One of the nuttier regenerative medicine trends of the last two years has been the explosion in physicians using what they call “amniotic” or “placental” stem cells. The problem with the concept is that none of these products contain any living cells, let alone stem cells, so now a few doctors who got the memo have renamed the products a few different ways. One of those is “stem cell factors.” Others claim that these products will make your older stem cells function better. Is there any science behind either of these claims, or are they as “out there” as claiming dead tissue is really a vial of millions of viable stem cells. Let’s take a look.
I’ve blogged a number of times on the fact that many websites and physicians will tell patients that a vial of amniotic fluid or membrane derived from the placenta of a baby is rich in stem cells. This is despite many of these products being gamma irradiated to kill every living thing or dehydrated and delivered in a powder. The Interventional Orthopedics Foundation (IOF), a 501c3 nonprofit, tested these products and found that none contained any living or viable cells, let alone stem cells.
Now some the physicians using this stuff, who have gotten the memo that it’s dead tissue, have reworked the messaging. The concept is that it’s really “stem cells factors” and those can assist older cells in functioning better. So let’s examine those claims as I would be all for using amniotic products if any of that were true.
The term “stem cell factors” seems to be a mishmash of two concepts—stem cells and growth factors. It doesn’t really exist in science, so it’s a made-up term. However, it implies that amniotic or placental tissue contains growth factors directly produced by stem cells. So let’s explore that idea.
What are growth factors? These are nature’s espresso shots for cells. They literally act a lot like caffeine in a person: they make tired cells work faster and harder.
Amniotic fluid is the stuff that surrounds the baby. In the last trimester it gets very diluted by the baby’s urine, hence my statement that if you ask an OB/GYN what’s in the amniotic fluid of a term baby, it’s really mostly baby piss. Be that as it may, in its most concentrated form (which is not what is being sold in these vials), during the second trimester, there is stem cell content of 0.5% to 1.5% of the total cells floating around in that fluid. A percentage that’s roughly equivalent to adult fat. In addition, since there are far fewer total cells in amniotic fluid than in bone marrow, the absolute number of stem cells is smaller than in bone marrow. Add in the dilution mentioned above due to third-trimester baby piss and you have a stem cell cocktail that is more feeble in numbers than the two most commonly used tissues in regenerative medicine today. So in reality, if we took fatty tissue or bone marrow and killed all the cells in them and put them in a bottle for resale, we could claim that they were “stem cell factors.”
So if these amniotic and placental products are “stem cell factors” because they contain large numbers of dead stem cells, right off the bat, the number of stem cells in them to start is smaller than what can be had by taking a sample of fat or bone marrow from a patient. Hence, we have a problem. However, maybe it’s either the amount of growth factors in them or their ability to stimulate growth or health in older stem cells that we should be focusing on, so let’s explore those two ideas.
Luckily, independent data from a third-party nonprofit is available on these products. The IOF tested many of them for growth factor (GF) content versus a control that was nonconcentrated pooled platelet lysate (pHPL). This is creating a dilute platelet rich plasma and exacting the growth factors out of the platelets that would have normally been released over 7–10 days. So how did these products fare? Not great.
The table above is the result of using multiplex, microarray ELISA on samples of Ovation (a chorion-derived product), BioDRestore (an amniotic-fluid product), and pooled platelet lysate as a control. The bold-face number represents the winner of that growth-factor contest. As you can see, the amniotic-fluid product loses all races in that it has the poorest growth-factor content of any of the products tested. How poor? For key growth factors thought to be important players in orthopedic healing, like PDGF, TIMP-1 and TIMP-2, TGF-beta, and VEGF, it has only a fraction of the growth-factor levels of a really weak platelet rich plasma derived from a combination of younger and older blood donors. The Ovation (which is a chorionic product pulled from the market due to having some living cells and being thus reclassified as a biologic drug), wins the race for bFGF, HGF, and IL-8. For orthopedic applications, the bFGF could be important for tendon and ligament healing. The high IL-8 levels could, however, be a problem as this chemokine is pro-inflammatory and is involved in tissue-debris cleanup. So depending on how that’s viewed, it could be bad or good.
In summary, the amniotic tissue product here has very weak growth-factor levels prepared to a weak PRP preparation. Hence, the idea that amniotic fluid can be called a bottle of “stem cell factors” has failed along two important tests. First, the actual stem cell content when the cells are living is very low. Second, the levels of growth factors present are also low. However, maybe it’s not the level of the growth factors that matters. Maybe it’s how they perform in stimulating the stem cells of a real patient. Let’s explore that a bit.
The below experiments were carried out by the IOF using older bone marrow mesenchymal stem cells that were placed in lab culture with either a serum-free (growth-factor poor) or pooled platelet lysate (growth-factor rich) media.
Caution: Now we’re leaving the more simple science of looking at growth-factor levels and moving into the more complex world of how stem cells grow and what we can measure about that growth. Two assays were done here. The first is a DNA quantification (PicoGreen). This is just a way to quantify how many cells grew by looking at the total DNA in the sample (more DNA equals more cells). The second is a reactive oxygen species (ROS) assay. These are nasty chemicals produced by older and sick cells that cause tissue damage. Younger cells don’t produce them and older cells, regrettably, do. So the lower the ROS level, the more the cell is behaving like a young cell, and the higher the ROS levels, the more the cell is behaving like an old cell. These tests were done due to the theory that adding in young amniotic- or placental-tissue products to older cells would cause the old cells to behave like young cells—a super-attractive concept if it were true.
So what happened? The good news is that two amniotic products, due to their growth-factor content, did stimulate the older stem cells to grow (see B above). The best was AmnioFix with BioDRestore putting in a good show. The other products were the same or less than a growth-factor-poor media, which isn’t saying much as stem cells won’t grow well in a growth-factor-poor media. How about when we use a growth-factor-rich media? That comparison is in D above. As you can see, when we add in a weak growth-factor cocktail that’s commonly used to grow stem cells (pooled platelet lysate), most of the effects of the growth factors in the amniotic products become swamped. Meaning that the effects we saw in growth-factor-poor serum are negated. Why is this happening? As we saw above, the amount of growth factors in amniotic products that would be important to stem cells (TGF-b, PDGF, VEGF) are much lower in these products than platelet preps. So the take-home is that while some of these products can help stem cells grow, they’re likely not as good at that as a simple PRP.
One of the more interesting ideas out there is that young tissue can make older cells perform better, more like younger cells. While this has been shown with live younger tissue, what happens when we add dead tissue, like that found in these amniotic and placental tissue products? Let’s look.
Before I review the data, it’s worth noting that at least one Texas website has fluorescent microscopy pictures on it that purport to show that by exposing bone-marrow mesenchymal stem cells (MSC) to its proprietary type of amniotic-tissue product, the cells just “look better” under a microscope. I personally can’t tell the difference, and I’ve seen many MSCs under a scope in my day. However, the data we’ll review now doesn’t require an eyeball of how the cells look, but it is measuring the bad stuff the cells produce.
Take a look at C above. This is the level of bad ROS chemicals these stem cells produce when exposed to the various products and controls. Take a look at the well-performing AmnioFix. It’s causing a huge spike in ROS “old man” nasty chemicals to be produced by the older stem cells. Ovation, which also had more GFs in some categories, is also causing high ROS levels. There may be a clue as to why this is happening in looking at the ROS levels of the complete culture media. This contained fetal-calf serum, which is often used to grow cells in culture. If I had to venture an educated guess, the reason why we’re seeing the biggest ROS reaction in both AmnioFix and the bovine serum is that both are producing a foreign-tissue reaction in the human stem cells. AmnioFix and Ovation are the most cellular of these products, hence they would have components that the stem cells are recognizing as “not self,” and it’s causing them to go into inflammatory defense mode. This is consistent with reports from colleagues who have used these products on human knees and witnessed severe inflammatory reactions.
Not so much.
Do these products help older stem cells act younger? Not based on this data. In fact, the amount of nasty ROS levels seen in using some products is concerning in that they’re doing the opposite—making cells behave older. It’s also notable that none of them reduced ROS levels (i.e., made stem cells look or behave younger).
The upshot? This data doesn’t reveal any compelling reason to use amniotic- or placental-tissue products. It also doesn’t support that calling them “stem cell factors” is appropriate as that suggests they have some special properties, like they’re growth-factor rich (they aren’t) or they help stem cells in some way (they don’t). So at this point, if you hear them being called that, it’s more marketing hype than any proven reality.
In the meantime, I want to remain open to someone proving that these products have their unique place in the regenerative medicine pantheon. So as I often do, I will leave an open challenge on the table. If some company believes it has the world’s best amniotic- or placental-tissue product that can act as a groundbreaking regenerative medicine product or truly help older stem cells, then bring it. We will spend our money testing it (we don’t take money from manufacturers), and we will all agree on what will be tested, and manufacturers can even supervise our staff running the tests so that they feel comfortable they’re getting a fair shake. The goal will be tests that show that what’s in the bottle is better than a Regenexx PRP preparation. If your product passes muster, we will be a huge customer if those results translate from the lab into real patients.
About the Author
Christopher J. Centeno, M.D. is an international expert and specialist in regenerative medicine and the clinical use of mesenchymal stem cells in orthopedics. He is board certified in physical medicine as well as rehabilitation and in pain management through The American Board of Physical Medicine and Rehabilitation.…