Stem Cytokine "Tissue of Origin" Issues (Part 4 of a Series)

In real estate, it’s “location, location, location”. It’s not much different in regenerative research into cytokine signaling networks. We like to call it “tissue of origin”. Because even though mesenchymal stem cells from many different tissues can be grown in culture, and their cytokines harvested, the resulting cocktail is really not the same. Different tissues have differing proclivities in terms of which cytokines they produce, how much they produce, and what else they produce along with those cytokines. The differences are not subtle.

Separating from Marrow from Fat

We will make our main focus the differences between adipose-derived mesenchymal stem cell (AD-MSC) and bone marrow-derived stem cell (AD-MSC) cytokines and related biochemicals. Adipose tissue (fat) has emerged as an alternate source for BM-MSC’s, because they are cheaper and easier to grow in culture.  Fat is thrown away daily in large quantities from liposuction procedures. The idea of recycling medical waste has appeal for some. You don’t have to pay for bone marrow donations, but fat is free. AD-MSC  are similar to BM-MSC in terms of morphology and surface marker expression. However, all stem cells (including MSC’s) differ in terms of gene expression based on their niche (where they live, and what are the other functions of that tissue).

The questions we want to address is this: what are the key differences between marrow- derived and fat-derived in terms of production of the cytokines and related chemicals important to healing and regeneration of skin? How do these cytokines add up in terms of the schema we presented in part 1, part 2, and part 3 of this series – in particular do they result in a balance that is inflammatory, neutral, or anti-inflammatory?  Are there other chemicals being produced that might cause concerns in regards to anti-aging?

We are going to start with some studies done in rats, which is fine because cytokines and their functions are remarkably conserved across mammals, and rat cytokines are 99.8% the same as our own and function just like ours. We have others done on the human proteome (cytokines and other proteins) with similar results.

Table 1 at left shows the difference in gene regulation comparing adipose with marrow derived MSC’s. The list includes cytokines and other chemicals. All show a difference of >10 times more (fat>marrow). Note that 16 of the genes have inflammatory molecules as their products, and 4 have anti-inflammatory qualities. Note the presence of the adipokine leptin, a fat hormone, and inflammatory mediator. Note that IL-1 is present at 38 times the amount expressed by BM-MSC’s.  IL-1 is one of the most potent inflammatory cytokines.  Note also the balance between upregulated inflammatory and anti-inflammatory proteins (more than 4:1 ratio in favor of inflammation).

Next we will do the same but in the opposite direction. Table 2 demonstrates gene expressions present in BM-MSC’s at more than 10 times the amount in AD-MSC’s.    Unlike the pro-inflammatory ratio found in table 1, the genes upregulated in BM-MSC’s are predominantly anti-inflammatory (by a ratio > 2:1).  Some were upregulated as much as 200 fold (200 times) the levels in AD-MSC’s.

Many of the other upregulated gene products are transport proteins and matrix proteins (for elastin and collagen production), as well as key enzymes involved in regeneration.

Unlike AD-MSC upregulation of the protease MMP-9, BM-MSC demonstrate upregulated MMP-12, but also an inhibitor of metalloproteases, TIMP-3. Thus there is a balance between the breakdown of collagen, and inhibition of same, not present in the AD-MSC upregulated gene profile.

The authors of this study conclude that “AD-MSC ‘s secrete significantly larger amounts of inflammatory cytokines than BM-MSC’s” and that this “…suggests the importance of selecting the appropriate cell type.”

We agree.

Are Adipose derived stem cells always inflammatory?

The evidence base is conflicted about whether AD-MSC’s in vivo have an inflammatory or anti-inflammatory effect. In some experiments, it seems that injected AD-MSC’s reduce inflammation in target tissues, while in other studies the opposite seems to be true. This conflict may reflect the fact that AD-MSC’s may not always be the same.  But how can that be? Recall that that there are several key variables that affect how stem function in the laboratory (in vitro) as well as if injected into the body (in vivo).  One of these is donor age.

Recall our discussion of age and inflammation. Aging is associated with progressive tendency to inflammatory cytokines. Now many chronic diseases, including obesity, are also inflammatory. Fat is an endocine organ, and many inflammatory mediators are higher in individuals who have too much fat.  Now recall also that AD-MSC’s tend to what to differentiate into fat cells. In fact even when still undifferentiated  stem cells, their expression of fat-related messenger proteins like adiponectin and leptin are far higher than seen from BM-MSC’s. Further, these same hormones are upregulated by obesity. The two effects (tissue source fat + condition obesity) amplify one another.

Age and adiposity of donor affects AD-MSC’s

typical liposuction vs marrow aspirate donor

Now comes a crucial piece of this puzzle. There is a significant difference in the average age of those individuals supplying AD-MSC’s for therapeutic purposes compared to those supplying BM-MSC’s. The average BM-MSC donor is a college student, often an athlete, between 18-22 years of age.  The average person undergoing liposuction is 40-60 years of age. The average college student is fit, healthy, vigorous, and screened closely to be sure they are disease free. The average person undergoing liposuction is (you guessed it) obese. So, BM-MSC’s are going to be pretty consistent across samples, while AD-MSC’s not nearly so. Some are going to exude anti-inflammatory cytokines in culture, some inflammatory. This may explain the discrepancies in the literature regarding AD-MSC’s while BM-MSAC’s show such remarkable consistency.

But you might say … wait, the cells have the same DNA, from youth to old age, and from fitness to fatness. When you grow them in culture, don’t you get the same responses independent of age? The answer is clearly no. Many studies have been done on donor aging and stem cells, including AD-MSC’s in particular and all suggest key differences between young and old. Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms which regulate gene expression without altering DNA sequence. These changes may remain through cell divisions for the remainder of the cell’s life and may also last for multiple generations. Many of the effects we see with aging are epigenetic. Obesity also creates epigenetic effects on cells that may be harvested from liposuction waste products.

So why does it matter if cytokines are inflammatory, or anti-inflammatory?

Back to our discussion at the beginning of this series, recall that inflammation is necessary and appropriate at the beginning phases of wound healing, but that a successful wound must quickly resolve that inflammation in order to fully transition to the proliferative and rebuilding phases. It is these latter phases that supplies what we seek for regeneration – new, healthy tissue. In the case of skin, we want new keratinocytes, and new, remodeled matrix (collagen and elastin and soluble factors like hyaluronic acid in abundance).  We may want to add volume, especially to the dermis. We certainly don’t want scarring (which leads to more wrinkles) and surface imperfections like spots, depressions, discolorations, and the like.

If you apply inflammatory cytokines to uninjured skin every day, what do you suppose would happen. It induces inflammation, as though it were injured. Now, perhaps this will cause your own skin to respond by mounting its own cytokine defense. That sound a lot like what happens with PICT or acid peels or other deliberate damages. But if you keep applying these inflammatory substances every day, you actually inhibit your skin’s ability to mount a counter response. Eventually you will attain a chronic inflammatory state. It might not even be visible, but over a long period of time the end result will be scarring at the dermal level. The proliferative phase will never take hold. Collagen will be broken down as quickly as it is being made.  Elastin will suffer even more.  In the long term, more damage than good will be achieved. Remember, inflammation and aging go hand in hand. Inflammation will drive your skin to age faster, not slower.

Lesson from copper peptides

You have probably heard of a phenomenon with copper peptides called “the uglies” where you skin looks worse; but then you are told to press on and it will eventually look better. The theory actually appeals logically. Copper peptides are potent inducers of MMP’s (metalloproteinases) which cause collagen and elastin to break down. This can cause skin to become more saggy. But there is a compensatory mechanism whereby TIMP’s (tissue inhibitors of metalloproteinases) are upregulated after a period of time (and by a complex mechanism), and the tide shifts. New collagen and elastin are constructed and bundled into healthy fibers. The dermis is strengthened. But there is one key difference here; although copper induces MMP-1, it is net anti-inflammatory. It downregulates TNF and a few other inflammatory cytokines. So, the net effect is not inflammatory at all.

Inflammation and tumors

Inflammation has consequences. A role for inflammation in tumorigenesis is now generally accepted, and it has become evident that an inflammatory microenvironment is an essential component of all tumors Inflammatory responses within tissues play decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis. Inflammation also affects immune surveillance and responses to therapy. Many environmental causes of cancer and risk factors are associated with some form of chronic inflammation, be it smoking, pollutants, or chronic infections (which causes chronic inflammation). Up to  35% of cancers may be attributable to dietary factors, and 20% of cancer burden is closely linked to obesity.

So, having developed an hypothesis about AD-MSC’s, and their link to donor characteristics (age & obesity), and having raised the issue of inflammatory cytokines as a baseline expression even without those factors,  we might next to the oncology literature. Is there any evidence that AD-MSC’s or their cytokine products  have any association with tumorogenesis? Recent studies have demonstrated that human ASCs, after prolonged culture in vitro, are capable of forming tumors in immunodeficient mice.  Further, mesenchymal stem cells derived from human adipose tissues favor tumor cell growth in vivo.  Data are accumulating to support a role for adipose-derived mesenchymal stem cells (AD-MSCs) in breast cancer progression. Adipose-derived stem cells can promote gastric cancer cell growth, migration and invasion. But are these effects only important in relationship to cellular therapies, or do cell-free cytokine cocktails carry possible risk?  There is a phenomenon called epithelial-mesenchymal transition that plays a role in the progression of certain cancers. This can be induced in the laboratory with the inflammatory cytokine TGFβ-1.  In a recent study by Zimmerlan at the University of Pittsburgh Cancer Institute, they found “The secretome profile of ASC resembled that reported for MSC, but included adipose-associated adipsin and the hormone leptin, shown to promote breast cancer growth”.

These studies are not in any way definitive, and do not mean that you should stop using any product derived from AD-MSC’s.  We bring them to your attention as illustrative of one of the basic principles we want to drive home: that chronic inflammation has many consequences.  The specifics will continue to be debated as more data is gathered. But the basic principle is well accepted.

On balance

This points to the idea that some inflammatory mediators are good, at least sat the proper stage in wound healing, and to some extent regeneration. But the overall balance is important. If you keep attracting inflammatory white cells to your face, day after day, you are not going to get the result you desire. You may notice better hydration and even some smoothing of lines. But don’t be fooled – inflammation brings with it something called edema (fluids excreted by and as a result of immune cells) which can puff up the skin.  But if they stay around long enough, they will do damage. Many common diseases are related to inflammation. Perhaps even aging itself.

Our summation is this: in regards to the beneficial effects of stem cytokines on skin, you need to know what cytokines are being applied. Are they net inflammatory, or net anti-inflammatory? How is the scale tipped?

Our conclusions, thus far

• AD-MSC’s are phenotypically different than BM-MSC’s
• AD-MSC’s excrete a different pattern of cytokines as a result of these differences
•  The cytokine profile of AD-MSC’s compared to BM-MSC’s are more likely to be inflammatory, because:
  • AD-MSC’s secrete adipokines in abundance, which are inflammatory, and/or
  • AD-MSC’s donors are significantly older and more likely to be obese
• Chronic inflammation has consequences, including:
  •  a predisposing factor for many diseases
  •  fibrotic rather than scar-free healing (TGFβ-1, TGFβ-2 overwhelming TGFβ-3).
  •  pro-aging rather than anti-aging
• A model of regeneration deriving from knowledge about phases of wound healing dictates that positive results will come from a balance of cytokines with a predominance of anti-inflammatory cytokines, if they are to achieve anti-aging benefit.

In the next installment, we tie back in the other issues that affect cell source – cytokine factory choices, look at some specific products in terms of cytokine composition,  and try to ties all these threads together into a cohesive concept.

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