STEM CELLS HAVE EXCITING potential for many areas of biomedicine as well as skin care. This potential derives from two important characteristics that distinguish them from other cells. First, stem cells can renew themselves indefinitely in an undeveloped or undifferentiated state and second, they can differentiate into different specialized cell types.

Mammals, including humans, have two types of stem cells. Embryonic stem cells are, as the name implies, derived from embryos. They are called pluripotent because they can generate any cell type in the body (i.e., >200 different cell types), including brain, heart, liver and skin. Adult stem cells are relatively undifferentiated, but they have committed to a specific differentiation pathway. They are also called multipotent because they can produce only a limited number of cell types within their own “family of cell types.” For example, skin stem cells only produce the various types of skin cells. Thus, cell differentiation involves a succession of stages. Embryonic stem cells develop into adult stem cells and then into progenitor cells, which are somewhat more developed, and finally adult cells.

Skin serves many functions. It maintains a healthy state for skin and other cells (called homeostasis), prevents infections and maintains temperature. Cells of the basal layer (basal cells) adhere to the basement membrane, which separates the dermis from the underlying dermis. Basal cells form a single layer of proliferative cells. Fewer than 1% of the basal cells are epidermal stem cells.¹ These stem cells must guarantee life-long tissue integrity by replenishing the layers of skin cells that undergo a program of terminal differentiation when they move outward and eventually shed from the surface of the epidermis.² Moreover, they are pivotal for an appropriate response to injury as they provide fresh proliferating and migrating cells. Because skin cells are exposed to the environment, they wear out and must be replaced. As the outer layer of skin dies and is sloughed off, adult skin stem cells are dividing in the deeper skin layers to form new skin cells that move up to the surface to replace the lost cells. In this way, skin stem cells renew our skin and repair wounds.

Experimental Design

Primary epidermal stem and progenitor cells were isolated from foreskin, a common source of skin cells for research, and incubated in medium that maintains progenitor cells in-vitro. They were cultivated with the test compounds for three days. Then, the cells were washed and exposed to a broad-spectrum UV lamp (simulating sunlight), and examined for the various characteristics of stem cells. For example, cell proliferation was monitored without exposure to UV light by counting cell doublings over time. Colony forming efficiency was determined by seeding cells at a low density (200–1000 cells per dish) and counting the colonies 14 days later. We also tested the ability to form a 3D skin or epidermis in-vitro.

Six products were screened. Three were uncharacterized compounds, and one was a control. Two were well-established Rahn cosmetic actives. Active 1 (INCI: Octyldodecanol, echium plantagineum seed oil, cardiospermum halicacabum flower/ leaf/vine extract, helianthus annuus (sunflower) seed oil unsaponifiables) is an optimized system for physical and biochemical protection and care of sensitive skin types. It effectively inhibits inflammation. Active 1 functions on three levels. Cardiospermum halicacabum is effective against itchy and allergic inflammation of the skin. Stearidonic acids, derived from echium oil, inhibit inflammation and protect and strengthen the barrier of the skin. Unsaponifiable sunflower oil has a soothing and relipidation effect. Active 1 was tested at a sub-toxic concentration of 0.000005%.

Active 2 (INCI: Helianthus annuus (sunflower) seed oil, ethyl ferulate, polyglyceryl- 5 trioleate, rosmarinus officinalis (rosemary) leaf extract, water, disodium uridine phosphate) protects cells and DNA. Its active ingredients were specially developed to treat UV-stressed skin. Carnosolic acid and ethyl ferulate neutralize radicals that damage cells and effectively prevent UV damage. Uridine monophosphate, used in DNA synthesis, supports DNA repair. Active 2 was tested at the sub-toxic concentration 0.00004%.

Importantly, the test substances were present only before and not during or after UV exposure. This aspect of our study avoided experimental artifacts that might jeopardize the results. In our tests, removing the test substances before exposure eliminated any concerns that the substances themselves might have quenched the UV light, an action that would have made our results uninterpretable.

Results and Discussion

Active 1 and Active 2 protected the stem cell potential/function of skin stem- and progenitor cells. Notably, Active 1 enhanced stem-cell potential. No other compound did.

Figure 1: Active 1, Active 2 and untreated stem cells show comparable cell proliferation. Skin stem- and progenitor cells were counted weekly. The number of cumulative population doublings is shown.

Cell proliferation: Stem cells are important for skin cell replacement and wound repair, but they are double-edged swords. With their long lifespans, adult skin stem cells are the one kind of cell that can accumulate enough mutations to potentially cause skin cancer.⁶ Cancers are formed by the uncontrolled proliferation of relatively undifferentiated cells. Because of this, stem-cell proliferation is tightly regulated and well balanced in-vivo, and cosmetics must not induce unregulated stem-cell proliferation. The proliferation rates of skin stem cells treated with Active 1 and Active 2 were similar to the normal levels of untreated cells (Fig. 1). So the two active ingredients do not seem to cause abnormal cell proliferation.

Figure 2: Pre-treatment with Active 1 stimulates colony forming efficiency by up to 300% compared to the untreated control. Pre-treatment with an inappropriate compound (Negative Control) had detrimental effects – CFE was totally lost. For each experiment, 200 cells were seeded.

Colony-forming efficiency: An effective cosmetic active ingredient should protect stem-cell function or potential. In our testing, we wanted to determine if the stem cells could still form new colonies to evaluate the preservation of stem-cell potential after UV exposure. Differentiated adult skin cells die in culture, so colony-forming efficiency is a measure of the proportion of undifferentiated cells. After the UV treatment, we plated the cells and allowed them to grow for a defined period. Then we counted the colonies and calculated the effect of the pre-treatment. We found that Active 1 protected colony-forming efficiency. Untreated cells had lower colony forming efficiency. Pre-treatment with Active 1 even increased colony numbers and colony size, actually pointing to an enhancement of the stemcell potential.

Upon UV stress, colony-forming efficiency was stimulated by up to 300% when cells were pre-treated with Active 1. Remarkably, treatment with another compound led to total loss of colony forming efficiency (Fig. 2). In the CFE assay, pretreatment with Active 2 did not bring an additional value.

3D Epidermis In-Vitro: Healthy and young-appearing skin has a complex 3D structure. Skin stem cells are a major resource for tissue repair, regeneration and rejuvenation and have the potential to fully restore the skin or epidermis. The formation of a structured epidermis in-vitro is a complex process. Thus, it is a sensitive test for stem cell damage.

After exposure to UV stress, cells were allowed to grow to confluence. The cultures were then brought to the air-liquid interface (lifted) so that 3D growth could begin. Histological tests were performed to determine the effects. Active 1 and Active 2 protected the ability of stem cells to restore a structured epidermis after UV stress. This potential was markedly lower in untreated control cells (Fig. 3). Skin stem cells are critical for preventing skin aging. They are involved in tissue repair, regeneration and rejuvenation. Protecting these valuable cells is important for maintain youthful looking skin, and the active ingredients of a cosmetic product should help to protect skin stem cells from loss or damage.

The treatment with Active 1 leads to a remarkable strengthening of skin stem cells: upon UV-irradiation the colony forming efficiency was even enhanced—as compared to untreated control cells— pointing to an increased number of undifferentiated and functional stem and progenitor cells. Furthermore, the ability to restore a structured 3D epidermis after exposure to UV light was maintained.

Active 1 provides resistance to the key cells of skin homeostasis and thus counteracts the appearance of irritated and stressed skin. Active 2 protects epidermal stem and progenitor cells against sunlight. Moreover, it maintains their ability to restore a structured 3D epidermis upon UV stress and thus protects the function of epidermal stem cells. Active 2 is therefore an excellent anti-aging ingredient that protects DNA and stem cells and should be part of any modern anti-aging product.

References:
1. Fuchs, E. and Nowak, J. A., Cold Spring Harb Symp Quant Biol 73, 333 (2008).
2. Blanpain, C. and Fuchs, E., Annu Rev Cell Dev Biol 22, 339 (2006).
3. Kaur, P., J Invest Dermatol 126 (7), 1450 (2006).
4. Watt, F. M. and Jensen, K. B., EMBO Mol Med 1 (5), 260 (2009).
5. Blanpain, C. and Fuchs, E., Nat Rev Mol Cell Biol 10 (3), 207 (2009).
6. Drummond-Barbosa, D., Genetics 180 (4), 1787 (2008)

About the Author
Stefan Baenziger Ph.D. is manager of development
of cosmetic actives for Rahn AG. More info:
Rahn AG, Dörflistr. 120, 8050 Zurich, Switzerland.
Tel: +41 44 315 42 00. Fax: +41 44 315 42 45.
E-mail: [email protected]
Website: www.rahn-group.com

Figure 3: Active 1 and Active 2 protect stem cells from UV stress: the stem cell’s ability to restore a three-dimensional epidermis upon UV stress is maintained. Experiments were performed in triplicates. One exemplary picture 18 days after UV exposure is presented.