al., Brain oedema in focal ischaemia: molecular pathophysiology and theoretical implications. Lancet Neurol, 2007. 6(3): p. 258-68.
28.Kahle, K.T., et al., Molecular mechanisms of ischemic cerebral edema: role of electroneutral ion transport. Physiology (Bethesda), 2009. 24: p. 257-65.
29.Emberson, J., et al., Effect of treatment delay, age, and stroke severity on the effects of intravenous thrombolysis with alteplase for acute ischaemic stroke: a meta-analysis of individual patient data from randomised trials. Lancet, 2014. 384(9958): p. 1929-35.
30.Muchada, M., et al., Impact of time to treatment on tissue-type plasminogen activator-induced recanalization in acute ischemic stroke. Stroke, 2014. 45(9): p. 2734-8.
31.Hacke, W., et al., Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med, 2008. 359(13): p. 1317-29.
32.Loane, D.J. and A.I. Faden, Neuroprotection for traumatic brain injury: translational challenges and emerging therapeutic strategies. Trends Pharmacol Sci, 2010. 31(12): p. 596-604.
33.Yasuhara, T., et al., Cell therapy for central nervous system disorders: Current obstacles to progress. CNS Neurosci Ther, 2019.
34.Wei, L., et al., Stem cell transplantation therapy for multifaceted therapeutic benefits after stroke. Prog Neurobiol, 2017. 157: p. 49-78.
35.Eckert, A., et al., Bystander Effect Fuels Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells to Quickly Attenuate Early Stage Neurological Deficits After Stroke. Stem Cells Transl Med, 2015. 4(7): p. 841-51.
36.Jian, W.H., et al., Glycosaminoglycan-based hybrid hydrogel encapsulated with polyelectrolyte complex nanoparticles for endogenous stem cell regulation in central nervous system regeneration. Biomaterials, 2018. 174: p. 17-30.
37.Bao, X., et al., Transplantation of human bone marrow-derived mesenchymal stem cells promotes behavioral recovery and endogenous neurogenesis after cerebral ischemia in rats. Brain Res, 2011. 1367: p. 103-13.
38.Andres, R.H., et al., Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain. Brain, 2011. 134(Pt 6): p. 1777-89.
39.Oki, K., et al., Human-induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain. Stem Cells, 2012. 30(6): p. 1120-33.
40.De Feo, D., et al., Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection. Curr Opin Neurol, 2012. 25(3): p. 322-33.
41.Boese, A.C., et al., Neural stem cell therapy for subacute and chronic ischemic stroke. Stem Cell Research & Therapy, 2018. 9(1): p. 154.
42.Sammali, E., et al., Intravenous infusion of human bone marrow mesenchymal stromal cells promotes functional recovery and neuroplasticity after ischemic stroke in mice. Sci Rep, 2017. 7(1): p. 6962.
43.Aguado, B.A., et al., Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers. Tissue Eng Part A, 2012. 18(7-8): p. 806-15.
44.Wahlberg, B., et al., Ex vivo biomechanical characterization of syringe-needle ejections for intracerebral cell delivery. Sci Rep, 2018. 8(1): p. 9194.
45.He, N., et al., Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment. Sci Rep, 2015. 5: p. 10902.
46.Sandvig, I., et al., Strategies to Enhance Implantation and Survival of Stem Cells After Their Injection in Ischemic Neural Tissue. Stem Cells Dev, 2017. 26(8): p. 554-565.
47.Baldari, S., et al., Challenges and Strategies for Improving the Regenerative Effects of Mesenchymal Stromal Cell-Based Therapies. Int J Mol Sci, 2017. 18(10).
48.Das, R., et al., The role of hypoxia in bone marrow-derived mesenchymal stem cells: considerations for regenerative medicine approaches. Tissue Eng Part B Rev, 2010. 16(2): p. 159-68.
49.Carrière, A., et al., Preconditioning by mitochondrial reactive oxygen species improves the proangiogenic potential of adipose-derived cells-based therapy. Arterioscler Thromb Vasc Biol, 2009. 29(7): p. 1093-9.
50.Canfield, S.G., et al., Marked hyperglycemia attenuates anesthetic preconditioning in human-induced pluripotent stem cell-derived cardiomyocytes. Anesthesiology, 2012. 117(4): p. 735-44.
51.Song, H., et al., Tissue transglutaminase is essential for integrin-mediated survival of bone marrow-derived mesenchymal stem cells. Stem Cells, 2007. 25(6): p. 1431-8.
52.Park, J.S., et al., Engineering mesenchymal stem cells for regenerative medicine and drug delivery. Methods, 2015. 84: p. 3-16.
53.Moshayedi, P., et al., Systematic optimization of an engineered hydrogel allows for selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain. Biomaterials, 2016. 105: p. 145-155.
54.George, P.M., et al., Engineered stem cell mimics to enhance stroke recovery. Biomaterials, 2018. 178: p. 63-72.
55.Ansari, S., et al., Hydrogel elasticity and microarchitecture regulate dental-derived mesenchymal stem cell-host immune system cross-talk. Acta Biomater, 2017. 60: p. 181-189.
56.Alemdar, N., et al., Oxygen-Generating Photo-Cross-Linkable Hydrogels Support Cardiac Progenitor Cell Survival by Reducing Hypoxia-Induced Necrosis. ACS Biomaterials Science & Engineering, 2017. 3(9): p. 1964-1971.
57.Shin, J., et al., Three-Dimensional Electroconductive Hyaluronic Acid Hydrogels Incorporated with Carbon Nanotubes and Polypyrrole by Catechol-Mediated Dispersion Enhance Neurogenesis of Human Neural Stem Cells. Biomacromolecules, 2017. 18(10): p. 3060-3072.
58.Mironi-Harpaz, I., et al., Photopolymerization of cell-encapsulating hydrogels: crosslinking efficiency versus cytotoxicity. Acta Biomater, 2012. 8(5): p. 1838-48.
59.Ophelders, D.R., et al., Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia. Stem Cells Transl Med, 2016. 5(6): p. 754-63.
60.Dabrowska, S., et al., Human bone marrow mesenchymal stem cell-derived extracellular vesicles attenuate neuroinflammation evoked by focal brain injury in rats. J Neuroinflammation, 2019. 16(1): p. 216.
61.Baker, E.W., et al., Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model. Sci Rep, 2017. 7(1): p. 10075.
62.Wakabayashi, K., et al., Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model. J Neurosci Res, 2010. 88(5): p. 1017-25.
63.Sheikh, A.M., et al., A Mesenchymal stem cell line (B10) increases angiogenesis in a rat MCAO model. Exp Neurol, 2019. 311: p. 182-193.
64.Zanier, E.R., et al., Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma. Crit Care Med, 2011. 39(11): p. 2501-10.
65.Mao, F., et al., Mesenchymal stem cells and their therapeutic applications in inflammatory bowel disease. Oncotarget, 2017. 8(23): p. 38008-38021.
66.Gutierrez-Fernandez, M., et al., Effects of intravenous administration of allogenic bone marrow- and adipose tissue-derived mesenchymal stem cells on functional recovery and brain repair markers in experimental ischemic stroke. Stem Cell Res Ther, 2013. 4(1): p. 11.
67.Zanier, E.R., et al., Bone marrow mesenchymal stromal cells drive protective M2 microglia polarization after brain trauma. Neurotherapeutics, 2014. 11(3): p. 679-95.
68.Ohtaki, H., et al., Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses. Proc Natl Acad Sci U S A, 2008. 105(38): p. 14638-43.
69.Ohab, J.J., et al., A neurovascular niche for neurogenesis after stroke. J Neurosci, 2006. 26(50): p. 13007-16.
70.Wu, C.C., et al., Human umbilical vein endothelial cells protect against hypoxic-ischemic damage in neonatal brain via stromal cell-derived factor 1/C-X-C chemokine receptor type 4. Stroke, 2013. 44(5): p. 1402-9.
71.Jain, R.K., Molecular regulation of vessel maturation. Nat Med, 2003. 9(6): p. 685-93.
72.Bartosh, T.J., 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): p. 13724-9.
73.Park, I.S., J.W. Rhie, and S.H. Kim, A novel three-dimensional adipose-derived stem cell cluster for vascular regeneration in ischemic tissue. Cytotherapy, 2014. 16(4): p. 508-22.
74.Bhang, S.H., et al., Transplantation of cord blood mesenchymal stem cells as spheroids enhances vascularization. Tissue Eng Part A, 2012. 18(19-20): p. 2138-47.
75.Guo, L., 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): p. 978-89.
76.Petrenko, Y., E. Syková, and Š. Kubinová, The therapeutic potential of three-dimensional multipotent mesenchymal stromal cell spheroids. Stem cell research & therapy, 2017. 8(1): p. 94-94.
77.Seda Tiğli, R., A. Karakeçili, and M. Gümüşderelioğlu, In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree. J Mater Sci Mater Med, 2007. 18(9): p. 1665-74.
78.Cheng, N.C., S. Wang, and T.H. Young, The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials, 2012. 33(6): p. 1748-58.
79.Chen, C.H., et al., Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels. Biomacromolecules, 2006. 7(3): p. 736-43.
80.Liang, H.F., et al., Novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate as a pH-sensitive hydrogel. Biomacromolecules, 2004. 5(5): p. 1917-25.
81.Lee, W.Y., et al., The use of injectable spherically symmetric cell aggregates self-assembled in a thermo-responsive hydrogel for enhanced cell transplantation. Biomaterials, 2009. 30(29): p. 5505-13.
82.Wang, C.C., et al., Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty. Stem Cells, 2009. 27(3): p. 724-32.
83.Huang, C.C., et al., Injectable cell constructs fabricated via culture on a thermoresponsive methylcellulose hydrogel system for the treatment of ischemic diseases. Adv Healthc Mater, 2014. 3(8): p. 1133-48.
84.Chen, D.Y., et al., Three-dimensional cell aggregates composed of HUVECs and cbMSCs for therapeutic neovascularization in a mouse model of hindlimb ischemia. Biomaterials, 2013. 34(8): p. 1995-2004.
85.Yu, C.P., et al., Enhancement of Subcutaneously Transplanted beta Cell Survival Using 3D Stem Cell Spheroids with Proangiogenic and Prosurvival Potential. Adv Biosyst, 2020. 4(3): p. e1900254.
86.Chen, C.H., et al., Construction and characterization of fragmented mesenchymal-stem-cell sheets for intramuscular injection. Biomaterials, 2007. 28(31): p. 4643-51.
87.Tasca, C.I., T. Dal-Cim, and H. Cimarosti, In vitro oxygen-glucose deprivation to study ischemic cell death. Methods Mol Biol, 2015. 1254: p. 197-210.
88.Llovera, G., et al., Modeling stroke in mice: permanent coagulation of the distal middle cerebral artery. J Vis Exp, 2014(89): p. e51729.
89.Tureyen, K., et al., Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques. J Neurosci Methods, 2004. 139(2): p. 203-7.
90.Swanson, R.A., et al., A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab, 1990. 10(2): p. 290-3.
91.Nih, L.R., et al., Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke. Nat Mater, 2018. 17(7): p. 642-651.
92.Clarkson, A.N., et al., Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke. Nature, 2010. 468(7321): p. 305-9.
93.Lee, W.-Y., et al., Core–shell cell bodies composed of human cbMSCs and HUVECs for functional vasculogenesis. Biomaterials, 2011. 32(33): p. 8446-8455.
94.Kenmuir, C.L. and L.R. Wechsler, Update on cell therapy for stroke. Stroke and Vascular Neurology, 2017. 2(2): p. 59.
95.Quittet, M.S., et al., Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat. Acta Biomater, 2015. 15: p. 77-88.