First thing that we do is we take a cofluent T 25 of umsl. So I'm just gonna wash with PBS and I'm just gonna add a few drops of TR on And let that sit for a couple minutes And I can see that the cells are cutting off the plate. So it's pretty much ready.
Okay, so now I'm gonna collect the trypsin eye cells and it's a good idea to trip to trigger rate your trypsin eye cells for just a moment to break up the colonies test and put them onto the UN plate. And this will just sit in the incubator for about 30 to 45 minutes. Now I'm just gonna spin down these cells to collect them and I am just gonna aspirate.
So I'm just gonna resus in a few minutes so I can count this off. So I resus suspended and now I've counted and now I'm taking out an aliquot and I'm gonna delete this aliquot to 50 mils and that's gonna gimme the appropriate concentration to get a hundred cells per drop just to make sure the cells are okay. So now we have a reservoir.
Just give these a little swish again, we pour these into our reservoir. Okay, so this is already programmed. There we go.
Now it's pretty straight. Turn it over and just set it aside. Every couple of plates, I just sort of just tilt it a little bit just so it s swishes around a bit.
Okay, so that's five plates. I'm gonna now just put these in the incubator. So these are our hanging drops that we played in two days Ago.
So to collect then you just shake the plates to collect the drops. So I'm just gonna collect everything and I try to do this gently so as not to disrupt the EVs too much, I'm just gonna take five or six meals of PBS and just wash the plates. And then we leave these for 10 minutes at least and the EBS will settle by gravity.
Once you've confirmed visually that your EBS have settled, then you remove the, the most of the super name, but not quite all of it because you don't wanna disrupt your, Okay. Okay, so now and then they shake until day six. So these are embryo bodies that were Prepared six days ago from mirroring embryonic stem cells and usually collect them, we swirl the plates a little bit.
When you swirl the plates, the eds sort of pull in the center of the dish and it's a little bit easier to correct them. So now you let the eds settle by gravity into the bottom of the tube and then what you do is you remove the media and then you wash once with 10 mils, 10 to 12 mils of PBS. Okay, so now we'll let these, wait a minute.
So now I'll just remove the P.So we add 250 microliters of our enzyme mix and we add a milli and then we just put them just a 37 degree water bath. And every now and then, maybe a couple times I'll come by and just flick them just to mix them up because the Ed's settled. So now we have this cell dissociation buffer that's enzyme free that we just get from gpro.
And we're gonna add about eight mils of this to each tube. And then we're just gonna iterate with a five mil pipet to disassociate the, as you iterate in this associate, it'll become very cloudy. And that's just sort of a visual cue that the cells are being dispersed in the media and making it very, now I'm gonna count the cells by tri exclusion, if you're trying to do an infection or you're calculating an MOI, it's very important to count.
So I'm just gonna prepare a large batch of virus plus media plus cells at the appropriate concentration plus protein sulfate. And, and then I just make sure the cells, and once again I'm paranoid about the cells outta suspension. So I'm always, and I also use a small, I try to be careful not to do too much splashing and how we spend for 2, 500 rm.
So I'm collecting our day 10 cells that have been, I save all the washes off. I used to use PI for P iodide, but seven is not as bright. And so it doesn't bleed into as many channels as much cleaner than.
So now we just Get variable and this is a good way to prevent loss in needle because volume. So I'm Shannon McKinney Freeman. I'm a postdoctoral fellow in George Families Life Attorney at Children's Hospital Boston.
So today we're going to go over how to derive a transplantable population of cells from mouse embryonic stem cells, Transplantable and hemato system. Why do We do this experiment? Well, we know that using this system we can derive, well first the ultimate protocol was developed in order to derive a population of cells from embryonic stem cells that had hematopoietic stem cell activity in the sense that they were capable of resing mice from lethal radiation, reconstituting their hematopoietic system for a long time, the lifetime of the organism and in all lineages of the peripheral blood.
So that is why the protocol was developed. Now we're doing further experiments to understand the phenotype of the population of cells within this expanded population that we derive that are actually mediating this hemato blood stem cell activity. cause we don't know, we know that the population's very heterogeneous and we know that the cell is actually rescuing the mice is extremely rare within this population of cells.
And so we're trying to basically define its phenotype so we can purify it from these very heterogeneous cells. And then we're most interested in understanding how it functionally compares to a bonafide and hematopoietic stem cell that would, one might derive from whole bone marrow. Does it have equal reconstituting ability developmentally, does it seem to reflect an adult bone marrow HSC?
Or perhaps it might be more like a hemato stem cell. You might find early development say in the a GM or the fetal liver. And so this is ultimately what we're trying to understand and we wanna understand these questions because we ultimately, we would like this there, this protocol to sort of inform similar work we're interested in doing with human embryonic stem cells.
And we feel like the information we learn with the math system will, you know, greatly benefit those future experiments. I think there's a couple things. First of all, there's just, it's a good in model of vitro, it's a good in feature model of development.
So we're trying to understand first of all the development of hematopoietic stem cells. And so it's a nice system that's very minimal to ectopic gene expression and looking at how these things influence hematopoietic stem cell development. So that is very useful from a basic science perspective.
But ultimately we do want to derive some sort of cell-based therapy that might be used clinically for deriving, you know, transplantable metabolic stem cells from human cells. But that Is probably some distance into the future. Well actually We've discovered that the most difficult part is not so much the derivation of the cells themselves, but we found through careful sort of experimentation, there was a period of time where we were having trouble getting the cells to reconstitute our recipient animals.
And what we eventually discovered was that we had to pay very close attention to the ablation of the recipients because these cells are not capable. So, so typically when you do a transplant in mice with bone marrow drive agec, you'll do 10 or 11 graves of a radiation to aate the mice and the bone marrow HC are quite capable of rescuing the animals from that. Well it turns out that these, this population of cells that we expand are not capable of rescuing animals from such a high dose of I radiation.
So what we had to do is very carefully tighter the dose of irradiation paying attention to the size of the animals. cause we found that if the animals were too big and we tried to use the same dose, they, they weren't effectively ablated as well. But once again, if you don't irradiate the animals enough, then the cells are not capable of competing with greater resistant hosts.
So really, once we figured out that this was a major variable, making it difficult for us to observe the ab activity of these cells and we, and we basically titered the irradiation dose and also realized that the age and weight of the animal is very significant, this is when our experiments began to work very reproducibly reproducibly. So what we've discovered is that if you use animals that are around 20 grams, 15 to 20 grams and you ablate them with 9.25 gray, that seems to be the perfect dose that allows, gives these cells the edge they need to reconstitute the mice. Yeah, and I think that's one was one of the most important variables.
The other, other, other significant thing would be the number of cells you're injecting. We have also titered using these particular conditions for radiating our model organism. We also titered the number of these unfractionated, this heterogeneous pool cells we were injecting into mice.
And we basically found that you need about a minimum dose, about 2 million to 5 million cells to rescue the animals. If you inject less than that, the animals aren't rescued. And I think that that speaks to the, probably the rare, you know, of the cell, it's actually mediating the rescue within this heterogene compartment.
So I think the most important variables are the number of cells you inject and being sure that whatever, you know, transplant model, you're using the tube, a blade at them sufficiently to give the cells the advantage they need to reconstitute, but not so much that they're just not capable of rescuing em. These things have been the big, the major variables.
