√ Seeing Isn’T Always Believing: A Cautionary Tale On Gfp Transfer When Trying To Restore Vision

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 4dukt 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

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√ Stem-Ish Cells: What’S The Real Thing?

Ain’t nothing like the real thing…right?

No, by the ‘real thing’ I’m not talking about Coke versus Pepsi or Coke versus Diet Coke, but rather stem cells. (On a side note there’s still some people who apparently believe the stem cell myth that there are stem cells in Pepsi).

I get a regular stream of emails from patients and sometimes phone calls asking questions about stem cells and especially stem cell treatments, but at times I’m not sure the treatment in question really involves stem cells. There are also thousands of papers published on “stem cells.”

Maybe those doing research on the cells in question or selling the cells at a clinic or writing about the cells as a journalist all think that the cells are stem cells, but how do we know? Even things that are mostly really stem cells have some progenitor or differentiated cells in the mix too that spontaneously pop up.

Many things are called “stem cells” when they aren’t the real thing or when they are a mixture of cell types of which only one is really a stem cell. For example, there has been a whole debate about MSCs or mesenchymal stem cells. Some have proposed changing the meaning of the MSC acronym to no longer include “stem” because MSCs as typically purified and used may only contain a minority population of actual stem cells unless they are carefully sorted. As a result, other names such as “mesenchymal stromal cells” and even “medicinal signaling cells” have been used or proposed to replace the standard meaning of the MSC acronym. One other issue with the MSC name is that it is an umbrella term that is often applied to very different preparations of cells.

This “not the real thing” reality of some MSCs not being pure stem cells isn’t necessarily a bad thing depending on context and how they are portrayed, but it does make life complicated. Each type of MSCs should be thought about for what they actually are in terms of research and then in addition in marketing by for-profits. It’s important for patients to know that generally the clinics selling MSCs are not using pure populations of actual stem cells with wide potency to address health issues throughout the body related to diverse tissues generally unrelated to MSCs.

The bottom line is that “MSCs” are often not going to be pure stem cells. I’d say they are generally stem-ish cells with the important exception that some researchers do purify stem cells that are referred to as MSCs.

A product called platelet rich plasma (PRP) is often portrayed as being the basis of stem cell therapies too. While PRP is frequently combined with MSCs and so the combined product might have some actual stem cells in it, PRP is largely an acellular fluid product with molecules from platelets that might have some useful properties. For this reason, I’d say that PRP is not even stem-ish even though it is sometimes even alone called a stem cell therapy.

Then there is the whole “birth-related” stem cell arena that is very tricky. There are an increasing number of clinic firms injecting stuff that is marketed as “stem cells” into customers, when in fact there not only aren’t stem cells in it but also there are no living cells at all. It’s just an extract from placenta or amniotic membrane.

As I discussed before, the irony here is that many clinics know that their product isn’t living stem cells, but they market it that manner anyway to attract customers even though if regulators were to come knocking the firms would probably often point to the fact that they aren’t really using living stem cells since that changes how regulators handle biologic products (see, “Wanted both dead & alive: amniotic stem cell clinics sell zombie cells?“). I’m still not even sure that living amnion has many stem cells to start with in utero.

Finally, there’s the special case of fat-derived stromal vascular fraction or SVF, which is generally portrayed as “stem cells”. Actually SVF is often kind of a mystery product that I’d guess has a dozen or more cell types in it, only maybe one or two of which are stem cells. For instance, many of the MSCs in SVF may not be stem cells. I suppose SVF is stem-ish and it is probably a useful product for some narrow applications, but not some stem cell-centric panacea.

So when we read, hear, write, or say, “stem cells”, we should think about what is being referred to and perhaps ask yourself, “is that the real thing or just stem-ish cells?”

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