Seeing isn’t always believing: a cautionary tale on GFP transfer when trying to restore vision

Valerie Wallace
Dr. Valerie Wallace, presenting her research, at the annual Till & McCulloch Meetings in Mont Tremblant, on Nov 6th.

By Samantha Yammine

When our TV stops working, it’s usually because one of the wires has come unplugged. We begrudgingly huff and puff over to the back of the TV, track down the loose culprit, plug it back in, and boom: back to Netflix.

If you multiply all those wires in the back of your TV by several billion, that will approximate what the wiring in your central nervous system is like. Your eyes, brain and spinal cord are made up of different types of cells that are connected together in a way that lets you see, think and move.

With many diseases and injuries, some of the cells in this interconnected web become damaged and no longer function properly. So why can’t we just plug new cells in like we do our TVs and move on with it already?

While a network of cells a billion times more complicated than your TV is certainly harder to fix, this is not as impossible a task as it may seem.

And it’s one that Canadian stem cell researcher Dr. Valerie Wallace, along with her lab, wanted to tackle in the context of vision loss. She reported on her team’s work at this year’s Till & McCulloch Meeting and this post is a summary of the key points of the presentation. Her lab asked whether it is possible to restore vision by transplanting new light-sensing photoreceptors into the retina – the dark sheet at the back of our eyes whose cells receive light and enable us to see.

The researchers tested this question in the expected manner: they took cells from a healthy mouse that were labeled with a green fluorescent protein (GFP) and transplanted them carefully into intact retinas that were not fluorescent. With only the donor cells marked green, they reasoned that any green cells found integrated into the host retina would be indicative of successfully integrated transplanted cells.

Three weeks after they injected the green donor cells, they checked under the microscope and saw some beautifully connected photoreceptors in the retina fluorescing green! An exciting and promising result you’d probably want to write home about…

…but it wasn’t what they had hoped. In fact, the more they tested this the more they realized that the GFP+ cells fluorescing green were not actually the cells they had transplanted, but rather the result of the host cells taking up GFP from the donor cells!

It is very common, in fact it is critical for our survival, for cells to signal to one another by transmitting proteins. But the transmission of a fluorescent protein like the one used in this study was definitely a surprise that had big implications on how the researchers interpreted their results.

To better test what was happening, they cleverly tried labeling the DNA of their donor cells instead. Proteins float around in the cell’s cytoplasm and so are transferred a bit more easily, but DNA doesn’t leave a cell unless the cell is dividing. Since none of theirs were thought to be dividing they assumed this would be a much more stable way to do their experiment.

Unfortunately, but interestingly, this confirmed their suspicions: they no longer saw labeled cells integrated in the retina when they fluorescently-labeled DNA instead of proteins. Their transplanted cells were not effectively integrating into the host retina, but just sharing their fluorescent contents with cells that were already intact in the circuitry.

They looked back at other reports where researchers had suggested they could get their transplanted cells to integrate and found that if you looked closely enough, the old results were not that different from their own. Dr. Wallace cautioned that we must be careful how we interpret older data for this reason, and be more clever with how we design controls for future experiments.

This strange transfer of cell materials remains elusive to study; it doesn’t seem to happen to cells in a dish that are a lot easier to observe, and so far they’ve only seen it happening between photoreceptor cells. Whether it may be a cause of false positive transplant results in other parts of the body remains to be seen, and will easily go unnoticed unless scientists do their experiments by labeling proteins as well as the DNA of the cells they’re transplanting.

It’s not all bad news though – in fact, it’s quite interesting. See many transplant studies, including those in other parts of the body, show that transplanting new cells can have a beneficial effect just by offering support to their host cells. These are called “paracrine” effects, and studying this type of protein transfer could improve our understanding of these indirect beneficial effects seen in the eye and other systems.

But for now, Dr. Wallace cautions: “Just because you see marked cells integrating after your transplant, doesn’t mean the cells you transplanted have successfully integrated!” Seeing isn’t always believing and it’s important we keep our minds open to new interpretations for the things we think we see.

An eye-opening talk. #punintended #sorrynotsorry

To read more, see the recently published paper here.

About the Author: Samantha Yammine is a PhD Candidate studying neural stem cell biology in Dr. Derek van der Kooy’s lab at the University of Toronto. Her research focuses on neural stem cell hierarchies in the developing mammalian brain, and activation of quiescent stem cells in the adult brain. She is also an avid science communicator on social media and can be found @SamanthaZY on Twitter and @Science.Sam on Instagram sharing the science we all love in new ways everyday.

Shared with permission from Signals Blog

4 thoughts on “Seeing isn’t always believing: a cautionary tale on GFP transfer when trying to restore vision”

  1. Great to have this phenomenon confirmed independently. Prof. Marius Ader at the Center for Regenerative Therapies Dresden reported the observation last year.

    Nat Commun. 2016 Oct 4;7:13028. doi: 10.1038/ncomms13028. Retinal transplantation of photoreceptors results in donor-host cytoplasmic exchange. Santos-Ferreira T, Llonch S, Borsch O, Postel K, Haas J, Ader M.

    1. Dear Ms. Yammine and Admin:

      Thanks for sharing and posting, respectively, this very important discovery. As indicated, given the widespread use of cytoplasmic fluorescent proteins as cell-specific markers in cell and tissue biology research, this phenomenon could have important implications for the accuracy of interpretations of many past studies. As for the erroneous conclusions due to unnoticed cell fusion in earlier years of stem cell transplantation research, control experiments will need to be developed to safeguard against this newly recognized cause of false operating assumptions. Kudos to the Wallace lab for detecting and reporting this important problem! If I still had the opportunity to teach and train STEM students and junior scientists, I would definitely add this report to my previous lectures and presentations on the importance of testing underlying crucial technical (and conceptual) assumptions during experimental design.

      James at Asymmetrex

  2. Paul,

    Thanks for pointing this out. I was unaware of the talk and will do a literature search. Is it back to boy-girl gender mismatch studies or the mouse marker studies? I would assume that this type of DNA data is still correct ?


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