General
1. What is the Allen Institute for Cell Science?
The Allen Institute for Cell Science is a division of the Allen Institute, an independent, 501(c)(3) nonprofit medical research organization dedicated to understanding and modeling cells: the fundamental units of life. To learn more about the Allen Institute for Cell Science, including our mission, news and career opportunities, visit cellscience.alleninstitute.org.
2. Is the Allen Cell Explorer (allencell.org) mobile-friendly?
While much of the site is mobile-friendly, several pages are currently optimized for desktop use. We recommend accessing the site through wi-fi since many of the resources are data heavy.
Biology
1. Why are human induced pluripotent stem cells (hiPSC) your model of choice?
Human iPSCs are non-transformed, diploid, and proliferative. They provide a distinct advantage for cell biological studies over the traditional transformed, often aneuploid, and non-clonal cell lines. The added benefit of hiPSCs is that they can be differentiated into many cell types, providing isogenic cell types from the same source of iPSCs.
2. What are the advantages of using this particular hiPSC line?
We are working with a WTC line obtained from Bruce Conklin at the Gladstone Institutes. This line was derived episomally from a healthy donor and has been fully sequenced, has a normal karyotype, differentiates into multiple cell types, and is amenable to gene editing. We are also able to make it available to the research community and sequence its genome: properties shared by a few other lines of which we know.
3. Does the Institute distribute the parental line?
No. The Allen Institute does not distribute the parental line. The WTC-11 line (GM25256) is banked and distributed directly by The Coriell Institute. Please refer to the Coriell protocol for specific instructions on how to thaw and recover these cells. Note that the required handling techniques differ significantly from those used for the Allen Cell Collection lines.
4. Will you expand your studies to other hiPSC lines?
Yes, but not at this time.
5. Why are you tagging these particular proteins in the Allen Cell Collection?
Our goal is to understand intracellular organization and dynamics using live cell imaging. The first step is to tag proteins that represent key cellular structures commonly studied by cell biologists while selecting for proteins (and taggable regions of those proteins) that minimize perturbation—or altering emergent properties of the cell. The GFP-tagged proteins thus serve as reporters for the locations of various structures in our imaging studies.
6. How are you deciding on which protein to tag and where to tag it?
We perform a literature review and engage with colleagues in the cell biology community who have direct experience with the structure of interest to determine which protein and protein-tagging strategy best represents that structure and works well for live-cell imaging.
7. Will you take suggestions for tagging other proteins/what are the criteria for adding a particular protein tag to the Allen Cell Collection?
Yes, we are open to suggestions but cannot do all of them. The protein must represent a key structure, complex or pathway that fits our overall mission.
8. How do you test that the tag does not adversely affect the function of the protein?
We perform several quality control assays to test for obvious adverse effects, including the localization of the protein to the correct structure, cell and organelle morphology, growth rate, and differentiation. However, we do not perform in-depth functional assays specific to each structure as this is beyond our expertise and the scope of our project. We encourage the research community to do so with our cell lines as part of their studies and to share their observations.
9. Why did you choose to use mEGFP rather than one of the other enhanced versions or another color fluorescent protein?
While there are many colors and variants of fluorescent proteins to choose from, some generate artifacts and others have not been used extensively by the cell biology community. Our goal was to use fluorescent proteins that were stable, well characterized biologically, and processed efficiently. Further, these proteins generated the fewest artifacts, had good quantum efficiency and photostability, and did not have intellectual property (IP) concerns that would limit open distribution. Our conversations with people in the community and confirmation experiments pointed to monomeric EGFP (mEGFP) as the best choice at the time we started our tagging strategy. We are currently testing several red fluorescent proteins to be able to apply our tagging strategy in a second color.
10. Will you use other tags (such as Flag, SNAP, Halo) in addition to fluorescence tags?
We have no plans in place yet. However, our protocols can be used to introduce any tag into the genome.
11. Will you share the CRISPR sequences and plasmids that resulted in successful editing?
The Cell Catalog page on our website (under Allen Cell Collection tab of our website's navigation menu) gives access to the CRIPSR sequences that resulted in successful editing as well as donor plasmids available from Addgene.
12. How can I obtain a cell line that you have created and at what cost?
We distribute our gene-edited hiPSC lines through the non-profit biorepository at the Coriell Medical Institute. The cost of $650 covers some of the storage and distribution costs.
13. What specific quality control (QC) tests do you perform on your edited cell lines?
We test genomic stability, off target activity, expression of the fusion protein, localization to the appropriate cellular structure, and pluripotency.
14. Do you perform deep sequencing? If so, will this data be shared?
Yes, we perform RNAseq and exome seq on some of our final gene edited clones. We no longer perform deep sequencing on all the clones, because we learned very little from these exhaustive efforts, but we do share the sequencing data for the ones we performed them on.
15. Why are you studying cardiomyocytes; will you study other cell types, as well?
We chose cardiomyocytes since there are existing protocols for robust differentiation in a relatively short time frame (2-3 weeks). We are now focusing most of our efforts on differentiating our hiPSC into endothelial cells and studying the epithelial to mesenchymal transition (EMT).
16. Do you plan to test the effect of disease mutations?
Yes. We are interested in suggestions from experts.
17. Do you plan to culture organoids?
Yes- We hope to move from cells plated on 2D surfaces to 3D cultures, including organoids in the future.
Analysis & modeling
1. Do you share your image processing/segmentation methods?
Microscopy
1. What are your imaging modalities?
Our main workhorse is the spinning disk confocal microscopy. We are also using Zeiss Airyscan FAST technology to permit us to rapidly image live cells in 3D at increased resolution. We will expand our modalities, including light sheet, in the future as needed.
2. Why don’t you use Lattice Light Sheet, FCS, NLO, etc.?
We started with spinning disk microscopes because they best meet our initial objective in a turnkey system and allow high sample throughput, ease-of-use, image quality, and optical sectioning. With these systems, we acquire high quality data in a reliable way. Other modalities may be introduced as our program progresses.
3. What software do you use to control your microscopes?
We believe that our systems are best controlled by software developed by the vendor. However, we are developing our own Python-based infrastructure to handle advanced automation needs.
We use the following vendor software packages:
We use the following vendor software packages:
- Carl Zeiss ZEN (blue edition)
Used for Carl Zeiss spinning disk and Zeiss confocal microscope LSM 800. The Open Application Development exposes some of the Python macro methods as com objects. We call these objects with the library win32com. - Carl Zeiss ZEN (black edition)
Used for Carl Zeiss confocal microscope LSM 880. - 3i SlideBook
Used for 3i spinning disk microscope.
4. What software do you use to store your images?
We store our images using a custom-built file storage service backed by file metadata stored in LabKey. LabKey Server is an open-source platform used by organizations to manage biomedical data. The LabKey database tracks information about all stored files, as well as all the cell lines and experiments they are associated with.
5. What software do you use to process your images?
We use a Python-based toolbox, the Allen Cell & Structure Segmenter, that was developed by our Assay-Development team to produce segmented images. Other Python libraries we use are scikit-image for classic image processing and PyTorch for deep-learning based image processing. We have also collaborated with CellProfiler, an open source high-content image analysis package developed by Anne Carpenter and her group at the Broad Institute of Harvard and MIT to expand the capabilities of this package from 2D to 3D image processing.
Tools & data
1. Do I need to buy special software to run your programs and view your images?
We provide a wide range of open-source software tools, most of which run directly online in your web browser with no specific requirements. Other more specialized tools will specify their requirements on their respective download pages. All our tools are freely accessible on pages under the Data & Digital Tools tab on our website's navigation menu.
2. How do you visualize and present large 3D images in a web browser?
Our Cell Feature Explorer takes advantage of a full-featured in-house volume viewer. The volume viewer uses WebGL ray marching to visualize the data in 3D. It can handle any number of channels and provides many options for adjusting the image. We down-sample all the 3D images to optimize them for interactive display in the browser. The original full-size images are easily downloadable from the viewer.
3. How do you plan to share the images/data?
The volumetric microscopy images are downloadable individually from the 3D Cell Viewer or in bulk as .ome.tif files, which then can be viewed and analyzed directly from your hard drive using a variety of popular academic and commercial software packages. Visit the Data Downloading page to get started.
4. Are you going to share your image processing/segmentation methods?
5. How do I use your online tools?
To access our online tools, please visit the Data & Digital Tools tab on our website's navigation menu. For guidance on how to best use the tools, please visit the Tutorials for digital tools section under the Support tab. If you have any additional questions, we have a dedicated support forum for this purpose.
Software engineering questions
1. Is your code open source? If yes, what licenses do you use and why?
With a goal of practicing and promoting open science, we use and develop open-source software whenever it is possible and practical. Licensing will be described as software is released. Although we prefer the most permissive BSD-style licensing, we are sometimes constrained to less permissive licenses due to imported software dependencies.
2. Do you ever accept pull requests for external contributions to your code?
Community contributions to our many open-source projects are welcomed. We regularly review pull requests for code that would be useful to merge into our work.
If you cannot find answers to your questions above, please visit the Allen Cell Discussion Forum, a meeting spot for the broader research community to interact with the multidisciplinary team that created our fluorescently tagged hiPSC collection.