Human Neurospheroid Arrays for In Vitro Studies of Alzheimer’s Disease

Neurospheroids are commonly used for in vitro disease modeling and drug screening. However, the heterogeneity in size of the neurospheroids mixtures available through current methods limits their utility when employed for basic mechanistic studies of neurodegenerative diseases or screening for new interventions. Here, we generate neurospheroids from immortalized neural progenitor cells and human induced pluripotent stem cells that are uniform in size, into large-scale arrays. In proof of concept experiments, we validate the neurospheroids array as a sensitive and robust tool for screening compounds over extended time. We show that when suspended in three-dimensional extracellular matrix up to several weeks, the stem cell-derived neurospheroids display extensive neurite outgrowth and extend thick bundles of dendrites outward. We also cultivate genetically-engineered stem cell-derived neurospheroids with familial Alzheimer’s disease mutations for eight weeks in our microarray system. Interestingly, we observed robust accumulation of amyloid-β and phosphorylated tau, key hallmarks of Alzheimer’s disease. Overall, our in vitro model for engineering neurospheroid arrays is a valuable tool for studying complex neurodegenerative diseases and accelerating drug discovery.

[1]  Z. Hall Cancer , 1906, The Hospital.

[2]  A. Baer The genetic perspective , 1977 .

[3]  G. Curt,et al.  The pharmacology and clinical use of methotrexate. , 1983, The New England journal of medicine.

[4]  M. Weinblatt,et al.  Efficacy of low-dose methotrexate in rheumatoid arthritis. , 1985, The New England journal of medicine.

[5]  R. Tanzi,et al.  Twenty Years of the Alzheimer’s Disease Amyloid Hypothesis: A Genetic Perspective , 2005, Cell.

[6]  Robert W Berry,et al.  Tau, tangles, and Alzheimer's disease. , 2005, Biochimica et biophysica acta.

[7]  Michael S. Krzemnicki,et al.  Challenges and perspectives , 2009 .

[8]  J. Götz,et al.  Animal models of Alzheimer's disease and frontotemporal dementia , 2008, Nature Reviews Neuroscience.

[9]  Krishanu Saha,et al.  Technical challenges in using human induced pluripotent stem cells to model disease. , 2009, Cell stem cell.

[10]  Ali Khademhosseini,et al.  Controlled-size embryoid body formation in concave microwell arrays. , 2010, Biomaterials.

[11]  G. Whitesides,et al.  Soft lithography for micro- and nanoscale patterning , 2010, Nature Protocols.

[12]  Shoji Takeuchi,et al.  A neurospheroid network-stamping method for neural transplantation to the brain. , 2010, Biomaterials.

[13]  Y. Hatano,et al.  Folate antagonist, methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina , 2010, Neuroscience Letters.

[14]  Hiroyuki Miyoshi,et al.  Self-formation of functional adenohypophysis in three-dimensional culture , 2011, Nature.

[15]  Sung-Ho Kim,et al.  Neurotoxicity of methotrexate to hippocampal cells in vivo and in vitro. , 2011, Biochemical pharmacology.

[16]  J. Chin,et al.  Selecting a mouse model of Alzheimer's disease. , 2010, Methods in molecular biology.

[17]  S. Leurgans,et al.  Cognitive decline in prodromal Alzheimer disease and mild cognitive impairment. , 2011, Archives of neurology.

[18]  Erika Pastrana,et al.  Eyes wide open: a critical review of sphere-formation as an assay for stem cells. , 2011, Cell stem cell.

[19]  A. Kriegstein,et al.  Development and Evolution of the Human Neocortex , 2011, Cell.

[20]  Kristopher L. Nazor,et al.  Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells , 2012, Nature.

[21]  R. Tanzi,et al.  iPSCs to the rescue in Alzheimer's research. , 2012, Cell stem cell.

[22]  L. Goldstein,et al.  Alzheimer's disease in a dish: promises and challenges of human stem cell models. , 2012, Human molecular genetics.

[23]  H. Okano,et al.  Establishment of Induced Pluripotent Stem Cells from Centenarians for Neurodegenerative Disease Research , 2012, PloS one.

[24]  Zhong Zhong,et al.  Prevention of β-amyloid induced toxicity in human iPS cell-derived neurons by inhibition of Cyclin-dependent kinases and associated cell cycle events. , 2013, Stem cell research.

[25]  W. Thies,et al.  2013 Alzheimer's disease facts and figures , 2013, Alzheimer's & Dementia.

[26]  J. Kessler,et al.  Stem cell derived basal forebrain cholinergic neurons from Alzheimer’s disease patients are more susceptible to cell death , 2014, Molecular Neurodegeneration.

[27]  Katsuhiro Yoshikawa,et al.  Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Aβ and differential drug responsiveness. , 2013, Cell stem cell.

[28]  Kun Zhang,et al.  The presenilin-1 ΔE9 mutation results in reduced γ-secretase activity, but not total loss of PS1 function, in isogenic human stem cells. , 2013, Cell reports.

[29]  Adrian Ranga,et al.  Drug discovery through stem cell-based organoid models. , 2014, Advanced drug delivery reviews.

[30]  Joana Galvao,et al.  Unexpected low‐dose toxicity of the universal solvent DMSO , 2014, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[31]  D. Selkoe,et al.  The familial Alzheimer's disease APPV717I mutation alters APP processing and Tau expression in iPSC-derived neurons. , 2014, Human molecular genetics.

[32]  Ottavio Arancio,et al.  Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors , 2014, PloS one.

[33]  Basavaraj Hooli,et al.  A three-dimensional human neural cell culture model of Alzheimer’s disease , 2014, Nature.

[34]  David L Kaplan,et al.  Bioengineered functional brain-like cortical tissue , 2014, Proceedings of the National Academy of Sciences.

[35]  S. Socorro,et al.  Paradoxical and contradictory effects of imatinib in two cell line models of hormone‐refractory prostate cancer , 2015, The Prostate.

[36]  R. Tanzi,et al.  γ‐Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[37]  K. Blennow,et al.  APP Metabolism Regulates Tau Proteostasis in Human Cerebral Cortex Neurons , 2015, Cell reports.

[38]  Diane Hoffman-Kim,et al.  Three-Dimensional Neural Spheroid Culture: An In Vitro Model for Cortical Studies. , 2015, Tissue engineering. Part C, Methods.

[39]  Young Hye Kim,et al.  Alzheimer's in 3D culture: Challenges and perspectives , 2015, BioEssays : news and reviews in molecular, cellular and developmental biology.

[40]  S. Conroy,et al.  Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9 , 2015, PloS one.

[41]  Young Hye Kim,et al.  A 3D human neural cell culture system for modeling Alzheimer's disease , 2015, Nature Protocols.

[42]  R. Tanzi,et al.  Recapitulating amyloid β and tau pathology in human neural cell culture models: clinical implications. , 2015, US neurology.

[43]  Jung Keun Hyun,et al.  Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as an in vitro model of Alzheimer's disease. , 2015, Lab on a chip.

[44]  DoYeun Park,et al.  Networked neural spheroid by neuro-bundle mimicking nervous system created by topology effect , 2015, Molecular Brain.

[45]  Norie Tooi,et al.  The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells. , 2016, Biochemical and biophysical research communications.

[46]  Christine Van Broeckhoven,et al.  The genetic landscape of Alzheimer disease: clinical implications and perspectives , 2015, Genetics in Medicine.

[47]  Madeline A. Lancaster,et al.  Dishing out mini-brains: Current progress and future prospects in brain organoid research , 2016, Developmental biology.

[48]  Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a M146I mutation in PSEN1. , 2016, Stem cell research.

[49]  G. Waldemar,et al.  Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1. , 2016, Stem cell research.

[50]  Marc Tessier-Lavigne,et al.  Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9 , 2016, Nature.

[51]  L. Tsai,et al.  Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer’s Disease Phenotypes , 2016, PloS one.

[52]  B. Strooper,et al.  Alzheimer's Disease Mechanisms and Emerging Roads to Novel Therapeutics , 2016 .

[53]  Madeline A. Lancaster,et al.  Stem Cell Models of Human Brain Development. , 2016, Cell stem cell.

[54]  P. Arlotta,et al.  The promises and challenges of human brain organoids as models of neuropsychiatric disease , 2016, Nature Medicine.

[55]  D. Price,et al.  Building brains in a dish: Prospects for growing cerebral organoids from stem cells , 2016, Neuroscience.

[56]  Han-Kyu Lee,et al.  Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells , 2016, PloS one.

[57]  Jessica E Young,et al.  Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer's Disease Mutations. , 2016, Cell reports.

[58]  R. Bateman,et al.  Stop Alzheimer’s before it starts , 2017, Nature.

[59]  Lin Cheng,et al.  Selective and effective targeting of chronic myeloid leukemia stem cells by topoisomerase II inhibitor etoposide in combination with imatinib mesylate in vitro , 2017, Cell biology international.

[60]  C. Revnic,et al.  THE EFFECT OF SERUM FACTORS FROM PATIENTS WITH/WITHOUT ESSENTIAL HYPERTENSIOIN ON MEMBRANE RECEPTORS FROM RAT CEREBRAL CORTEX VASCULAR AND CARDIAC MUSCLE CELLS , 2017, Alzheimer's & Dementia.

[61]  Jonathan A. Bernstein,et al.  Assembly of functionally integrated human forebrain spheroids , 2017, Nature.

[62]  B. Cullen,et al.  Update on Alzheimer's Disease Therapy and Prevention Strategies. , 2017, Annual review of medicine.

[63]  Clinical Implications. , 2017, Hypertension.

[64]  Mehdi Jorfi,et al.  Three‐Dimensional Models of the Human Brain Development and Diseases , 2018, Advanced healthcare materials.