NEW YORK (GenomeWeb) – New stem cell lines featuring GFP-tagged structures may help researchers see the cell as they've never seen it before.
Developed and engineered over the past year by the Allen Institute for Cell Science, these human induced pluripotent stem cell (iPSC) lines feature GFP fused to a protein of interest for one of five cellular structures — the nucleus, mitochondria, cell-cell junctions, microtubules in the cytoskeleton, and adhesions — all made possible by CRISPR/Cas9.
"We're asking the cell to produce a fusion protein so we can do live imaging of these cells," Ru Gunawardane, director of stem cells and gene editing at the Allen Institute, told GenomeWeb. "CRISPR will allow us to really spy on the cell without manipulating it too much."
The institute announced the Allen Cell Collection earlier this week and will distribute them through the Coriell Institute, essentially at cost, to any lab that's interested in using them. Moreover, the cells are derived from the wild type C (WTC) line developed by Bruce Conklin at the Gladstone Institutes and the University of California, San Francisco, which are clear of any human subject consent or intellectual property reachthrough issues that could affect the work done with them. "Researchers are completely free to make discoveries and know they will be theirs," and not anyone else's, Conklin said.
The Allen Institute expects they'll be of interest to biologists of many stripes, including stem cell biologists who want to have access to [GFP-tagged structures] as well as scientists who want to start using stem cells, said Susanne Rafelski, director of assay development. "It's a big goal of ours to get regular cell biologists to use these cells."
These are the first five of 20 planned cell lines in development. They're an early step in the even more expansive Allen Cell Explorer project, an initiative to generate image and movie data that will be used to develop computational models of cell function.
Gunawardane joined the Allen Institute last year and began immediately on a pilot experiment, but the full effort to develop these cell lines only began in January. In less than a year she's evaluated several parentage lines, picked one, developed a method for CRISPR/Cas9 editing in iPSCs, and led development of a battery of assays used in quality control.
"We've spent a lot of resources on this, because it's not just for us," she said. "We're saving other researchers a lot of effort."
Gunawardane said she picked the WTC line for several reasons. It's well characterized, genomically stable, has been differentiated into at least seven mature cell types (including cardiomyocytes, neuronal cells, and liver cells), and had been successfully gene edited before.
Conklin, a member of the Allen Institute for Cell Science's Stem Cell and Gene Editing Advisory Council, has used the cell in his own work, which merges stem cells and gene editing. He estimated that the cells are being used in at least 40 labs around the world.
Conklin has published extensively on making point mutations in the cells, but the Allen researchers wanted to insert a large region to make the GFP-fusions. That required some more background research on how best to edit the cells. They settled on using ribonucleoproteins pre-complexed with a two-part, synthetic guide RNA system from GE Dharmacon, featuring separate tracrRNAs and crisprRNAs. Gunawardane added that stem cells notoriously don't do homology-directed repair as well as cancer cell lines, so she designed long homology arms, about 1 kilobase on either side flanking the GFP sequence, to increase efficiency.
That still only leads to gene editing efficiency of about 10 percent, she said. Because the Institute intends for the cell lines to be a major research platform, they then run them through a gauntlet of assays to ensure everything is in its right place.
The process starts with 100 clones, but "right off the bat, we get rid of half," Gunawardane said. "Some just don't have it." Others have plasmid integration, an "underappreciated problem," she said. Other QC tests include fragment analysis — essentially mass spectrometry but for DNA, a three-part PCR fragment analysis, karyotyping, and both Sanger and next-generation sequencing.
NGS analysis takes up approximately the last month of a six-month process and is the last of about 10 tests. Only two out of the original 100 clones even make it this far.
While it costs tens of thousands of dollars to produce them, the Allen Institute is offering cell lines through the Coriell Institute for about $600, to cover distribution costs, which is less than most iPSC lines, GFP-tagged or not, Gunawardane said.
The Allen Institute has already given the lines to collaborators at the University of Washington. Conklin said he's eagerly awaiting a batch of the mitochondria-tagged cells for use in his lab. One possible experiment would be to differentiate them into cardiomyocytes and then insert a CRISPR interference construct to screen for changes in mitochondrial behavior, which he could watch in real-time.
"The markers they have chosen cover many of critical types of cellular structures," Conklin said. "Any change in cell biology will often result in one or more of these [structures] being altered."
Soon, there will be even more lines featuring tags for other structures. Among those next in line to be released are —in no particular order — actin filaments, the endoplasmic reticulum, and the nucleolus. Lines of GFP-tagged cardiomyocytes are also in the Allen Institute's plans.
The Allen team said they would share protocols online and at conferences.