Mark Tuszynski

Axonal Sprouting and Regeneration in the Central Nervous System

Going to talk with you about neural stem cells, which we hadn’t worked with much until recently.  My colleague Paul Lu came to me with a plan to look at neural stem cells . . . I discouraged him, but he was right, and his work has changed the direction of what we do in our lab.

Giving credit to funders, including the VA, CIRM,  and the Roman Reed foundation.

Showing a 9-year-old injured cord. What keeps it broken?

No permissive substrates in the lesion cavity, not enough neurotrophic support, and inhibition.  (My translation of this for the non-scientists would be: nothing for the axons to lie on as they grow, not enough fertilizer to help them grow, and too much toxic shit in the way.)

Inhibitors include myelin-asociated inhibitory proteins, like nogo, mag, omgp, semaphorin, netrin, and ephrin. (That’s some of the toxic shit.)

Then there are neuron-intrinsic factors like regeneration-related gene expression and or production of proteins (this isn’t really toxic shit . . . it’s shit that relates to the way your genes magically know when they’re allowed to help neurons regenerate and when they’re forbidden to do that.  Neurons in an embryo can regenerate, but neurons in an infant can’t.  Same cells, different rules coming from your genes.)

This talk is going to be about that last item . . . if you take an embryonic neural stem cell that IS growing and put into the adult spinal cord injury, will it grow?

Many years of efforts say, not really.  BUT, if you take E14 (E14 is shorthand for a 14-day-old embryo) cells from a developing rat and isolate the stem cells, they’re pluripotent in terms of neural cell types. (Meaning, they can still be turned into all three kinds of  neural cells: neurons, astrocytes, and glial cells.)  Hans Keirstead was able to take human stem cells and grow them in such a way that he got pure oligodendrocytes, but we’re not doing that .  . we’re taking them before they’re differentiated and putting them into the site.

They put trackers on their cells so they could see what happened to them once they were transplanted.

We grafted them two weeks post, because we wanted as much clinical relevance as possible. (“Clinical relevance” means being able to actually do this in human beings.)  We spent more than a year figuring out how to include a gel — a fibrin/thrombin matrix — that would let the cells survive and stick together, and we added some growth factors. (So, they had a good surface for the axons to lie on while they were growing across empty space, and they added some good fertilizer along with their undifferentiated, embryo-style neural cells.)

We used a T3 complete transection . . . six weeks later, the transplanted cells had completely filled the injury site.  Our tissue stains let us see what the cells had become.  A lot of them were neurons.  (Showing a slide with the section lit up)  Some cells turned into astrocytes, and others into oligodendrocytes.  All 3 types were there . . .

The truly remarkable thing was that axons emerged at very high densities over extremely long distances.  Until this experiment, the best we’d ever seen was about a hundred.  We have a thousand times more than that.

Not only that, but they’re growing much further than anything we’d ever seen, all the way up to c4 and down to lumbar.  When we did c5 hemisection models, the cells went all the way to the brain stem and then back down to the t8 level.  Some axons even grew out of the roots to exit the cord and head into the nerve roots.

We could follow the rate of growth over time . . . growth started right away and went at a rate of 1-2 millimeters per day, same rate as peripheral nerves.

This wasn’t just a few extra good results — it’s 80% of the time.  The stem-cell derived axons form connections with host motor neurons.  And they’re being remyelinated as they go.

So . . . a lot of axons grow out and grow for long distances — something I never thought I’d see.

But that’s only half the story.  You need axons to grow into the site as well, and that’s what we saw.  The host axons from the brain stem can penetrate the graft.  This means they could be forming new relays, new connections between the brain stem and the motor neurons.

We measure whether that’s happening, and it is.  Take a t3 graft, stimulate at c7, measure at t6 . . . and got a measurable electrical response, not quite normal, but real.  (To make sure they weren’t fooling themselves, they cut the cord again, just above the graft — and saw what would be expected, namely that the response vanished.)

The BBB score went from about 2 to about 7 with the grafts.  All of which supports the idea that you’re getting functional new relays across the injury.

These were the most potent results we’d ever seen.  So we looked at two human stem cell lines, one from neural stem cells and another from neuronal cells grown at Harvard from human stem cells.

(okay, to be clear . . . he’s saying that they started by putting rat embryo neural cells into transected rat cords, and then did the same experiment two more times, once with human embryo-derived neural cells put into transected rat cords, and then with human neural cells grown in the lab from human stem cells, again placed in rats.)

When we did the human neural cells, it looked a lot like what we saw with the rat neural cells when we put those cells into transected rats.  I have to say that for someone who has worked in this field for a very long time, these results are simply astonishing.

When we did the human stem-cell-based neural cells, we again got axons growing over very long distances, and lots of them.

As part of the CA consortium about 10 years ago, we set up a primate lab, so that was in place for the next phase of testing.

We did C7 hemisection lesion on the primates, and again delayed the graft for 2 weeks.  This time we used the human neural stem cells and have shown — again — axons growing robustly through white matter and gray matter.

This work with the monkeys is just beginning; it’s very important to do it.  The gels we used in the rats didn’t work in the monkeys . . . we have to devise a whole new matrix to fill that much larger cavity.  95% of rat-to-human models fail.  (95%!)

We need to see this work if (when!) we get to human trials.  We’re seeing many scientists from other labs come to see what we’re doing in the hope that it can be extended.

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3 Comments on “Mark Tuszynski”

  1. Kyle Says:

    Pardon my ignorance, what are ‘BBB’ scores?

  2. […]  Dr. Mark Tuzynski talking about axons growing abundantly across the injury site in rats with cords transected two […]

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