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Authors: Ruilin Tian, Jason Hong, Martin Kampmann
Group: Neurodegeneration Method Development Community, KampmannLab

Proper citation: Ruilin Tian, Jason Hong, Martin Kampmann 2019. iPSC editing with TALENs. protocols.io https://dx.doi.org/10.17504/protocols.io.8dahs2e Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Summary: 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.

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Screening Edited iPSC Clones. protocols.io https://dx.doi.org/10.17504/protocols.io.yazfsf6 Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: 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:// Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Fatma Ayhan, Genevieve Konopka
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: This protocol outlines our preparation of single-nuclei suspension from surgically acquired fresh human adult brain tissue.

Proper citation: Fatma Ayhan, Genevieve Konopka 2018. Nuclei Isolation from Human Brain Using Sucrose Gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.scneave Copy   

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Authors: Cortina Chen, Florian T Merkle
Group: Neurodegeneration Method Development Community
Summary: This protocol is about Preparing hPSC-derived neurons for single-cell RNA sequencing.

Proper citation: 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 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Preparation of feeder-free iPSCs culture. protocols.io https://dx.doi.org/10.17504/protocols.io.x83fryn Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: 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/ .

Proper citation: 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 Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural progenitor banking. protocols.io https://dx.doi.org/10.17504/protocols.io.x89frz6 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: 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 Copy   

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Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes feeding of T75 flask, T150 flask, or the back up 6 well plate.

Proper citation: Andrea Argouarch 2020. Feeding T75, T150, and 6wp Backup. protocols.io https://dx.doi.org/10.17504/protocols.io.8gkhtuw Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: 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 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural Rosette Formation and Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.x86frze Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural progenitor expansion. protocols.io https://dx.doi.org/10.17504/protocols.io.x88frzw Copy   

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