Recent studies have found that the conditioned media from skin-derived adipose mesenchymal stem cells (CM-ADMC) penetrate intact human skin and induce wound healing when topically applied. Exosomes are one mechanism by which the molecules penetrate the skin. Likewise, in animal models, CM-ADMC reduces inflammation and promotes wound healing when topically applied to intact skin.

I’ve developed products from the molecules that stem cells release (Maguire, 2013) that can be topically applied to have effects in the epidermis and dermis. This penetration of the molecules means that even the stratum corneum and the tight junctions in the epidermis are not barriers to the stem cell released molecules (SRM). We have much evidence for how these topically applied molecules penetrate and act throughout the skin’s layers to provide many benefits.

In the 1990s when I was a professor at the University of California, San Diego we were using a type of stem-cell genetically modified in order to have a living cell constituently secrete Nerve Growth Factor (NGF) into degenerating neural tissue to rescue the neurons and other cells from dying. In the process of studying the genetically modified cells, we discovered that the control stem cells that were not genetically modified to secrete NGF were working as well or better than the genetically modified fibroblast.  I realized that normal stem cells were releasing numerous molecules to repair and prevent neural degeneration, and that was an epiphanic moment for me – that we could use stem cells as cellular factories to produce these beneficial molecules. And this meant that if you use the stem cells to produce the molecules in the laboratory, you wouldn’t have to inject or otherwise administer stem cells themselves to the tissue. Rather you could culture and stimulate the stem cells in the laboratory to optimize the output of the molecules that the stem cells release for maximum therapeutic benefit. Many studies in the ensuing years have provided evidence that it is the release of molecules from stem cells that provide most of the stem cell’s therapeutic benefit.  Using the molecules themselves without the cells as a therapeutic is much easier and more straightforward, and more efficacious than injecting or administering stem cells to the patient where we don’t know the number of stem cells accruing in the injured area and where we don’t know if the stem cells are working correctly. That is, one doesn’t know if the cells are making and releasing the molecules into the injured area. Whereas, using the molecules means that you apply a defined, optimal dose of molecules directly to the injured tissue. Importantly, the molecules released from the stem cells, and not molecules artificially extracted from the stem cells, are critical for two key reasons. First, the molecules need to be fully formed for them to work properly, and it is the released molecules, not extracted molecules, that are fully formed. Not waiting for the molecules to be released means that extracted molecules may not have fully formed and may be misfolded, causing them to be ineffective and potentially dangerous. Second, the released molecules are packed into exosomes that are natural protection and penetration devices for the released molecules. Extracted molecules are not packaged into the exosomes.

Back in the 1990s and into the early 2000s stem cell therapeutics was mainly focused on embryonic stem cells. Embryonic stem cells were all the rage because those cells could fully differentiate, that is turn into or transform themselves into almost any cell type in the body. The idea was to use embryonic stem cells to make new tissues. The thought of using adult stem cells was carried forth by only a few of us during that time, and funding was tight for anything other than embryonic stem cells. The adult stem cells could not turn into any tissue in the body and had limited potential to differentiate into other cell types – and this was an anathema to academia as well as the investment community. Adult stem cells are tissue specific and have restricted lineage fates. Instead of developing an organism, as embryonic stem cells do, adult stem cells have partially matured (differentiated) into a phenotype that is used by a particular tissue to maintain and heal itself. The adult stem cells found in our tissues have evolved to maintain and heal our tissues, doing so mainly through the release of molecules (Maguire, 2013). At the time, when I proposed not only using adult stem cells as a therapeutic but also using just the molecules released from adult stem cells, there was little interest and sometimes downright bashing of my proposal. Despite zeitgeist focusing on embryonic stem cells, in the 1990s we began to use the stem cell released technology for repairing brain tissue (Maguire et al, 2019). Because we had been using genetically modified adult stem cells derived from the skin to begin our studies of repairing the brain, we realized that using these adult stem cells from skin might be used to heal the skin. This would yield proof of concept safety and efficacy studies that were less expensive and more quickly accomplished than having to deliver the molecules into the brain and measure the results in an organ that is much less accessible than the skin. This is how I began studying skin. The more I looked at the skin, the more fascinated I became, especially given we began to see very encouraging results using the molecules to heal wounds. With a beautiful, layered structure, constant turnover of stem cells, such as the keratinocytes, and powerful innate and adaptive immune systems, studying the skin became a labor of love. When we were injecting these molecules into the brain, it was easy to understand how they penetrated through the tissue. But when we began working on the skin, and the molecules were not only working in wounded skin with a degraded barrier, but were also working on intact skin with a normal barrier – we were surprised. I was taught, and indeed I taught my students that these large proteins we were working with would not penetrate skin barriers.

But the molecules were penetrating intact skin. We saw it, and so did others (Kim et al, 2017). Within 3 hours following application to the skin, the exosomes are penetrating the epidermis, at 18 hours they are deep in the epidermis, and within 3 days they have begun to increase the production of collagen and elastin in the dermis. How are they penetrating the skin? The simple answer is exosomes, a liposome-like structure. But the exosomes are more complicated than liposomes and have some extra features that seem to enable them to better penetrate tissue than a liposome. While having a more flexible structure than a liposome, allowing them to squeeze through closely packed structures, the exosome also has proteases and glycosidases contained on its surface (either attached or as transmembrane proteins), as well as on its inside (Sanderson et al, 2019). Those proteases and glycosidases are known to break down barriers, including tight junctions (Lin et al, 2020) and matrix molecules that would otherwise prevent the exosome’s penetration through that part of the tissue. So as the naturally flexible exosome is squeezing through structures in the skin, the proteases and glycosidases are temporarily breaking punctate structures that prevent their penetration. We now understand that cells in the skin use exosomes to send their signals to other cells (Cicero et al, 2015; Nasiri et al, 2020), including to directly modify immune cells (Zhou et al, 2020), and that these stem cell derived exosomes can be safely used for skin therapy (Maguire and Friedman, 2020). Work continues to further develop these technologies – stay tuned.

References

Kim YJ, Yoo SM, Park HH, Lim HJ, Kim YL, Lee S, Seo KW, Kang KS. Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin. Biochem Biophys Res Commun. 2017 Nov 18;493(2):1102-1108.

Lin Y et al (2020) Exosomes derived from HeLa cells break down vascular integrity by triggering endoplasmic reticulum stress in endothelial cells, Journal of Extracellular Vesicles, 9:1.

Cicero, A., Delevoye, C., Gilles-Marsens, F. et al. (2015) Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat Commun 6, 7506.

Maguire G. Stem cell therapy without the cells. Commun Integr Biol. 2013 Nov 1;6(6):e26631. doi: 10.4161/cib.26631. 

Maguire G, Friedman P. (2020) The safety of a therapeutic product composed of a combination of stem cell released molecules from adipose mesenchymal stem cells and fibroblasts. Future Sci OA. 6(7):FSO592.

Nasiri, G., Azarpira, N., Alizadeh, A. et al. (2020) Shedding light on the role of keratinocyte-derived extracellular vesicles on skin-homing cells. Stem Cell Res Ther 11, 421..

Sanderson RD, Bandari SK, Vlodavsky I. Proteases and glycosidases on the surface of exosomes: Newly discovered mechanisms for extracellular remodeling. Matrix Biol. 2019 Jan;75-76:160-169.

Zhou X et al (2020) Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing. ACS Nano: 14, 10, 12732–12748