Liveyon, according to its website, bills itself as “the exclusive worldwide distributor of a regenerative medicine product that is derived from umbilical cord. This product contains cells, stem cells and growth factors which may serve as a therapy for various degenerative diseases/disorders.” Today, I’m going to provide a review of a white paper the company distributes entitled “Regenerative Medicine Product Report: Independent Third Party Studies”. However, the bigger message here is that physicians need a resource to learn how to interpret viability testing, not just on Liveyon’s products but on any product.
Physicians are constantly bombarded with marketing materials from companies wanting us to purchase their products, and most physicians have a hard time sorting the good from the bad. I’m just using Liveyon as a great example here because its report was in the last batch of marketing material making the physician rounds, and this report purported to show that their regenerative medicine product contains many live mesenchymal stem cells (MSCs).
Before I begin this Liveyon review, you need to understand FDA registered and FDA approved.
FDA Registered Does Not Equal FDA Approved
FDA-registered is not the same as FDA-approved. Establishments that work with cell and tissue products are required under section 361 by the FDA to register. This is an online process: you go online, you provide your information, and you pay your fee. FDA approved is much more involved, requiring a great deal of time and a great deal of money. Approval requires a drug process with three-phase clinical trials, and it costs a couple hundred million dollars over a number of years.
So all of these birth tissues (e.g., cord, amniotic, placenta) are FDA registered only, and yet distributors are claiming they have live viable cells, which is technically not appropriate because if they really had live viable cells or even if that’s only the marketing, they would have to be FDA approved.
Wanted: “Live” Stem Cells…Some Imagination Required
While we continue to see these FDA-registered birth-tissue vendors pushing reports supposedly supporting that their products have live and viable cells, the truth is, so far, none of these products that we’ve tested have actually had live cells. I really do have an interest in this—I’d love to find an amniotic or cord-tissue company that can actually demonstrate excellent clinical viability of at least 500K of mesenchymal stem cells per vial. Our network would be their largest customer by far. So I care deeply about whether the claims on these white papers produced by these companies hold water. But I won’t purchase an amniotic “stem cell” product without testing it first.
Why Birth-Tissue Stem Cells Aren’t Passing the Test
Every single company’s amniotic or cord cell product that we have lab tested or whose white papers we’ve reviewed fails along three basic lines:
- Only simple live/dead testing with no actual testing of true clinical viability
- Awful cell survival rates out of cryopreservation
- Poor flow cytometry that’s really incapable of identifying stem cells
Let’s jump into the Liveyon review data. Please watch the video above to view detailed graphs and other content that follow along with my write-up here.
Simple Live/Dead Testing
The disturbing thing about simple live/dead testing is that we see 59% and 63% viability, which is pretty awful. More importantly, simple live/dead testing doesn’t tell you whether or not the cell is 1) alive and healthy, 2) on its deathbed because it’s been badly beaten up, or 3) already dead. The best you can get from this test is that the cells that fall under 1 and 2 are live cells, and the cells that fall under 3 are dead cells. So this test is too broad for a product that has low viability. Where live/dead stains come in handy is when you’ve got a known live cell process and you’re trying to fine tune viability.
Cryopreservation Survival Rates
Normal recovery rates out of cryopreservation for cells that are properly handled are 90% or above. So if you’re only getting recovery rates of 50–60% for hardy fetal cells like this, the cells have likely been through a cell version of Dante’s Inferno—they’ve undergone a serious beating. So right off the bat, you’d have serious concerns that any of the recovered cells would live to be viable.
Despite this, Liveyon makes no attempt to test beyond just the simple live/dead test. It doesn’t test for apoptotic markers (meaning the cells are dying) or to culture the cells to see what will grow.
Liveyon says: “The remainder of the product was evaluated for flow cytometry using BD Accuri C6 Glow Cytometer (Fig 2). Briefly, the cells in the Liveyon regenerative medicine product were stained with FITC or PE directly conjugated antibodies to CD34, CD45, CD90, HLA-DR and HLA-ABC. These markers were chosen as they are standard markers used for MSCs.” This is basically telling us that they’ve chosen their cell markers, which is what flow cytometry tests for, based on standard markers used for mesenchymal stem cells. Which paper does Liveyon reference to support its statement? THE position paper, put out a number of years ago, on determining if you have mesenchymal stem cells: “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy [ISCT] position statement” (Cryotherapy. 2006;8(4):315–7).
The ISCT paper says to identify an MSC, you need these three markers that are on the surface of the cell: CD73+, CD105+, and CD90+. The cells also need to lack expression of CD45-, CD34-, CD14- or CD11b-, CD79alpha- or CD19-, and HLA-DR-. Then, and only then, does the flow cytometry say that there are probably MSCs.
If these criteria all exist, the cells must also be grown out in plastic adherent culture, and the cells must differentiate to osteoblasts, adipocytes, and chondroblasts in vitro, or in the lab. Neither of these things were done in Liveyon’s case. All that was done was the flow cytometry testing.
Liveyon Review: How Did the White Paper Do on Flow Cytometry Testing?
Not too good. While Liveyon tested for CD 90, for some reason, it left out CD105 and CD73. Liveyon ultimately proved that its cells lacked expression of CD45 and CD34, but it completely skipped over CD14 or CD11. They did test HLA-DR. So though Liveyon references the ISCT paper, the “stem cells” weren’t tested using the ISCT standards.
Let’s break this down a bit. Liveyon’s “MSCs” had CD90…but what else has this marker?
- Neuronal cells
- A subset of fetal liver cells
- Fetal thymocytes
- Activated endothelial cells
Its “MSCs” do have HLA-ABC, but this is expressed by all human nucleated cells and is central in cell-mediated immune response and tumor surveillance. So no big deal there.
Liveyon’s “MSCs” didn’t have HLA-DR, a common T-cell receptor, which is good. They also didn’t have CD34 and CD45, so we know they’re not HSCs (hematopoietic stem cells), and we know they’re not lymphocytes (lymphocyte common antigen), at least the subset that they tested.
So what cells are CD90+, HLA-ABC+, and CD34-, CD45-, and HLA-DLR-? Well, they’re certainly not MSCs, meaning not the flow cytometry signature of MSCs. There may be MSCs in that subset, but that can’t be determined by the poor job of flow cytometry that was done here that didn’t follow the ISCT guidelines.
Since Liveyon didn’t determine if the cells were plastic adherent in culture and they didn’t try to differentiate them, we really have no idea whether or not there were MSCs in this sample.
The growth factors in Liveyon don’t fare any better than the flow data. Be sure to watch the video to view comparison charts between Liveyon’s growth factors and our testing of 1X PRP, serum, and bone marrow concentrate (BMC). The graph shows comparisons of four things:
- VEGF (vascular endothelial growth factor)
- FGF (fibroblast growth factor)
- SCF (stem cell factor)
- IL-1ra (interleukin 1 receptor antagonist)
VEGF for Liveyon was less than 35; our lowest concentration 1X PRP was 56. FGF for Liveyon was about 5; our 1X PRP was 61.4. SCF for Liveyon was just over 5; human serum—forget about PRP, this is just the serum— was 1,000. IL-1ra for Liveyon was just under 2,000; Bone Marrow Concentrate was 13,482 in one study. Hence I found nothing in the chemical signature that impressed upon me that this is a stem cell or regenerative medicine product that’s better than PRP, serum, or bone marrow concentrate.
- The viability rates are so dismal that it’s highly likely all of this product is dying on the thaw.
- This white paper makes no attempt to determine if these cells are functionally viable (i.e., if they have apoptotic markers or culturing them).
- The flow cytometry data doesn’t have the ability to identify an MSC. And Liveyon didn’t do the other two tests for MSCs either.
- The growth factor levels are far less than PRP, serum, or BMC.
Though this Liveyon review wasn’t a good one, if someone can find a single amniotic or cord blood cell product that isn’t a massive disappointment and one that can top a $200 PRP kit, I’m all in. Until then, as long as I continue to get flawed data on birth-tissue “stem cell” products, I’ll continue to tear it all apart. Why? Physicians need to know what they are purchasing for their patients.