In this series, we are focusing on the role cytokines play in skin healing and regeneration. In part 2 we began compiling a compendium of key cytokines, and examined them from the perspective of how each operates in relationship to the temporal stages of healing a wound, and in terms of their function as stimulators or soothers of inflammation within tissues.
Early on in our genesis we wrote about human stem cells and stem cell as sources of cytokines both in normal physiology and (when augmented) as therapeutics in skin care. We promised you then to reveal more details as to the inner workings, but got distracted with other projects. We will use this opportunity (talking about cytokines in depth) to add to what we have already disclosed about how such products work.
Mesenchymal stem cells live within most tissues in the human body. They provide a source of new progenitor cells, that means they can differentiate (transform) into new cells specific to that tissue. Bone marrow-derived stem cells can become bone or cartilage cells. Adipose tissue derived (fat) stem cells can become new adipocytes (fat cells). Ditto for heart, kidneys, and even skin. But because these cells are pluripotent (part of what defines them as stem cells) they can also cross over; in other words, marrow-derived stem cells can also become heart muscle cells. This why you are constantly seeing in the news reports of mesenchymal stem cells being used as experimental therapies for heart attacks, strokes, and many other diseases. The type of stem cell most often used is the mesenchymal stem cell from the bone marrow compartment. There is a population of cells there that act as our body’s 911 system, send cells to respond to emergencies, by supplementing the local tissue cells and tissue-resident stem cells. These specialized bone marrow stem cells (we call them BM-MSC’s for short) are the only ones naturally appointed to this physiologic purpose. They actually mobilize, do their work, and then in order to conserve them, some return to their base station in the bone marrow. Like a paramedic team returning to the fire station.
While there are mesenchymal stem cells in other tissues, and those stem cells behave in many ways just like BM-MSC’s,
they are not phenotypically identical. That means they may have different genes expressed, and thus have different functions. In terms of differentiation, stem cells from bone marrow prefer to become bone cells (osteoblasts), while those from adipose tissue (AD-MSC’s) tend to become adipocytes (etc. for other tissues). When we grow these cells in the laboratory we observe this because each type of MSC excretes chemicals unique to its particular tissue of origin (sort of like family of origin – you can’t shake the influence). So adipose-derived stem cells, despite being true mesenchymal stem cells (e.g. as defined by surface markers of stemness), export different chemicals, including cytokines, in comparison to mesenchymal stem cells from other tissues.
We will detail some of these differences in cytokine/protein excretion later, as they become important when we begin to pull all this together in the context of products derived from stem cell cytokines. First let us look at some things that mesenchymal stem cells can accomplish.
Transplantation of mesenchymal stem cells (MSCs) may be an effective therapy for many diseases including blood disease, several different types of cancer, acute respiratory distress syndrome, spinal cord injury, liver injury, heart disease, neurologic disease, arthritis, autoimmune disorders, diabetes, Alzheimer’s, Parkinson’s, paralysis . Positive results from clinical trials are being reported daily. MSC’s can be taken from an individual, grown in the laboratory, and expanded into sheets or on scaffolds to create new tissues. In the future, entire organs many be created; true organ regeneration is on the horizon. One of our colleagues the stem cell institute recently created a retina from stem cells. Cures for blindness, deafness, and many other disabilities are imagined. Moreover, the vast majority of clinical trials conducted with MSCs in regenerative medicine applications have not reported major health concerns.
MSCs delivered into an injured or diseased tissue may secrete cytokines and growth factors that stimulate recovery in a paracrine (local messenger protein) manner. MSC’s can also modulate the “stem cell niche” of the host by stimulating the recruitment of endogenous stem cells to the site and promoting their differentiation along the required lineage pathway. MSC’s provide antioxidants chemicals, free radical scavengers, and chaperone proteins at an ischemic site. Exciting studies have suggested that transplanted bone marrow–derived MSCs can deliver new mitochondria to damaged cells, thereby rescuing aerobic metabolism. It may develop that similar studies in ASCs will uncover a comparable ability to contribute mitochondria. MSC’s also provide excellent vehicles for gene therapy.
Skin can be engineered in the lab. MSC’s are placed on scaffolds, and induced to growth in sheets. Engineered skin can be made from an individual’s own harvested stem cells, removing concerns about immune compatibility.
MSC’s as cells in a liquid or gel can be placed on, or injected into, wounds. MSC treatment of acute and chronic wounds results in accelerated wound closure with increased epithelialization (new surface cells), granulation tissue formation and angiogenesis (new blood vessels). Although there is evidence for MSC’s in the wound differentiating into skin cells, most of the therapeutic effects are due to MSCs releasing soluble factors that regulate local cellular responses to cutaneous injury.
MSC’s can be grown in the laboratory, and cytokines and other biochemicals can be extracted from what is called “conditioned medium”. This is the nutrient rich fluid in which cells grow. MSC’s are a type of cell that needs to grow on a surface, e.g. on the plastic of flasks or beads in the lab (and attached to other tissues in the body). When there is space available, and the medium provides all the cells need for growth (called expansion in culture). When they begin to grow to a point where they sense crowding, they slow down. In the lab, this marks the time for a “passage” – splitting each culture into two. Or in a bioreactor, add more beads (they manage to colonize new “bead planets” all on their own). While growing MSC’s talk to one another, and do so with cytokines. This is how the medium becomes “conditioned”.
Conditioned medium from mesenchymal stem cells, rich in cytokines (the remnants of conversations between cells) and other biochemical, is a therapeutic substance unto itself. It turns out that much of the benefit of MSC’s in vivo (e.g. injected in the body) is due to these very chemicals. But in this case, the chemicals are used, and the MSC’s that made them (the cells) are left behind. However, some laboratories will follow a different protocol, and will “lyse” (crack open) the cells so you get also the intracellular contents tossed in as well. There are many thousands of biochemical inside all human cells, and things like DNA, RNA, etc, so this approach it is a bit of a catch all. It might even serve to dilute the cytokines in the conditioned medium. Certainly it is a very random sort of thing to do in cellular therapeutics, as there is no convincing evidence that it provides any additional benefit.
Conditioned media of MSC’s has been used successfully on skin, in both wound healing (repair), and esthetic (regenerative) paradigms. Wound healing is accelerated with MSC’s of differing tissue origins (BM-MSC’s and AD-MSC’s are the most studied).
You might think that conditioned medium is easy to make. After all, you just need to grow MSC’s in the lab, and they crank it out. But this is a gross oversimplification – it turns out that the cytokine composition of that conditioned medium changes depending on many factors. Everything from temperature to light to O2 and CO2 concentration to the presence or absence of key “additions” to the culture can dramatically alter its composition. It actually changes with every passage (generation). Those who simply create conditioned medium but don’t bother to measure what is in it (which key cytokines) cannot really make claims about what their product will do, because it won’t be anything like what another lab produced under different, highly specified, conditions (and then published). In fact it could be quite random, resulting in a product that is all over the map in terms of its effects, and/or their magnitude. It’s Coke one day, and 7-up the next, and Mountain Dew the next. Then they batch it all together. Witches’ brew.
MSC’s that are grown in the lab that are not “autologous” (i.e. not from the same person to whom they are being therapeutically applied) are donated by other humans. Humans vary in terms of age. Old age is a major risk factor for many diseases and disorders. Decline in the function of stem cells (like other cells of the body) occurs with age. The increasing age of the donor thus diminishes the effectiveness of MSCs transplantation for age related diseases.
In terms of wound healing, the proliferative and anti-inflammatory abilities of MSCs from young donors is better than aged MSCs. Age related increased expression of apoptotic (cell death) and senescent genes with concomitant decrease in key gene expressions may play an inhibitory role on stem cell functionality. This would be true regardless of tissue origin of the MSC’s.
Next up: in part 4 of this series, we take all the information from parts 1-3 and weave it into a clear and compelling case for MSC-derived stem cytokines in healing and rejuvenation. We revisit the basic paradigm of a crucial balance between inflammatory and anti-inflammatory cytokines and growth factors, and demonstrate where it is possible to go wrong. We will reveal in clear detail the differences between BM-MSC’s and AD-MSC’s. We explore the results when a net inflammatory cytokine mixture is applied to skin over time, using examples from the literature.
We will detail cytokine and protein expression differences between MSC’s of different tissue origins, and the impact of that. We re-examine the variable of donor age, and of tissue of origin, looking for synergistic impacts, and re-examine the risks and benefits of stem cytokine based rejuvenation. We will look at specific products in terms of claimed cytokine composition, and show you how to evaluate these (sort of like reading a nutrition facts label). We will reveal more about our own research, including cytokine measurement.
Choudhery MS, Khan M, et. al. Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation, and anti-apoptosis capabilities. Cell Biol Int. 2012 Feb 22.
Spees JL, Olson SD, Whitney MJ, Prockop DJ. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A. 2006;103:1283–1288.
Jeffrey M. Gimble, Adam J. Katz and Bruce A. Bunnell. Adipose-Derived Stem Cells for Regenerative Medicine. Circ. Res. 2007;100;1249-1260
Lindroos, B.,Suuronen, R., & Miettinen, S. The Potential of Adipose Stem Cells in Regenerative Medicine. Stem Cell Rev and Rep DOI 10.1007/s12015-010-
Alt E, Senst C, Murthy SN, Slakey DP, et.al. Aging alters tissue resident mesenchymal stem cell properties. Stem Cell Res. 2012 Mar;8(2):215-25.
Mantovani C, Raimondo S, Haneef MS, Geuna S, Terenghi G, Shawcross SG, Wiberg M. Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages. Exp Cell Res. 2012 May 29
Mimeault M, Batra SK. Aging of tissue-resident adult stem/progenitor cells and their pathological consequences. Panminerva Med. 2009 Jun;51(2):57-79.
Raicevic G, Najar M, Stamatopoulos B, De Bruyn C, Meuleman N, Bron D, Toungouz M, Lagneaux L. The source of human mesenchymal stromal cells influences their TLR profile as well as their functional properties. Cell Immunol. 2011;270(2):207-16.
Lee SH, Jin SY, Song JS, Seo KK, Cho KH. Paracrine effects of adipose-derived stem cells on keratinocytes and dermal fibroblasts. Ann Dermatol. 2012 May;24(2):136-43.
Walter MN, Wright KT, Fuller HR, MacNeil S, Johnson WE. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res. 2010 Apr 15;316(7):1271-81.