Original articles
Cheng Chen, Xin Dong, Kai-Heng Fang, Fang Yuan, Yao Hu, Min Xu, Yu Huang, Xixiang Zhang, Danjun Fang, Yan Liu. Develop a 3D neurological disease model of human cortical glutamatergic neurons using micropillar-based scaffolds[J]. Acta Pharmaceutica Sinica B, 2019, 9(3): 557-564

Develop a 3D neurological disease model of human cortical glutamatergic neurons using micropillar-based scaffolds
Cheng Chena,b,c, Xin Donga,b, Kai-Heng Fanga,b, Fang Yuana,b, Yao Hua,b, Min Xua,b, Yu Huangc, Xixiang Zhangd, Danjun Fangb, Yan Liua,b,e
a Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China;
b Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China;
c Department of Biological Engineering, Utah State University, Logan, UT 84322, USA;
d Physical Sciences and Engineering Division(PSE), King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia;
e Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
Abstract:
Establishing an effective three-dimensional (3D) in vitro culture system to better model human neurological diseases is desirable, since the human brain is a 3D structure. Here, we demonstrated the development of a polydimethylsiloxane (PDMS) pillar-based 3D scaffold that mimicked the 3D microenvironment of the brain. We utilized this scaffold for the growth of human cortical glutamatergic neurons that were differentiated from human pluripotent stem cells. In comparison with the 2D culture, we demonstrated that the developed 3D culture promoted the maturation of human cortical glutamatergic neurons by showing significantly more MAP2 and less Ki67 expression. Based on this 3D culture system, we further developed an in vitro disease-like model of traumatic brain injury (TBI), which showed a robust increase of glutamate-release from the neurons, in response to mechanical impacts, recapitulating the critical pathology of TBI. The increased glutamate-release from our 3D culture model was attenuated by the treatment of neural protective drugs, memantine or nimodipine. The established 3D in vitro human neural culture system and TBI-like model may be used to facilitate mechanistic studies and drug screening for neurotrauma or other neurological diseases.
Key words:    3D culture    Cortical glutamatergic neurons    Human pluripotent stem cells    Cell differentiation    Disease modeling    Traumatic brain injury    Neural protective drugs    Drug screening   
Received: 2018-12-24     Revised:
DOI: 10.1016/j.apsb.2019.03.004
Funds: This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA16010306), the National Natural Science Foundation of China Grants (91849117 and 81471301), Key Research and Development Program of China (2016YFC1306703), The National Jiangsu Outstanding Young Investigator Program (BK20160044, China), Jiangsu Province's Innovation Person (China), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China Project (Grant No. 17KJB180010).
Corresponding author: Danjun Fang, Yan Liu     Email:djf@njmu.edu.cn;yanliu@njmu.edu.cn
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Cheng Chen
Xin Dong
Kai-Heng Fang
Fang Yuan
Yao Hu
Min Xu
Yu Huang
Xixiang Zhang
Danjun Fang
Yan Liu

References:
1. Anderson SA, Marín O, Horn C, Jennings K, Rubenstein JL. Distinct cortical migrations from the medial and lateral ganglionic eminences. Development 2001;128:353-63.
2. Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamura K, Keleher J, et al. Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J Comp Neurol 2000;424:409-38.
3. Wilson SW, Rubenstein JL. Induction and dorsoventral patterning of the telencephalon. Neuron 2000;28:641-51.
4. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861-72.
5. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-7.
6. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917-20.
7. Tao Y, Zhang SC. Neural subtype specification from human pluripotent stem cells. Cell Stem Cell 2016;19:573-86.
8. Petros TJ, Tyson JA, Anderson SA. Pluripotent stem cells for the study of CNS development. Front Mol Neurosci 2011;4:30.
9. Yamanaka S. A fresh look at iPS cells. Cell 2009;137:13-7.
10. Li XJ, Zhang X, Johnson MA, Wang ZB, LaVaute T, Zhang SC. Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells. Development 2009;136:4055-63.
11. Shi Y, Kirwan P, Smith J, Robinson HP, Livesey FJ. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci 2012;15:477-86.
12. Shi Y, Kirwan P, Smith J, MacLean G, Orkin SH, Livesey FJ. A human stem cell model of early Alzheimer's disease pathology in Down syndrome. Sci Transl Med 2012;4:124ra29.
13. Cao SY, Hu Y, Chen C, Yuan F, Xu M, Li Q, et al. Enhanced derivation of human pluripotent stem cell-derived cortical glutamatergic neurons by a small molecule. Sci Rep 2017;7:3282.
14. Frega M, Tedesco M, Massobrio P, Pesce M, Martinoia S. Network dynamics of 3D engineered neuronal cultures:a new experimental model for in-vitro electrophysiology. Sci Rep 2014;4:5489.
15. Tang-Schomer MD, White JD, Tien LW, Schmitt LI, Valentin TM, Graziano DJ, et al. Bioengineered functional brain-like cortical tissue. Proc Natl Acad Sci U S A 2014;111:13811-6.
16. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cellmatrix adhesions to the third dimension. Science 2001;294:1708-12.
17. Bosi S, Rauti R, Laishram J, Turco A, Lonardoni D, Nieus T, et al. From 2D to 3D:novel nanostructured scaffolds to investigate signalling in reconstructed neuronal networks. Sci Rep 2015;5:9562.
18. Lu HF, Lim SX, Leong MF, Narayanan K, Toh RP, Gao S, et al. Efficient neuronal differentiation and maturation of human pluripotent stem cells encapsulated in 3D microfibrous scaffolds. Biomaterials 2012;33:9179-87.
19. Carlson AL, Bennett NK, Francis NL, Halikere A, Clarke S, Moore JC, et al. Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds. Nat Commun 2016;7:10862.
20. Zhang X, Han F, Syed A, Bukhari EM, Siang BC, Yang S, et al. Fabrication of highly modulable fibrous 3D extracellular microenvironments. Biomed Microdev 2017;19:53.
21. Yuan F, Fang KH, Cao SY, Qu ZY, Qi Li, Krencik R, et al. Efficient generation of region-specific forebrain neurons from human pluripotent stem cells under highly defined condition. Sci Rep 2015;5:18550.
22. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods 2011;8:424-9.
23. Hinzman JM, Thomas TC, Burmeister JJ, Quintero JE, Huettl P, Pomerleau F, et al. Diffuse brain injury elevates tonic glutamate levels and potassium-evoked glutamate release in discrete brain regions at two days post-injury:an enzyme-based microelectrode array study. J Neurotrauma 2010;27:889-99.
24. Daschil N, Humpel C. Nifedipine and nimodipine protect dopaminergic substantia nigra neurons against axotomy-induced cell death in rat vibrosections via modulating inflammatory responses. Brain Res 2014;1581:1-11.
25. Patel TP, Ventre SC, Geddes-Klein D, Singh PK, Meaney DF. Singleneuron NMDA receptor phenotype influences neuronal rewiring and reintegration following traumatic injury. J Neurosci 2014;34:4200-13.
26. Choi SH, Kim YH, Hebisch M, Sliwinski C, Lee S, D'Avanzo C, et al. A three-dimensional human neural cell culture model of Alzheimer's disease. Nature 2014;515:274-8.
27. Jensen G, Morrill C, Huang Y. 3D tissue engineering, an emerging technique for pharmaceutical research. Acta Pharm Sin B 2018;8:756-66.