Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System

Summary Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4–5 months, even without astrocyte feeder layers.

[1]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[2]  Thomas Vierbuchen,et al.  Direct conversion of fibroblasts to functional neurons by defined factors , 2010, Nature.

[3]  Derek H. Oakley,et al.  Accelerated High-Yield Generation of Limb-Innervating Motor Neurons from Human Stem Cells , 2013, The Journal of Neuroscience.

[4]  H. Okano,et al.  Modeling human neurological disorders with induced pluripotent stem cells , 2014, Journal of neurochemistry.

[5]  D. Geschwind,et al.  Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture , 2015, Nature Methods.

[6]  Andreas Fouras,et al.  Flow Characterization of a Spinner Flask for Induced Pluripotent Stem Cell Culture Application , 2014, PloS one.

[7]  D. Melton,et al.  Generation of Functional Human Pancreatic β Cells In Vitro , 2014, Cell.

[8]  Daniele Linaro,et al.  Pyramidal Neurons Derived from Human Pluripotent Stem Cells Integrate Efficiently into Mouse Brain Circuits In Vivo , 2013, Neuron.

[9]  Yoshiki Sasai,et al.  Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. , 2008, Cell stem cell.

[10]  Su-Chun Zhang,et al.  Differentiation of spinal motor neurons from pluripotent human stem cells , 2009, Nature Protocols.

[11]  Yan Liu,et al.  Annals of the New York Academy of Sciences Human Stem Cells as a Model of Motoneuron Development and Diseases , 2022 .

[12]  F. Gage,et al.  Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes. , 2011, Molecular therapy : the journal of the American Society of Gene Therapy.

[13]  T. Bourgeron,et al.  Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening , 2013, Translational Psychiatry.

[14]  D. Roberts,et al.  Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages , 2009, Nature.

[15]  Shane R. Crandall,et al.  High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1 , 2014, Nature Communications.

[16]  Madeline A. Lancaster,et al.  Cerebral organoids model human brain development and microcephaly , 2013, Nature.

[17]  M. Gerstein,et al.  FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders , 2015, Cell.

[18]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.

[19]  Athar N. Malik,et al.  Activity-dependent regulation of inhibitory synapse development by Npas4 , 2008, Nature.

[20]  F. J. Livesey,et al.  Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks , 2012, Nature Protocols.

[21]  Fred H. Gage,et al.  Induced pluripotent stem cells: the new patient? , 2012, Nature Reviews Molecular Cell Biology.

[22]  Michael E. Greenberg,et al.  Activity-dependent neuronal signalling and autism spectrum disorder , 2013, Nature.

[23]  Takeshi Imai,et al.  SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction , 2013, Nature Neuroscience.

[24]  G. Daley,et al.  Use of differentiated pluripotent stem cells in replacement therapy for treating disease , 2014, Science.

[25]  Ya-min Wu,et al.  Directed differentiation of embryonic stem cells into motor neurons by gene , 2004 .

[26]  A. Kriegstein,et al.  Cerebral Organoids in a Dish: Progress and Prospects , 2013, Cell.

[27]  Y. Sasai.  Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. , 2013, Cell stem cell.

[28]  C. Henderson,et al.  Motor neuron diversity in development and disease. , 2010, Annual review of neuroscience.

[29]  Y. Ben-Ari.  Excitatory actions of gaba during development: the nature of the nurture , 2002, Nature Reviews Neuroscience.

[30]  Lixia Yue,et al.  Specification of Region-Specific Neurons Including Forebrain Glutamatergic Neurons from Human Induced Pluripotent Stem Cells , 2010, PloS one.

[31]  T. Graf.  Faculty Opinions recommendation of Induction of pluripotent stem cells from adult human fibroblasts by defined factors. , 2007 .

[32]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[33]  K. Eggan,et al.  Constructing and Deconstructing Stem Cell Models of Neurological Disease , 2011, Neuron.

[34]  M. Eiraku,et al.  Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues , 2011, Nature Protocols.

[35]  P. Ross,et al.  Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD , 2014, Front. Cell. Neurosci..

[36]  B. Strulovici,et al.  Induced pluripotent stem cells — opportunities for disease modelling and drug discovery , 2011, Nature Reviews Drug Discovery.

[37]  David G Hendrickson,et al.  Genome-wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy. , 2015, Cell stem cell.

[38]  S. Anderson,et al.  Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. , 2013, Cell stem cell.

[39]  N. Nakatsuji,et al.  A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production , 2014, Stem cell reports.

[40]  Daniel H. Geschwind,et al.  The Human Brain in a Dish: The Promise of iPSC-Derived Neurons , 2011, Cell.

[41]  Hynek Wichterle,et al.  A functionally characterized test set of human induced pluripotent stem cells , 2011, Nature Biotechnology.

[42]  M. Greenberg,et al.  A Biological Function for the Neuronal Activity-Dependent Component of Bdnf Transcription in the Development of Cortical Inhibition , 2008, Neuron.

[43]  L. Rubin,et al.  A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. , 2013, Cell stem cell.

[44]  M. Tomishima,et al.  Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling , 2009, Nature Biotechnology.

[45]  H. Wichterle,et al.  Directed Differentiation of Embryonic Stem Cells into Motor Neurons , 2002, Cell.

[46]  Steven W. Flavell,et al.  Genome-Wide Analysis of MEF2 Transcriptional Program Reveals Synaptic Target Genes and Neuronal Activity-Dependent Polyadenylation Site Selection , 2008, Neuron.

[47]  F. Gage,et al.  2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size , 2016, Cell stem cell.

[48]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors , 2008 .

[49]  Madeline A. Lancaster,et al.  Human cerebral organoids recapitulate gene expression programs of fetal neocortex development , 2015, Proceedings of the National Academy of Sciences.

[50]  M. Johnson,et al.  Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells , 2009, Development.