A new online discussion forum offers a meeting spot for the gene-editing and stem cell research community to interact with one another and with the scientists that created the Allen Institute's fluorescently tagged human induced pluripotent stem cell (hiPSC) line collection. If you can't find answers to your questions below, please visit the Discussion Forum.
General questions
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, and is a 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.
Is the Allen Cell Explorer mobile-friendly?
While much of the site is mobile-friendly, several resources are currently optimized for desktop use. We are working on mobile optimization across the site and plan to launch this feature by early 2018. We recommend accessing the site through wi-fi since many of the resources are data heavy.
The Allen Institute for Cell Science is a division of the Allen Institute, an independent, 501(c)(3) nonprofit medical research organization, and is a 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.
Is the Allen Cell Explorer mobile-friendly?
While much of the site is mobile-friendly, several resources are currently optimized for desktop use. We are working on mobile optimization across the site and plan to launch this feature by early 2018. We recommend accessing the site through wi-fi since many of the resources are data heavy.
Biology questions
Why did you choose to study hiPSCs?
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 iPSCs is that they can be differentiated them into many cell types, providing isogenic cell types from the same source of iPSC cells.
Why this particular iPSC line?
We are working with the 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 few other lines of which we know.
Do you 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 Coriell and requires different handling techniques for thaw and recovery from those used for the Allen Cell Collection. Please refer to the Coriell protocol for instructions.
Will you expand to use other iPSC lines?
Yes, but not at this time.
Why are you tagging these particular proteins?
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. The GFP-tagged proteins thus serve as reporters for the locations of various structures in our imaging studies.
How are you deciding on which protein to tag and where to tag it?
We look in the literature 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.
Will you take suggestions for tagging other proteins/what are our criteria for tagging a particular protein?
Yes, we are open to suggestions but cannot do all of them. The protein has to represent a key structure, complex or pathway that fits our overall mission.
How do you test that the tag doesn’t 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.
Why did you choose to use mEGFP rather than one of the other enhanced versions or another color FP?
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 well characterized biologically, generated the fewest artifacts, were processed efficiently, were stable, had good quantum efficiency and photostability, and did not have intellectual property (IP) concerns that would effect open distribution. Our conversations with people in the community and confirming experiments pointed to monomeric EGFP (mEGFP) as the best choice at the time we started our tagging strategy. We are currently also testing several red fluorescent proteins to be able to apply our tagging strategy in a second color.
Will you use other tags (such as Flag, SNAP, Halo) in addition to fluorescence tags?
We do not plan to use them in the near future. However, our protocols can be used to introduce any tag into the genome.
Will you share the CRISPR sequences and plasmids that resulted in successful editing?
Yes. We share the CRIPSR sequences that resulted in successful editing on our cell line catalog page. The donor plasmids are available from Addgene and can be accessed through the Cell Catalog.
How can I obtain a cell line that you have created and at what cost?
We are distributing our line through the non-profit biorepository at the Coriell Medical Institute. The cost is $650, which covers some of the storage and distribution costs.
What type of quality control (QC) do you do on your edited cell lines?
We test for genomic stability, off target activity, expression of the fusion protein, localization to the appropriate cellular structure, and pluripotency.
Do you perform deep sequencing? If so, will this data also be shared?
Yes, we perform RNAseq and exome seq of our final gene edited clones. We are currently looking into options for efficiently and effectively sharing this information.
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 may differentiate into other cell types in the future.
Do you plan to test the effect of disease mutations?
Yes. We are interested in suggestions from experts.
Do you plan to do organoids?
Yes- We hope to move from cells plated on 2D surfaces to 3D cultures, including organoids in the future.
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 iPSCs is that they can be differentiated them into many cell types, providing isogenic cell types from the same source of iPSC cells.
Why this particular iPSC line?
We are working with the 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 few other lines of which we know.
Do you 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 Coriell and requires different handling techniques for thaw and recovery from those used for the Allen Cell Collection. Please refer to the Coriell protocol for instructions.
Will you expand to use other iPSC lines?
Yes, but not at this time.
Why are you tagging these particular proteins?
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. The GFP-tagged proteins thus serve as reporters for the locations of various structures in our imaging studies.
How are you deciding on which protein to tag and where to tag it?
We look in the literature 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.
Will you take suggestions for tagging other proteins/what are our criteria for tagging a particular protein?
Yes, we are open to suggestions but cannot do all of them. The protein has to represent a key structure, complex or pathway that fits our overall mission.
How do you test that the tag doesn’t 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.
Why did you choose to use mEGFP rather than one of the other enhanced versions or another color FP?
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 well characterized biologically, generated the fewest artifacts, were processed efficiently, were stable, had good quantum efficiency and photostability, and did not have intellectual property (IP) concerns that would effect open distribution. Our conversations with people in the community and confirming experiments pointed to monomeric EGFP (mEGFP) as the best choice at the time we started our tagging strategy. We are currently also testing several red fluorescent proteins to be able to apply our tagging strategy in a second color.
Will you use other tags (such as Flag, SNAP, Halo) in addition to fluorescence tags?
We do not plan to use them in the near future. However, our protocols can be used to introduce any tag into the genome.
Will you share the CRISPR sequences and plasmids that resulted in successful editing?
Yes. We share the CRIPSR sequences that resulted in successful editing on our cell line catalog page. The donor plasmids are available from Addgene and can be accessed through the Cell Catalog.
How can I obtain a cell line that you have created and at what cost?
We are distributing our line through the non-profit biorepository at the Coriell Medical Institute. The cost is $650, which covers some of the storage and distribution costs.
What type of quality control (QC) do you do on your edited cell lines?
We test for genomic stability, off target activity, expression of the fusion protein, localization to the appropriate cellular structure, and pluripotency.
Do you perform deep sequencing? If so, will this data also be shared?
Yes, we perform RNAseq and exome seq of our final gene edited clones. We are currently looking into options for efficiently and effectively sharing this information.
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 may differentiate into other cell types in the future.
Do you plan to test the effect of disease mutations?
Yes. We are interested in suggestions from experts.
Do you plan to do organoids?
Yes- We hope to move from cells plated on 2D surfaces to 3D cultures, including organoids in the future.
Analysis & modeling questions
Are you going to share your image processing/segmentation methods?
Yes. The methods will be shared online and the code will be provided when ready. Please see Software engineering questions below for more details.
Yes. The methods will be shared online and the code will be provided when ready. Please see Software engineering questions below for more details.
Microscopy questions
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.
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.
What software do you use to control your microscopes?
We believe that our systems are best controlled by software developed by the vendor. The software packages that come with these systems allow for some automation, but in many cases we require additional features that are not provided by the vendors’ solutions. Therefore, we are developing our own Python-based infrastructure to handle advanced needs. Our software communicates through a thin layer with the vendor software. We will have more details on the website as our automated control matures.
We use the following vendor software packages:
Can I get your microscopy automation software?
Our goal is to make all tools we develop publicly available. At this time, our software is not in a state that would make it feasible for other users to implement. Please see Software engineering questions below for more details.
What software do you use to store your images?
We store our images using a custom-built file storage service back 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.
What software do you use to process your images?
We use matlab code and the Python-based Allen Cell Structure Segmenter tool box developed by the Assay-Dev team to produce our segmentation. 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.
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.
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.
What software do you use to control your microscopes?
We believe that our systems are best controlled by software developed by the vendor. The software packages that come with these systems allow for some automation, but in many cases we require additional features that are not provided by the vendors’ solutions. Therefore, we are developing our own Python-based infrastructure to handle advanced needs. Our software communicates through a thin layer with the vendor software. We will have more details on the website as our automated control matures.
We use the following vendor software packages:
- Carl Zeiss ZEN Blue
Used for Carl Zeiss spinning disk and Zeiss confocal microscope LSM 800. The Open Application Development (http://forums.zeiss.com/microscopy/community/) exposes some of the Python macro methods as com objects. We call these objects with the library win32com (https://sourceforge.net/projects/pywin32/) - Carl Zeiss ZEN Black
Used for Carl Zeiss confocal microscope LSM 880. We can communicate through the VBA interface provided by this software package. - 3i SlideBook
Used for 3i spinning disk microscope. We can communicate through the MatLab interface provided by this software package. 3i developed custom code for us to control some of the spinning disk functionality through MatLab.
Can I get your microscopy automation software?
Our goal is to make all tools we develop publicly available. At this time, our software is not in a state that would make it feasible for other users to implement. Please see Software engineering questions below for more details.
What software do you use to store your images?
We store our images using a custom-built file storage service back 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.
What software do you use to process your images?
We use matlab code and the Python-based Allen Cell Structure Segmenter tool box developed by the Assay-Dev team to produce our segmentation. 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 questions
Do I need to buy special software to run your programs and view your images?
All of the software we currently provide runs directly online in your web browser, so you do not need special software to run our programs or view our images. Older web browsers and Internet Explorer may not work properly, so we recommend using Chrome, Firefox, Edge, or Safari to explore images online using the 3D Cell Viewer.
How do you visualize and present large 3D images in a web browser?
We use the open-source BisQue bioimage management and analysis system developed by the Center for Bio-Image Informatics at the University of California, Santa Barbara. This server based system converts the large 3D images into a compressed format for display in any WebGL capable browser. We have extended the software to create the 3D Cell Viewer.
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 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.
Are you going to share your image processing/segmentation methods?
Yes. The methods will be shared online and the code will be provided when ready. Please see Software engineering questions below for more details.
How do I use your online tools?
For questions about how to use the 3D Cell Viewer, Cell Catalog, Interactive Plotting Tool or data download, please visit the pages under the Support tab of the navigation menu.
All of the software we currently provide runs directly online in your web browser, so you do not need special software to run our programs or view our images. Older web browsers and Internet Explorer may not work properly, so we recommend using Chrome, Firefox, Edge, or Safari to explore images online using the 3D Cell Viewer.
How do you visualize and present large 3D images in a web browser?
We use the open-source BisQue bioimage management and analysis system developed by the Center for Bio-Image Informatics at the University of California, Santa Barbara. This server based system converts the large 3D images into a compressed format for display in any WebGL capable browser. We have extended the software to create the 3D Cell Viewer.
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 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.
Are you going to share your image processing/segmentation methods?
Yes. The methods will be shared online and the code will be provided when ready. Please see Software engineering questions below for more details.
How do I use your online tools?
For questions about how to use the 3D Cell Viewer, Cell Catalog, Interactive Plotting Tool or data download, please visit the pages under the Support tab of the navigation menu.
Software engineering questions
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.
Do you ever accept pull requests for external contributions to your code?
In the coming year, we will begin to post code for various projects into an open-source repository. We will periodically review pull request for code that would be useful to merge into our work and will make decisions on a case-by-case basis.
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.
Do you ever accept pull requests for external contributions to your code?
In the coming year, we will begin to post code for various projects into an open-source repository. We will periodically review pull request for code that would be useful to merge into our work and will make decisions on a case-by-case basis.
If you don't see your question answered above, please contact us.