Transplantation of Whole Kidney Marrow in Adult Zebrafish

My name is Jocelyn Blank. I'm a technician in Lauren Z's lab And so I'm studying hematopoiesis and zebrafish. So we're trying to learn more about hematopoiesis especially.

So this is a general hematopoiesis sort of schematic. And so you start with a long term meta stem cell which can self-renew and it can also make a short term stem cell, which then can make progenitors a lymphoid progenitor and a myeloid progenitor. And then these respectively make lymphoid or myeloid cells.

And so we're very interested in this beginning part of how HSC can make for Genders, which then make the differentiated cells. I radiate wild type Fish at 25 gray two days before transplantation. And so then we have a clean system and we can transplant in donor marrow from fluorescent transgenic donors and then we can, this way we can watch the cells That we transplant in and see if they can repopulate the blood on day of transplant Sacrifice the appropriate number of fluorescent donors in trican.

Just wait until the, can't see the gills move anymore. So I just put this some on a, just a dish like this, a platform and then make a small cut right below its belly and then I can insert the scissors and cut down the belly.This? Yes, so now it's cut all the way down here and sort of hold the fish open.

And then with the other one, pull out the other organ. So the kidney is along the back of the fish. Can I take my good forcep and sort of grab it at the top and then it peels off and down and sort of strip that have there and put it into the, so now I'm just gonna transfer it into a conical tube and I'm just gonna put that up and down.

To break up the kidney pieces, we'll break up with 10 mil syringe and 18 and a half gauge needle transfer to a tube and fill up with PBS solution. Spin down at 5, 000 RRP M about this for p bsm suspend or you micron cell strainer. I want to strain the strainer and I won't catch any big pieces that you have.

Pour out Resus suspend in one mil PBS plus count cells. Calculate number of cells to transplant, Sacrifice Fish and tri coat, pipe tip with heparin. Stab through gill with pipette tip, add blood to PBS solution.

Spin down resus suspend spin down again. Filter through 40 micron filter count cells as with the whole kidney marrow. So I'm gonna fill the syringe up with cells to Five microliter, making sure that there's no bubbles in the syringe.

The fish fish will be anesthetized in trica before injection. For the injection, inject five microliters of the whole kidney marrow and peripheral blood solution into the heart of each recipient using a Hamilton syringe. And then I'm gonna insert the syringe a little ways back.

The heart is about here, so that's where I want to end up. I'm gonna poke it in, staying very shallow. And then I'm gonna poke around right here where the heart is.

And sometimes you see a burst of blood, which means you hit the heart. Sometimes you don't. I'm not seeing any blood, so I'm just gonna inject.

That's it. Keep Fish off flow for one week to avoid infection. Keep about five to 10 fish per cage, removing dead each day and manually change Water each day.

So the kidney marrow is where the, where we think that the Stem cells reside and that's where they make their progeny. So it's sort of like the bone marrow in humans. So when we do bone marrow implants in humans, it's the same exact idea when we transplant them into the heart of the animal.

This just, we've tried different ways. This is the most effective way to get the cells into circulation. So the kidney marrow is where the HSP can form all of its proteiny.

And so the HSEs have to find their way to the kidney marrow and so they can do this through the circulation. If we do get repopulation of the marrow, then we know that we transplant it in a long term hemato stem cell because that's the only cell that can repopulate and make all of these Cells. That's what we're most interested in.

So I would definitely say the actual injections, that takes a lot of Practice, a lot of fish will die. And basically trying to get it into the heart, which you're not always sure if you are getting it into the heart, it's very hard to see. And also just not killing the fish with the injection.

So when you're injecting you could hit other internal organs, which can cause death or they can get an infection after the injection. So one thing we're trying, so usually a lot of the fish will die the day after the injection. And so what we're trying now is to keep everything as sterile as possible so we don't put the fish back onto the flow, back into the system water because there could be, they could get infections in the water that way.

So we keep them separate, we change the water every day and we're thinking about maybe adding some antibiotics to the water, stuff like that just to keep them healthier during those, their crucial Time right after the transplant. So we can use it to study mutants. If we think a mutant Has a defect in their stem cells, we can transplant in marrow from them into wild type fish.

Or we can transplant in wild type marrow into those fish and see if there's some sort of problem with the stem cells themselves or the stem cell niche. So for some reason, the stem cells can't find their way to a kidney marrow, then that would be a problem. And so we could measure that based On whether the fish can graft or not.