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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: Human iPSCs are an ideal system for studying human biology due to their rapid proliferation, genomic stability, and ability to differentiate into many somatic cell types. Historically, specialized culture practices and costly reagents have hindered widespread adoption of iPSCs by the cell biology community. In recent years, however, development of new culture techniques and improved media formulations have dramatically simplified iPSC culture and reduced costs.The protocols described in this unit are adapted from a collection of publications that establish optimal practices for the maintenance of human iPSC cultures (Beers et al., 2012; Ludwig et al., 2006; Chen et al., 2011). While these publications provide useful guidelines for the stem cell novice, here we distill the fundamental procedures necessary for maintaining iPSCs in a pluripotent state and highlight critical steps that may need to be optimized for individual applications. In practice, iPSC lines of interest are usually maintained in an undifferentiated state in small cultures (1 to 3 wells of a 6-well plate) to reduce reagent use before being expanded as needed for experimentation.Essential 8 (E8) is the simplest defined medium for hiPSC culture. E8 may be prepared from its components by the consumer (Table 1; Chen et al., 2011) or purchased as a preformulated kit. Other commercially available media may be substituted, such as mTeSR1 or StemFlex. A volume of 12 ml of medium should be added to each 10-cm tissue culture dish or distributed evenly across each standard multiwell plate (i.e., 2 ml/well for a 6-well plate). E8 should be aspirated and replaced with fresh medium daily, although a double volume may be added at low confluency to permit an extra day of culture without medium changes. StemFlex and E8 Flex contain components that stabilize the recombinant growth factors present in the medium, permitting medium exchange every other day as a general practice. Some iPSC lines (e.g., WTC11) tolerate every-other-day medium changes of standard E8 medium without loss of pluripotency or cell death, further reducing costs of medium and consumables. Finally, mTeSR1 may promote cell survival in stressful conditions better than E8, especially for finicky iPSC lines, although supplementation with a ROCK inhibitor (RI) is also recommended in such scenarios. Use of standard E8 will be assumed throughout this basic protocol.Additionally, this protocol uses Matrigel-coated tissue culture plates. Matrigel works well for iPSC culture and has been widely adopted for research applications. However, since Matrigel is derived from murine sarcoma cells, it is not chemically defined and exhibits batch-to-batch variability. Alternative defined coatings include recombinant laminin or vitronectin, although these substrates are typically more costly. Notably, downstream neural differentiation described in these protocols occurs in fully defined conditions (see Basic Protocols 5 to 8), so the choice of iPSC substrate is of minimal scientific importance to all but clinical-grade applications.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. MAINTENANCE CULTURE OF iPSCs (Basic Protocol 1). protocols.io dx.doi.org/10.17504/protocols.io.48vgzw6 Copy
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 dx.doi.org/10.17504/protocols.io.menc3de Copy
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 dx.doi.org/10.17504/protocols.io.x83fryn Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. ASSESSING RTTA ACTIVITY (Support Protocol 6) . protocols.io dx.doi.org/10.17504/protocols.io.5w8g7hw Copy
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 dx.doi.org/10.17504/protocols.io.scneave Copy
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 dx.doi.org/10.17504/protocols.io.54rg8v6 Copy
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 dx.doi.org/10.17504/protocols.io.8dahs2e Copy
Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community
Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters. protocols.io dx.doi.org/10.17504/protocols.io.8g5hty6 Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Following Day 3 replating, i3Neurons should be cultured for at least 1 week before lysis, fixation, or other experimental endpoint, and they remain viable for at least 1 month with proper maintenance. This protocol covers medium conditions, coating of tissue culture dishes with synthetic polymers, recommended plating densities, and maintenance procedures for long-term culture and for specific experimental applications.Cortical Neuron Culture Medium (CM) is sufficient to promote the maturation and longterm maintenance of i3Neurons in culture. i3Neurons express general markers of cortical neurons, as well as specific pre- and postsynaptic markers of glutamatergic excitatory cortical neurons. Since these cells are post-mitotic after 3 days of differentiation (see Basic Protocol 5) and prefer neuron-conditioned medium to fresh medium, maintenance conditions for these cultures are minimal. Generally, half-medium changes every 7 days with fresh, pre-warmed CM are sufficient for culturing beyond d10 (7 days after replating). Neuron attachment and growth also require a strongly adhesive substrate. Coating plates with synthetic polymers such as poly-L-ornithine (PLO) is necessary for i3Neuron attachment, viability, and successful outgrowth.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. CULTURING i3NEURONS (Basic Protocol 6). protocols.io dx.doi.org/10.17504/protocols.io.5jpg4mn Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Live imaging permits visualization of molecular and organellar dynamics within the neuron. While a standard confocal microscope is sufficient for short imaging experiments, extended imaging applications (>1 hr) are best served by a 37 °C live imaging chamber outfitted onto the microscope. CM should also be changed to Hibernate A Low Fluorescence Medium (BrainBits LLC, cat. no. SKU#HAPR) for extended imaging.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. LIVE IMAGING OF i3NEURONS (Support Protocol 5) . protocols.io dx.doi.org/10.17504/protocols.io.5w7g7hn Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocols included in this collection describe cell isolation from human dura mater, culturing, and banking.
Proper citation: Andrea Argouarch 2020. Dural Cell Isolation and Culturing - Collection. protocols.io dx.doi.org/10.17504/protocols.io.8m2hu8e Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Serial Dilution of Nucleofected iPSC Pools. protocols.io dx.doi.org/10.17504/protocols.io.x76frre Copy
Authors: Jennifer B Treweek, Ken Y Chan, Nicholas C Flytzanis, Bin Yang, Benjamin E Deverman, Alon Greenbaum, Antti Lignell, Cheng Xiao, Long Cai, Mark S Ladinsky, Pamela J Bjorkman, Charless C Fowlkes, Viviana Gradinaru
Group: Optical Clearing of Tissue, Neurodegeneration Method Development Community
Summary: To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.
Proper citation: Jennifer B Treweek, Ken Y Chan, Nicholas C Flytzanis, Bin Yang, Benjamin E Deverman, Alon Greenbaum, Antti Lignell, Cheng Xiao, Long Cai, Mark S Ladinsky, Pamela J Bjorkman, Charless C Fowlkes, Viviana Gradinaru 2019. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. protocols.io dx.doi.org/10.17504/protocols.io.8xehxje Copy
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 dx.doi.org/10.17504/protocols.io.x8sfrwe Copy
Authors: Marcos Otero-Garcia, Inma Cobos
Group: Neurodegeneration Method Development Community
Summary: - Protocol optimized to isolate single neuronal somas with cytoplasmic protein aggregates from postmortem fresh frozen brain- Works for the isolation of neuronal somas with neurofibrillary tangles from Alzheimer disease's brains- Note that cell membranes are highly disrupted in tissues that were previously frozen. Thus, cytoplasmic transcripts are largely lost. This protocol is therefore useful for the isolation of specific cell populations using antibodies against cytoplasmic antigens, but not for the profiling of cytoplasmic mRNA.
Proper citation: Marcos Otero-Garcia, Inma Cobos 2019. Isolation of single somas from postmortem fresh frozen human brain and immunostaining for AT8 and MAP2. protocols.io dx.doi.org/10.17504/protocols.io.6uqhevw Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Thawing iPSC Plate. protocols.io dx.doi.org/10.17504/protocols.io.x8ufrww Copy
Authors: Jacob Marsh, Rj Martinez, Celeste Karch
Group: Neurodegeneration Method Development Community
Proper citation: Jacob Marsh, Rj Martinez, Celeste Karch 2020. Thawing, Expanding, and Freezing Human Fibroblasts. protocols.io dx.doi.org/10.17504/protocols.io.bgunjwve Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol for feeding dural cells after outgrowth from tissue in a 6 well plate.
Proper citation: Andrea Argouarch 2020. Feeding 6wp. protocols.io dx.doi.org/10.17504/protocols.io.8gihtue Copy
Authors: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru
Group: Neurodegeneration Method Development Community
Summary: We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.
Proper citation: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru 2019. Systemic AAV vectors for widespread and targeted gene delivery in rodents. protocols.io dx.doi.org/10.17504/protocols.io.84ahyse Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes banking dural cells into cryovials for long term storage in liquid nitrogen.
Proper citation: Andrea Argouarch 2020. Freezing/Cell Banking. protocols.io dx.doi.org/10.17504/protocols.io.8gmhtu6 Copy
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