Umbilical Cord Stem Cell Treatments: Liveyon Product Review II
POSTED ON 8/12/2017 IN Research BY Christopher Centeno
A while back I posted a review of an umbilical cord product called Liveyon. A white paper issued by the company had been sent by a colleague, and he wanted my opinion. Like every white paper I had seen written by an amniotic or cord-blood vendor, it has serious scientific flaws as it purported to show that the product contained live stem cells. Hence, I performed my own scientific review of the document, posted it on the blog, and forgot about it. Then, a few months ago, I began hearing that a critique of my review had been posted by the company, and I was just too busy to try and locate it. This week a reader of my blog got ahold of it and sent it along. So here's my review of that critique.
What Is Cord Blood, and What Is Liveyon?
Cord blood is what it sounds like: it's the blood taken from the umbilical cord of a baby during or after a live birth. This has been the mainstay of an entire industry that stores umbilical cord blood, and that product has been used to treat pediatric cancer. In fact, right now, the only FDA approved indication for cord blood is cancers of the blood, like leukemia and lymphoma. Cord blood contains mostly blood-producing stem cells with very, very few mesenchymal stem cells. In fact, the FDA website has this warning concerning its use:
“Because cord blood contains stem cells, there have been stem cell fraud cases related to cord blood,” says Wonnacott. “Consumers may think that stem cells can cure any disease, but science doesn't show this to be the case. Patients should be skeptical if cord blood is being promoted for uses other than blood stem cell regeneration.”
I first became aware that cord-blood banks were marketing to physicians to use cord blood for orthopedic treatments when I was contacted by a local cord-blood bank. First, I knew enough about the regulatory hurdles to know that using cord blood in orthopedic conditions wasn't allowed. The salesperson I spoke to pushed back, saying it was fine, but when I asked to speak to the medical director of the cord-blood facility, he admitted that an FDA approval would be needed to start using his product as part of an approved trial. I also knew enough to know that this trial would take 5–10 years and cost hundreds of millions of dollars. When I asked him where the research was that showed that his cord-blood product could help orthopedic problems, he admitted there was none. I hung up.
My First Liveyon Review
My first review of the Liveyon cord-blood product and its white paper is here in video form:
In this review, I noted that the product was the result of a simple online 361 tissue registration. To learn more about how that differs from an FDA approved product, see the video below: I then looked at the reported viability rates for Liveyon and remarked that they were much lower than for fresh tissue, such as bone marrow concentrate, and that this was a concern. I also pointed out that the flow cytometry data the company used to try to find mesenchymal stem cells (MSCs) in the product referenced a methods paper, but then didn't follow those methods. Finally, I pointed out that the growth factor and cytokine levels reported in the white paper were low compared to commonly used orthobiologics.
Does Cord Blood Have MSCs?
Before I get into my critique of the Liveyon white paper, it's probably worth spending two minutes to watch this video on whether the scientific literature has been able to find MSCs in cord blood: [Spoiler alert: cord blood contains very, very few MSCs.]
My Review of the Critique
OK, let's do this! I'll take the Liveyon critique part by part and give my response. First, there will be the Liveyon response to my last blog post (in italics) and then my answers.
FDA Registered vs FDA Approved
Liveyon: Centeno contends that in order to have live cells in a product that the product must be FDA approved and fall under paragraph 351 which requires Stage 1, 2, and 3 clinical trials. False: Simply reading the guidelines for paragraph 361 you can see this is not true. Under 361 guidelines for “minimal manipulation”, the guideline specifically states; “For cells or non-structural tissue, the processing does not alter the relevant biological characteristics.” The definition acknowledges cells. Under the “homologous use portion” of 361 the FDA defines homologous use as the; “replacement or supplementation of a recipient's cells or tissue with an HCT/P. (Human cells, tissues, and cellular and Tissue-Based Products). The definition clearly acknowledges the presence of live cells in the 361 guidelines.
My Response: Liveyon has left out a critical part of the 1271 regulation. Under 21 CFR 1271 subpart A section 4(i) it states: "The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function." The regulation here is referring to donor cells, which means it applies to cord blood. In addition, living mesenchymal stem cells, which are being claimed by Liveyon in its white paper, do have systemic effects and are dependent on their metabolic activity for their primary function. Hence, the claim that Liveyon has MSCs pushes it out of a simple, online tissue registration (361 regulation) and into the 351 drug regulations. As a cell drug, Liveyon would require clinical trials and full FDA approval before the product can be sold or marketed. However, that's solely based on the marketing claim that the product has MSCs. Now let's delve into whether the Liveyon white paper proved that it has any living and functional cells, including MSCs.
None of the product has live cells
Liveyon: The gold standard for laboratory testing of live vs. dead cells in the research world is using AO/PI stain as explained by Dr. Rafael Gonzalez, Ph.D, who has more than 15 years of laboratory experience working with and testing stem cells.
My Response: As I have published online many times, a simple live/dead test tells us very little about the overall viability and the ability of the cells in this product to function. Basically, a live/dead test is inadequate. Please see the video below for more details on that topic:
The white paper reported awful viability rates for Liveyon
Liveyon: Awful survival rates out of cryopreservation. Gives the example of not knowing whether the cells are 'live and healthy' or 'in the ICU' because no testing was done for clinical viability. When using AO/PI stains to test cell viability, any cell that is not live and healthy will begin to have cell wall permeability and the AO/PI stain will count it as a dead cell. Also, clinical viability is well known in both the research world and the clinical world for cells derived from UC blood.
Centeno: Huh? Nothing like answering a challenge to the answer by just repeating the previous answer? This is just a regurgitation of how simple live/dead stains work (see video above) without any answer to the question of whether the low viability of the Liveyon product means that the cells are dying or severely dysfunctional. This question could have been answered with data from an apoptotic assay or culture data on the product.
Fresh tissue or properly cryopreserved cells typically have viability rates of >90%.
Liveyon: Normal recovery rates out of cryopreservation are approximately 90%. True: In the lab it is normal to have recovery rates of 90% and our lab has had rates as high as 93% and 94% but Liveyon specifically asked the lab to treat the cells in a way similar to clinical conditions. The cells were sent to the lab on dry ice, the lab removed them from the dry ice and placed them in a -80C freezer for over a week until they were ready to test them. They then removed the cells, thawed them in the palm of their hand, to simulate a clinical setting. Literature reports a survival rate of 50% to 70% in a clinical setting.
Centeno: Huh? Again, the issue is that cells that are properly cryopreserved and thawed under controlled conditions have viability rates that are typically >90% versus the 50–70% reported by Liveyon. Here, the answer states that while Liveyon can get higher rates of recovery from fresh tissue, when their product is sent to a doctor and shock-thawed in his or her office, they get much poorer viability. This was the point of the original review, so not sure how this answer changes anything other than to confirm poorer viability rates for this product.
The low survival rates mean the cells are beat up
Liveyon: This statement is proven false by the findings of substantial concentrations of CD factors, growth factors, interleukins, and chemokines in the product.
Centeno: Again, this one could have been answered with flow cytometry studies looking for apoptotic markers or data on culturing the cells in the Liveyon product to see if any survive. However, their answer is instead a statement about growth factors and cytokines? Huh? These are chemicals that exist in cord blood whether or not there are dead cells. These chemicals can survive harsh conditions, such as storage and freezing; hence, their presence tells us nothing about how many cells are functional despite the low viability of the product.
There was no testing for apoptotic markers
Liveyon: False: A cardinal sign that a cell is undergoing apoptosis is cell wall permeability, so any cell that is no longer healthy and moving towards apoptosis will positively test as a dead cell and will be counted as a dead cell.
Centeno: Again, Liveyon again makes the cardinal mistake of confusing apoptosis with cell death. Apoptosis means the cell is dying. While Liveyon contends that apoptotic cells have incompetent membranes, that's not what can be found in the peer-reviewed literature. For example, "…apoptosis is ATP-dependent and is characterized by cell shrinkage, maintenance of plasma membrane integrity…" (Curr Protoc Pharmacol. 2004 September 1; 0 12). Note that apoptosis is characterized by an intact cell membrane. This whole discussion is important because all Liveyon tested for was the presence of broken cell membranes (dead cells) and not apoptotic cells (dying cells). This again could have easily been answered with data from a simple apoptotic assay on the Liveyon product, such as the commonly used flow cytometry assay using Annexin to bind to the phosphatidylserine marker.
There was no testing of the Liveyon cells to see if they could survive to function and proliferate in culture.
Liveyon: True: The scope of this paper was meant to show live cell viability and measure the TNC's in the product, not to re-prove what 50 years of stem cell study has already proven.
Centeno: Huh? Fifty years of research on the Liveyon product? On cord blood that's shipped and used for orthopedic purposes after being shock-thawed in a doctor's office? The response in nonsensical.
Liveyon didn't follow the ISCT guidelines it quoted in trying to identify MSCs.
Liveyon: While it is true that all 3 components make up an MSC, research has shown that CD90 is the most clinically significant and we are testing for clinical application, not lab analysis. The purpose of this paper was not to do an in-depth lab analysis of the cells to show all markers, or to show differentiation into osteoblasts, adipocytes, or chondrocytes. Rather, the white paper was written to show the presence of clinically important makers, growth factors, and chemokines. UC blood has already been tested extensively for many years to show all the factors at the laboratory level.
Centeno: LOL. As I have discussed, in order to identify an MSC via flow cytometry, you need a defined subset of cell-surface markers to be present on the cells and some that need to be absent. Liveyon quoted the definitive paper on what those markers need to be, but then ignored the paper. So instead of testing for all of the markers, they left out about half of them. The response now seems to be to pick one of the many markers that need to be present and absent and dig in on just that one. They chose CD90, a marker found on lots of cell types, only one of which is an MSC. However, it doesn't by itself identify an MSC. The response claims that CD90 is the most "clinically relevant"? Based on what research? None is quoted. However, to back up that statement, Liveyon would need studies with their product showing that the number of CD90+ cells was associated with a positive outcome in orthopedic therapies. After a thorough review of the US Library of Medicine this morning, no such research exists.
The growth factor content of Liveyon is less than common orthobiologics like PRP and BMC.
Liveyon-What he fails to mention is that VEGF, FGF-2, and SCF in PRP will only function for one half-life, or approximately 12 to 72 hours because there are no live cells in PRP. Whereas, in the UC product, there are documented live cells that will continue to produce these growth factors for weeks and possibly months. He also has no controls in his studies and is not comparing to the same volume to or cell counts.
Centeno: Second LOL. Not sure where to begin with this ridiculous and nonsensical answer. Here goes:
- The half-life of any substance is the length of time it takes for the level of that substance to drop to half of its original level. This is because it degrades or it is eliminated (i.e., by the kidney or liver).
- The claim is that growth factors secreted by platelets only function for one "half-life." Since a half-life is a measure of time and the serum level of the chemical, this makes no sense, as the concept of a half-life and acting at cell receptors are not the same. Any chemical can have effects beyond its half-life depending on the original level of that substance in the area, the number of receptors for the chemical on the target cells, the amount of time the chemical sticks around until it's deactivated, etc...
- There are no live cells in PRP. Platelets are living cell fragments that respond to their microenvironment. They actively secrete growth factors for 7–10 days after they are activated by exposed collagen. So the idea that growth factors are only present for 12–72 hours after PRP is administered is not accurate.
- Based on what I've discussed, there may be no live and functional cells in Liveyon, so comparing the ability of these cells to secrete growth factors (a test not done in the Liveyon white paper) to PRP, which has been extensively tested to release growth factors over time, is nonsensical.
- There are live cells in Liveyon which will continue to excrete growth factors for weeks and possibly months. See the discussion above. The Liveyon white paper didn't show that the product had live and functional cells capable of secreting live growth factors. That would have required a test of the cells in culture, something Liveyon didn't do.
- No controls and different volumes. Controls for what? The volumes studied in the papers showing that PRP, human serum, and BMC all have higher growth factor and cytokine levels than those reported by Liveyon all used clinically meaningful volumes that are used in common orthobiologic treatments.
My comparison of IL-1ra in the Liveyon product to BMC is not clinically relevant.
Liveyon: Yes, if you take bone marrow from a healthy 20 or 30-year-old, you will have extensive amounts of IL-1ra. The problem is that most of the target audience that needs these types of cells is more like 50, or 60, or 70 and beyond AND, many of them have an autoimmune disease that further diminishes these levels of IL-1ra. Also, note that Centeno did NOT compare VEGF, FGF-2, and SCF in BMAC to UC cells. Sometimes what is NOT said is quite telling.
Centeno: The critique here is that I didn't account for declining GF levels in older patients as compared to younger patients. While it's true that older patients have lower growth factor and cytokine levels, that's easily adjusted by just increasing the concentration of PRP. We've studied this extensively with mesenchymal stem cells and tenocytes, showing that increasing the concentration of PRP not only dramatically increases growth factor levels, but also the cellular proliferation of MSCs and tenocytes.
A second issue is that Liveyon believes most patients being treated with PRP have autoimmune diseases. Huh? The company is marketing this product for use in orthopedic injuries, and only a tiny fraction of these patients have been diagnosed with an autoimmune disease.
Yes, I didn't compare the growth factors listed to BMC because BMC has both platelets and serum and the comparison to both PRP and serum had already been made.
The upshot? I have been hearing about the Liveyon response to my review for months and, frankly, I expected a bit more science. Instead, as shown above, most of the responses either just reiterate the white paper talking points or don't make scientific sense. In the meantime, might Liveyon or any other low-viability cord-blood product help orthopedic injuries? Maybe. We just don't have any data that shows that's the case. In addition, as I've shown above, by Liveyon or any of these companies arguing that their products have living cells that have metabolic activity (here that the cells live for weeks or months in the donor and excrete growth factors that will impact the body systemically), the company is arguing for the FDA to take its product off the market and reclassify it as a 351 drug. Despite that regulatory faux pas, nothing about the Liveyon white paper demonstrates the presence of live and functional MSCs.
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