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| Name | Authors | DOI | Group |
Summary |
Associated Publications |
RRIDs used | ||||||
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iPSC editing with TALENs Resource Report Resource Website |
Ruilin Tian, Jason Hong, Martin Kampmann | DOI:10.17504/protocols.io.8dahs2e | Neurodegeneration Method Development Community, KampmannLab | University of California, San Francisco, University of California, San Francisco, University of California, San Francisco | 1 | 2019 | Ruilin Tian, Jason Hong, Martin Kampmann 2019. iPSC editing with TALENs. protocols.io https://dx.doi.org/10.17504/protocols.io.8dahs2e | 2021-04-15 09:16:34 | ||||
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Screening Edited iPSC Clones Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.yazfsf6 | Neurodegeneration Method Development Community | Screening Edited iPSC ClonesExpanding and gDNA Extraction from iPSC ClonesNote: It takes approximately 1 week for iPSC picked into 96 well plates to be sufficiently confluent for freezing and screening. For screening purposes, a fraction of the cells picked into one well of a 96 well plate will be saved for DNA Extraction and the remaining will be kept in culture or frozen down. | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Screening Edited iPSC Clones. protocols.io https://dx.doi.org/10.17504/protocols.io.yazfsf6 | 2021-04-15 09:16:19 | |||
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mRNA Synthesis and Transfection Resource Report Resource Website |
Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying | Neurodegeneration Method Development Community | Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology; Hugo W. Moser Research Institute at Kennedy Krieger, Department of Physiology and Biophysics, Howard University, College of Life Sciences, Northwest University, Department of Neurology, Johns Hopkins University School of Medicine; Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine; Adrienne Helis Malvin Medical Research Foundation, Hugo W. Moser Research Institute at Kennedy Krieger; Department of Neurology, Johns Hopkins University School of Medicine, Department of Neurology, Johns Hopkins University School of Medicine; Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine; Adrienne Helis Malvin Medical Research Foundation; Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Physiology, Johns Hopkins University School of Medicine, Department of Neurology, Johns Hopkins University School of Medicine; Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine; Adrienne Helis Malvin Medical Research Foundation; Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Hugo W. Moser Research Institute at Kennedy Krieger; Department of Neurology, Johns Hopkins University School of Medicine; Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Oncology, Johns Hopkins University School of Medicine, Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Hugo W. Moser Research Institute at Kennedy Krieger; Department of Neurology, Johns Hopkins University School of Medicine | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/ | 1 | 2020 | Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. mRNA Synthesis and Transfection. protocols.io https:// | 2021-04-15 09:16:19 | ||||
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Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.54rg8v6 | Neurodegeneration Method Development Community | Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i3Neurons) or lower motor neurons (i3LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3Neurons or i3LMNs, and we present neuronal culture conditions for various experimental applications. © 2018 by John Wiley & Sons, Inc. | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.54rg8v6 | 2021-04-15 09:16:18 | ||
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Nuclei Isolation from Human Brain Using Sucrose Gradient Resource Report Resource Website |
Fatma Ayhan, Genevieve Konopka | DOI:10.17504/protocols.io.scneave | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | This protocol outlines our preparation of single-nuclei suspension from surgically acquired fresh human adult brain tissue. | Neurocrine Biosciences, UT Southwestern Medical Center | 1 | 2018 | Fatma Ayhan, Genevieve Konopka 2018. Nuclei Isolation from Human Brain Using Sucrose Gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.scneave | 2021-04-15 09:16:19 | |||
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Preparing hPSC-derived neurons for single-cell RNA sequencing Resource Report Resource Website |
Cortina Chen, Florian T Merkle | DOI:10.17504/protocols.io.bpcbmisn | Neurodegeneration Method Development Community | This protocol is about Preparing hPSC-derived neurons for single-cell RNA sequencing. | Wellcome-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, CB2 0QQ, United Kingdom, Wellcome-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, CB2 0QQ, United Kingdom | 1 | 2020 | Cortina Chen, Florian T Merkle 2020. Preparing hPSC-derived neurons for single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bpcbmisn | 2021-04-15 09:16:21 | |||
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Preparation of feeder-free iPSCs culture Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x83fryn | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Preparation of feeder-free iPSCs culture. protocols.io https://dx.doi.org/10.17504/protocols.io.x83fryn | 2021-04-15 09:16:57 | ||||
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RNA Imaging with MERFISH - Design of Oligonucleotide Probes Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.menc3de | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | The first step in any MERFISH experiment is the design of the oligonucleotide probes that will be used to label individual RNA species. In our current implementation of MERFISH, each oligonucleotide encoding probe consists of three basic components as illustrated in Figure 2. The first region is a 30-nt targeting region that is complementary to a portion of the sequence of the RNA to which it is designed to bind. The second region is a set of sequences that are called readout sequences, which were designed to be complementary and hence only bind to MERFISH readout probes and not other nucleic acid in the cell. Finally, the third region is a set of priming regions used in the construction of these probes, which will be discussed in detail in Probe Construction. In addition to the nucleotide sequences for each of these components, a codebook —the specific set of binary barcodes that will be used and their association with different RNA species of interest—must also be designed. In this section, we provide protocols to design these sequences and to build a codebook. Example code to perform these steps can be found at http://zhuang.harvard.edu/merfish/ . | Harvard University, Cambridge, Massachusetts, Harvard University, Cambridge, Massachusetts | https://www.ncbi.nlm.nih.gov/pubmed/27241748 | 1 | 2018 | Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Design of Oligonucleotide Probes. protocols.io https://dx.doi.org/10.17504/protocols.io.menc3de | 2021-04-15 09:16:32 | ||
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GENOTYPING OF iPSCS WITH GENE INSERTIONS (Support Protocol 1) Resource Report Resource Website |
Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward | DOI:10.17504/protocols.io.5u9g6z6 | Neurodegeneration Method Development Community | This protocol will use genomic DNA isolated from the purified iPSC clones with CLYBL or AAVS1 gene insertion to determine if integration of the transgene has occurred correctly, in a heterozygous or homozygous fashion, and if the floxed selection genes are present. Primer sequences, amplicon sizes, PCR mix composition, and thermal conditions are included in the steps. | National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland | https://doi.org/10.1002/cpcb.51 | 1 | 2019 | Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. GENOTYPING OF iPSCS WITH GENE INSERTIONS (Support Protocol 1). protocols.io https://dx.doi.org/10.17504/protocols.io.5u9g6z6 | 2021-04-15 09:16:33 | ||
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Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells Resource Report Resource Website |
Ethan Lippmann, Hannah Wilson, Emma Neal | DOI:10.17504/protocols.io.bd6ei9be | Neurodegeneration Method Development Community | Human induced pluripotent stem cell (iPSC)-derived developmental lineages are key tools for in vitro mechanistic interrogations, drug discovery, and disease modeling. iPSCs have previously been differentiated to endothelial cells with blood-brain barrier (BBB) properties, as defined by high transendothelial electrical resistance (TEER), low passive permeability, and active transporter functions. Typical protocols use undefined components, which impart unacceptable variability on the differentiation process. We demonstrate that replacement of serum with fully defined components, from common medium supplements to a simple mixture of insulin, transferrin, and selenium, yields BBB endothelium with TEER in the range of 2,000-8,000 Ω × cm2across multiple iPSC lines, with appropriate marker expression and active transporters. The use of a fully defined medium vastly improves the consistency of differentiation, and co-culture of BBB endothelium with iPSC-derived astrocytes produces a robust in vitro neurovascular model. This defined differentiation scheme should broadly enable the use of human BBB endothelium for diverse applications. | Department of Chemical Engineering, Vanderbilt University, Nashville, TN, USA, Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA, Department of Chemical Engineering, Vanderbilt University, Nashville, TN, USA | 2 | 2020 | Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bd6ei9be | 2021-04-15 09:17:01 | |||
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Neural progenitor banking Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x89frz6 | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural progenitor banking. protocols.io https://dx.doi.org/10.17504/protocols.io.x89frz6 | 2021-04-15 09:16:50 | ||||
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Preparation of tissue culture plates for neural rosettes and neural progenitors Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x85fry6 | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Preparation of tissue culture plates for neural rosettes and neural progenitors. protocols.io https://dx.doi.org/10.17504/protocols.io.x85fry6 | 2021-04-15 09:17:01 | ||||
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Feeding T75, T150, and 6wp Backup Resource Report Resource Website |
Andrea Argouarch | DOI:10.17504/protocols.io.8gkhtuw | Neurodegeneration Method Development Community | Protocol includes feeding of T75 flask, T150 flask, or the back up 6 well plate. | University of California, San Francisco | 1 | 2020 | Andrea Argouarch 2020. Feeding T75, T150, and 6wp Backup. protocols.io https://dx.doi.org/10.17504/protocols.io.8gkhtuw | 2021-04-15 09:16:48 | |||
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iPSC Restriction Digest: For Screening Edited Clones Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x8sfrwe | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC Restriction Digest: For Screening Edited Clones. protocols.io https://dx.doi.org/10.17504/protocols.io.x8sfrwe | 2021-04-15 09:17:03 | ||||
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Neural Rosette Formation and Selection Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x86frze | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural Rosette Formation and Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.x86frze | 2021-04-15 09:17:07 | ||||
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Neural progenitor expansion Resource Report Resource Website |
Celeste Karch, Rita Martinez, Jacob Marsh | DOI:10.17504/protocols.io.x88frzw | Neurodegeneration Method Development Community | Washington University in St Louis, Washington University in St Louis, Washington University in St Louis | 1 | 2019 | Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural progenitor expansion. protocols.io https://dx.doi.org/10.17504/protocols.io.x88frzw | 2021-04-15 09:17:09 |
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