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Episode 17 – Liveyon Pure Cast – A REVIEW of the Liveyon Pure® Series
Dr. Alan Gaveck: Welcome back to the Liveyon Pure Cast Podcast. I’m Dr. Gaveck, Director of Medical Education for Liveyon. And today I have with me a very special guest. Dr. Margaret Coutts, Chief Scientific Officer for Liveyon. Welcome.
Dr. Margaret Coutts: Thanks, great to be here.
Dr. Alan Gaveck: Dr. Coutts, you have a very impressive resume starting at Boston University, getting your PhD at Tufts I believe, doing post-grad work at Harvard, Columbia, UC Irvine, I mean, that’s very impressive. We are very privileged to have you at Liveyon as our Chief Scientific Officer. But even what we’re here about today is you just recently wrote a white paper on the comparison of birth tissues.
Dr. Margaret Coutts: Well, it was really kind of inspired by my own curiosity. A physician had asked, “How do you pick the best product?” That’s a really hard question to answer, because most manufacturers don’t give you very much information at all.
Dr. Alan Gaveck: True. That’s perfect for you, right? Ask me a question, and you’ll go make some paper and figure it out. So what we’re going to do today is go through your study. I have to keep a couple notes because it’s fairly complex, and I really don’t want to miss anything for all of our viewers out there. But we’re going to take it as simple as we possibly can, so let’s start out really from the top. Define a birth tissue.
Dr. Margaret Coutts: Those are the tissues that support a baby in the nine months that it’s growing inside the mom. It’s the umbilical cord, the placenta, the umbilical cord blood, and the amniotic tissues.
Dr. Alan Gaveck: So you’re testing here umbilical cord blood, amniotic membrane that was in the fluid form, and then two different forms of Wharton’s jelly right?
Dr. Margaret Coutts: That’s correct.
Dr. Alan Gaveck: So when you did this analysis, you tested them in several different categories, right. Because we all know that each of them kind of sometimes have their own little thing that’s really good, but in order to really level the playing field, you tested them in multiple different categories. The first thing you did was a microscopic evaluation. And you took images of those microscopic evaluations and we have up here. And I would really love you to be able to explain to us exactly what we’re seeing. So we start in the top-left hand corner with the Pure Pro, and it looks like kind of Christmas decoration, though they don’t like very much red. What’s going on here with the Liveyon Pure Pro product?
Dr. Margaret Coutts: All of these products were thawed and prepared as the manufacturers directions. I then added a green dye and a red dye. So, the live cells will glow green and the dead cells will be red.
Dr. Alan Gaveck: And we’re going to talk about that red and green dye in a minute. But let’s go on to the next slide over to the right, which is the amniotic membrane. It looks like a couple islands out in the Pacific with a little red in them. What’s happening there?
Dr. Margaret Coutts: You can see in the lower right corner, there are two red dead cells. But this is just a ground up tissue, it kind of looks like debris ground up membranes.
Dr. Alan Gaveck: Don’t see any green cells in there.
Dr. Margaret Coutts: No. It looks mostly like debris.
Dr. Alan Gaveck: So let’s go to the lower panel. Below the Christmas decorations and look at WJ-1. Also, a lot of formations out there with a lot of red formations. But again, we don’t see much green.
Dr. Margaret Coutts: No, they’re just red cells in that field. Again, fibrous material crushed up membranes.
Dr. Alan Gaveck: Then the last one on the far lower right-hand corner, it’s kind of strange because this is also a Wharton’s jelly product, but we just see kind of two maybe three little separate islands out there, but just not a lot of anything out there.
Dr. Margaret Coutts: No, there weren’t a lot of cells in it. All the cells were dead. There’s a very fine debris in that product, it’s probably fragments of dead cells that have just basically died and turned into dust.
Dr. Alan Gaveck: So, this is an accepted method for identifying live versus dead cells?
Dr. Margaret Coutts: Absolutely. And just looking at these pictures, you can see there’s obvious differences between the products.
Dr. Alan Gaveck: Absolutely. Next you did a cell count. You used the research grade cell counter, and we look at those values, cut that one second. So next you did a cell count using a research grade … we’re not going to two yet. Hold on one more second, James. So next you did the cell count with the research grade cell counter, and you used that red and green dye, and here’s probably a good time to talk about the red and green dye. Because you had identified them as cells. So, a red and green dye are the AO/PI staining in your industry, acridine orange and-
Dr. Margaret Coutts: Propidium iodide.
Dr. Alan Gaveck: … propidium iodide. So they’re the ones that give you the idea what’s alive and what’s dead. How do they work? How do you get a green live and a red dead?
Dr. Margaret Coutts: Well, the propidium iodide is a big molecule, so it can only get into dead cells that are leaky or damaged. The green dye is a very small molecule, and it can easily pass through the cell membranes.
Dr. Alan Gaveck: So by doing that, if you have a live cell, then you’re going to fluoresce green and if you have a dead cell, it’s going to show up as red.
Dr. Margaret Coutts: Exactly.
Dr. Alan Gaveck: So you use a research grade cell counter, right, are all cell counters created equal?
Dr. Margaret Coutts: No, there’s a big range. Some are much more accurate than others. They obviously vary a lot in price. The way that which dyes you use and how the machines capture the images is different.
Dr. Alan Gaveck: So, you’re using that cell counter with the AO and PI staining system, is that considered a standard to the industry?
Dr. Margaret Coutts: It’s a high standard.
Dr. Alan Gaveck: Okay. So we know that there’s got to be cheaper ways out there. A simple cell counter using some trypan blue and that sort of thing. Are these good? Are they as consistent as your AO/PI staining?
Dr. Margaret Coutts: No. But certainly sitting there on the front of a microscope with a little hand counter and counting blue cells is, you know, I’m sure it’s been done for, I don’t know, probably 80 years. But it has some problems. First the subjectivity, like how blue is that cell, is it light blue? Is it dark enough to call it a dead one? If the cells sit in the dye for a long time, they all turn blue. Red blood cells are darker, so that could be scored as a cell.
Dr. Alan Gaveck: Gotcha. So what you’re looking for is consistency and the AO/PI gives you that capability.
Dr. Margaret Coutts: Consistency and also not a subjective judgment. I mean, the machine software is programed to recognize something that’s the right size and shape for a cell, and it will focus on what it sees. It will count for different microscopic fields and average the different fields.
Dr. Alan Gaveck: Perfect. Table one of your graph is a cell count. Basically with your cell counter, you’re getting total number of cells, total number of live cells, and then that percentage of either viability or live cells.
Dr. Margaret Coutts: Yeah, it’s just simple math. Yeah. The machine does the counting and does the simple math.
Dr. Alan Gaveck: So we see we compared the Liveyon Pure Pro product with the amniotic membrane, and the Wharton’s jelly 1 and 2. They all have similar volume according to them. The Pure Pro had 31,900,000 live nucleated cells in a 30 million cell product. Is that what you do in the lab over produce them a little bit?
Dr. Margaret Coutts: Yes, we do intentionally overfill about 10% on the count.
Dr. Alan Gaveck: Because you know there’s going to be some cell death.
Dr. Margaret Coutts: Correct.
Dr. Alan Gaveck: So then say the total cell live count is still 27,900,000, so we have a viability of about 87.5%. Is that consistent or would that be like wow, we just struck the lottery, this is the biggie?
Dr. Margaret Coutts: No, this is something that we check with every single batch, what the viability is.
Dr. Alan Gaveck: So your validation studies have shown that you are consistently in that area.
Dr. Margaret Coutts: Yes.
Dr. Alan Gaveck: Now, when we go down to the amniotic membrane, we see what? 132,000 live cells? Not a whole lot of … or 132,000 cells, but not live cells. Is it shocking to you or is this within range?
Dr. Margaret Coutts: I had no idea what to expect from the product. So I was kind of surprised it was that low.
Dr. Alan Gaveck: But you really went in purely just wondering, “I’m wondering what I’m going to find out here, I’m going to learn some information.”
Dr. Margaret Coutts: Right. Because the manufacturer didn’t supply any-
Dr. Alan Gaveck: Any information.
Dr. Margaret Coutts: You know, but we have the tools in the lab to look at these things.
Dr. Alan Gaveck: The last two are the Wharton’s jelly 1 and the Wharton’s jelly 2. This is from the same manufacturer, which I find it interesting because we have over 1.7 million total cells in the Wharton’s jelly 1, but only 20,000 total cells in Wharton’s jelly 2. Yet, when you look at the live cells, they’re about the same. A million cells produced 3,500 live cells, and 20,000 cells produced 3,400. I guess my big thing is, how can you have the same product from the same manufacturer have such a huge variation and not only total cell count, but actual live cells that are in that product.
Dr. Margaret Coutts: Well, I think the products start with the same raw material that start with Wharton’s jelly, there’s really no description on how it’s processed, whether a cryopreservative is added. Again, I just didn’t know what to expect, but these were the number that came out.
Dr. Alan Gaveck: But when you look at an umbilical cord blood product, there are specific protocols in place for how to process and how to process those cells, correct?
Dr. Margaret Coutts: Exactly.
Dr. Alan Gaveck: Perfect. The next thing we want to look at is the differential count. You did a diff count on this blood. A physician understands why they’re going to do a CBC and a diff count, because they’re looking for some information in this blood data that can give them clues to a diagnosis or to maybe even confirm a diagnosis. Why do you do a diff count?
Dr. Margaret Coutts: Well, it’s to characterize the product both with the cord blood when we first receive it, make sure it’s healthy and normal, that the proportions of the different cells are healthy and normal.
Dr. Alan Gaveck: Such as if you had a huge amount out of whack WBCs you may go, “Maybe this isn’t something we want to use.”
Dr. Margaret Coutts: That would go immediately into the red biohazard bag. Yes. Yeah.
Dr. Alan Gaveck: So, characterize it to make sure we have safety margins, that’s a good way to look at it.
Dr. Margaret Coutts: But we also use the hematology counts during and at the end of the process to make sure that the products meet our standards.
Dr. Alan Gaveck: Okay. Perfect. So let’s take a few of these through, and I think something I want to point out right away, that we can get some clarity on, all of these cell counts are times a million. So when we look at the WBCs, white blood cells in the Liveyon Pure Pro product, there are over 38 million WBCs, and the product we just saw on the slide before only has 31,900,000 cells. How do you have 38 million of these when we only had 31 total?
Dr. Margaret Coutts: You caught that. So, the hematology counter uses a laser, uses electrical impedance, it uses a variety of fluorescent dyes. But those cells are passing through a laser, it doesn’t miss anything. The cell counts that we looked at previously are based on images.
Dr. Alan Gaveck: Gotcha.
Dr. Margaret Coutts: So it doesn’t bother me that the two numbers don’t agree. And you can see that we use the more conservative number to fill the vials.
Dr. Alan Gaveck: Right. One is just much more specific than the other. So as we look at that, we look at that count and we look at white blood cells, do white blood cells play an important component in what we’re dealing with here with this umbilical cord blood product?
Dr. Margaret Coutts: Absolutely. They’re one of the active ingredients. They’re supplying a lot of growth factors and cytokines.
Dr. Alan Gaveck: So, over 38 million, and there are nil in the amniotic fluid, and really a small amount in both the Wharton’s jelly 1 and 2. Is that what you would expect to see in those products?
Dr. Margaret Coutts: Wharton’s jelly typically does not have a lot of cells in it, but again, I had no idea what would be in these products.
Dr. Alan Gaveck: So the next category is red blood cells. They’re all basically less than detectable or an absolute zero. Why is that important?
Dr. Margaret Coutts: Well, a very important part of our process is to remove the red blood cells, because they have the A blood type, B blood type antigens on them, so we want to remove the red blood cells. That is something that really sets our product apart is that it doesn’t have a red color. We have to go through a multi-step process to get rid of the RBCs, which as you know outnumber white blood cells about 1,000 to one in the incoming blood. And we remove them to the level that they’re not detectable.
Dr. Alan Gaveck: So actually having a negligible level in an amniotic blood product is a positive.
Dr. Margaret Coutts: Right.
Dr. Alan Gaveck: And in the amniotic fluid or the Wharton’s jelly you wouldn’t really expect to see red blood cells anyway, would you?
Dr. Margaret Coutts: No.
Dr. Alan Gaveck: So platelets are the next biggie. 100 million platelets. Tell us about platelets, what’s the function that they’re going to help us with in this product?
Dr. Margaret Coutts: They’re small but they’re powerful. They’re filled with a variety of different growth factors.
Dr. Alan Gaveck: So it’s important to have the platelets in there?
Dr. Margaret Coutts: Yes.
Dr. Alan Gaveck: And that’s one of the reasons you put it on there. As we look down to the amniotic fluid and the Wharton’s jelly, again, I mean, amniotic fluid has 3 million cells, but one of the Wharton’s jelly has 22 million and the other only has 2 million. Again, we see that wide variation [inaudible] the consistency in the product. Then, of course, we go to the last category, not the last one, other cells. I found it was interesting that you put other cells in there, because all the others don’t really have much, but amniotic membrane has about a half a million of other cells. What is an other cell?
Dr. Margaret Coutts: They couldn’t be identified. So I mean, I feel this is really important that we are, we can really thoroughly categorize our product, we know what’s in it.
Dr. Alan Gaveck: So, it’s uncategorized cells, so to speak.
Dr. Margaret Coutts: Yeah. I’m sure we could do additional tests, a lot of different histology and staining to figure out what they were, but-
Dr. Alan Gaveck: Right, right. So, hemoglobin, obviously we’re on hemoglobin because we’re back in the red blood cell [inaudible 00:15:09]. So the next thing that you did was did an analysis with growth factors. I think it’s important to point out here that growth factor analysis is a very specific type of testing. So you send it out to a third party company that specializes in growth factor analysis. I think looking straight at it, the first question I would have is, wow, umbilical cord blood product, your Pure product, has basically the same growth factor profile as amniotic membrane. Kind of shocking to me, you’re probably well aware of this sort of thing, but kind of shocking to me that they would have the same profile. Do they act the same way?
Dr. Margaret Coutts: Well, they’re the same growth factors. But, in the amniotic membrane product, you would get a one-time dose of this, because all the material is dead. With the Pure Pro product, the cells are alive, so they’ll continue to secrete growth factors for their lifetime.
Dr. Alan Gaveck: So really having a live nucleated cell is important.
Dr. Margaret Coutts: Yes, it’s beneficial, yes.
Dr. Alan Gaveck: You know, the other two categories in the growth factors are the Wharton’s jelly 1 and the Wharton’s jelly 2. In a total of four slides here, we have three of them that show that their factors are strangely different across, whether it’s concentration of cells, concentration of live cells, and what their components are in a diff count. Now we’re on a growth factor analysis, and they don’t even look like the same type of product at all.
Dr. Margaret Coutts: I was surprised.
Dr. Alan Gaveck: Not something that you were already prepared to look at.
Dr. Margaret Coutts: I’m not going to go into depth, but it was just … a manufacturer might report on X, Y and Z, but to see the products compared side by side on the same analysis for me was really helpful.
Dr. Alan Gaveck: The other thing we want to talk about that you went over in the paper was … flow cytometry. Flow cytometry is a word that’s really kicked out [inaudible] a lot in this industry.
Speaker 3: [crosstalk] start that, giving the full sentence again? The next thing I want to talk about-
Dr. Alan Gaveck: Yeah, yeah, yeah. The next thing that you speak about in the white paper is flow cytometry. Flow cytometry is a big, big phrase in the stem cell world, because everybody uses it. And when you speak about it in there, you talk about a gating process that you can add or you can include, or exclude, certain things in order to make it. But that you use no gating process, which included allowing in the dead cells because you really wanted to have not only a fair unbiased testing, but in reality the products that don’t have many live cells, they wouldn’t have shown much of anything.
Dr. Margaret Coutts: There were too few in number to be evaluated. I mean, the power of flow is you can look at individual cells, but you’re collecting data on the whole population. Hundreds of thousands, or millions of cells. But there weren’t enough live cells in some of the other products too to only look at live cells.
Dr. Alan Gaveck: We also know, and only because I had this discussion with you a few weeks ago, because I wouldn’t have really known about them, is that you can use a gating technique to maybe skew the numbers just a little bit. I remember, I was reading an article about one of the companies that had a Wharton’s jelly product, and they were claiming 90-some plus percent mesenchymal stem cells, and I came to you and I said, “Is this possible?” And you said, “Well, no. Not really in the natural product. But there are ways that it can be done that flow cytometry can show that.” And you had spoken to an expert in flow cytometry, Dr. [inaudible 00:19:05]. And he’d explained to you the process. Can you just put it in kind of a really simple, synthesized, how can you take something, we know these birth tissues have single digit, right, stem cells, CD90 so to speak. How do you make a product that you say we have 90%?
Dr. Margaret Coutts: Gating is a legitimate thing to do in flow analysis, but you just have to be honest that your … let’s say looking at a subpopulation of a subpopulation of a subpopulation. So typically, you exclude dead cells, because they’re typically not of clinical or diagnostic value. So, you’re cutting out that population. And you might say, “Well, I don’t want to look at the white blood cells. So I’m going to exclude them or gate them out. And I’m only going to look at this subpopulation.” So you might go through two, or three, or four, five or six gates, and your final panel would say, “Of the cells that we have here, 90% of them are positive for the marker of interest.” So that’s true, but it’s less than a half truth if you don’t explain how you got to that number.
Dr. Alan Gaveck: So, that’s good to know about flow cytometry. One of the other things that I would like to talk about is really your analysis on the flow cytometry. You really picked out five CDs differentiators and you listed them out there. If you could briefly, I don’t expect you to give a full analysis on every one of the [inaudible] differentiators, but just give us an idea, what does this mean? What are we looking at CD19?
Dr. Margaret Coutts: CD19 is a very specific marker, it identifies B cells. CD34 is a widely accepted marker for hematopoietic stem cells and other type of stem cells. C45 and the gray bar on this graph, you can see that the Y axis had to be broken so we could jump from 2 million to 25 million. So there’s a high number of white blood cells here, which verifies what we saw with the hematology counter. Now, CD73 is a marker on mesenchymal stem cells, but it’s also on some endothelial, epithelial cells, endothelial and epithelial. CD90 is another mesenchymal stem cells marker. But it’s also on thymocytes, the immature T cells in your thymus and on neural cells too. So, the markers can be used to characterize a single cell, it can be used to characterize the overall population, but you have to be a little careful not to over interpret it, because every CD90 positive cell is not necessarily a mesenchymal stem cell.
Dr. Alan Gaveck: Perfect. In your concluding remarks, you spoke that manufacturers, many manufacturers, make many claims, but they really don’t put down the data to support that. Part of the reason for doing this study was to create that data and really for multiple reasons, to put that data out there and allow physicians to really have an informed, to make an informed decision, and I think that’s a big part of this paper.
Dr. Margaret Coutts: Yeah, that’s a really important question. I mean, physicians and their patients should be able to make an informed decision. But it was also to satisfy my curiosity too, to be honest, because some of these products come labeled one mil, and I’m just, “One mil of what?”
Dr. Alan Gaveck: One mil of product, right?
Dr. Margaret Coutts: Yeah.
Dr. Alan Gaveck: I think you were very fair in your closing that you said that all of the birth tissues do have the potential to help in the healing process, but we know that the growth factor is only going to last for a certain period of time. Even the live nucleated cells, the growth factors are going to have a finite life. But like you said earlier, just the growth factor without a nucleated cell is pretty much a one and done shot. And you get your way to go. So the bottom line comes down to live cells do matter.
Dr. Margaret Coutts: Yes. I mean, they’ll continue to secrete those growth factors. They can influence or help the recipient to cell-cell interactions. They can also secrete exosomes throughout their lifetime.
Dr. Alan Gaveck: Then, see now we’re going, now we have a complete new topic that we can talk on down the future. It’s been such a pleasure to have you here today and to be able to talk with you. Any closing arguments? Any words of wisdom?
Dr. Margaret Coutts: I think the important point was that live cells are going to be a better value to the recipient and the fact that many manufacturers make claims about stem cells, but if all the cells in your product are dead, you just don’t have the potential to have stem cell activity.
Dr. Alan Gaveck: Thank you so much for not only your expertise, but your integrity. I respect the fact that you wrote the paper because you were excited to find out that that learning itch was there. We look forward to definitely having you back in the future. But this is Dr. Margaret Coutts. I’m Dr. Gaveck with Liveyon Pure and we will see you next time.
Dr. Margaret Coutts: Thank you very much.