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5-2008 English Edition International Journal for Applied Science • Personal Care • Detergents • Specialties D. Schmid, C. Schürch, P. Blum, E. Belser, F. Zülli: Plant Stem Cell Extract for Longevity of Skin and Hair CO S M ET I C S ST E M C E L L S D. Schmid, C. Schürch, P. Blum, E. Belser, F. Zülli* Plant Stem Cell Extract for Longevity of Skin and Hair Keywords: stem cells, anti-aging, plant cell culture, senescence, hair follicles Introduction diabetes. Regenerative medicine will re- cells and adult stem cells. Embryonic sult in an extended healthy life span. Stem stem cells are pluripotent and thus can What are Stem Cells? cell research is now a fascinating, hot differentiate into all cell types of the Stem cells are unprogrammed cells that topic that is already used in cosmetics. body. The role of adult stem cells is to can differentiate into cells with specific Amatokin, a face care product line of Voss maintain and repair tissues. They are functions. Stem cells have therefore a Laboratories, is claimed to stimulate the more restricted and normally able to dif- big potential in regenerative medicine. stem cells in the skin. And Dior’s Capture ferentiate only into the different cell Regenerative therapies are used to stimu- R60/80 XP range are anti-wrinkle prod- types of the tissue in which they are late healing and might be used in the fu- ucts whose mechanism of action is based found. But there exist also adult stem ture to treat Parkinson’s, Alzheimer’s or on protection of the life force of stem cells with a pluripotent differentiation cells. potential. They are the focus of the So what exactly are stem cells? The main newest research because there are no principles that define stem cell popula- ethical concerns with adult stem cells. Summary tions include: (I) the capacity to replen- Each cell, whether stem cell or differen- ish themselves through self-renewal and tiated cell, has the same DNA, meaning (II) the ability to generate differentiated the same genes. So what makes a stem S tem cells of an old apple cul- cells. There are two types of stem cells in cell a stem cell? Their characteristics de- tivar with very good storage animals and humans: embryonic stem pend on signals from the microenvirone- properties were produced by applying plant cell culture techni- que. The extract of the cultured apple stem cells was shown to en- hance the viability of umbilical cord blood stem cells, to reverse Epidermal layer senescence signs in human fibrob- last cells and to increase lifespan Basal layer of isolated human hair follicles. PhytoCellTech™ Malus Domestica, Sebaceous gland Outer root sheath a cosmetic ingredient based on encapsulated extract of cultured Inner root sheath apple stem cells, was tested in a Hair shaft Hair bulge clinical trial over 4 weeks with 20 stem cells subjects. This new ingredient was found to significantly reduce wrin- Hair follicle matrix Dermal papilla kles in the crow’s feet area. Fig. 1 The hair bulge contains multipotent stem cells that contribute to the lin- eages of the epidermis, the sebaceous gland and the hair follicle 30 SÖFW-Journal | 134 | 5-2008 CO S M ET I C S ST E M C E L L S ment, i.e. from the neighbouring cells The basal layer of the epidermis contains cells in plants is also dependent upon that form a kind of niche. But principal- two different types of cell populations signals from the microenvironment and ly there are signals inside the cell that (Fig. 2): (I) the slowly dividing epidermal on similar epigenetic control as in mam- control their fate. These signals are called stem cells and (II) their progeny that are malian stem cells (3). Unlike humans, adult epigenetic signals. In brief, these are tags rapidly dividing cells in order to supply plants contain totipotent stem cells with on the DNA or the surrounding histone new cells to replace those that get lost the potential to regenerate a whole plant. proteins that regulate the switching on by desquamation. After a limited num- The plant tissue culture technique is or off of genes. ber of divisions, they detach from the based on propagation of plant stem cells basement membrane and start the dif- either to produce a whole plant, only tis- ferentiation program leading finally to sue or just single cells in culture to har- The Role of Stem Cells in the Skin stratum corneum cells. Newest studies vest plant metabolites. This practice al- showed that the stem cells might com- lows the production of plant material The skin is actually the largest organ in prise 2 – 7% of the basal layer cells. The under sterile and standardized condi- the human body. It is covered by the epi- fact that the self-renewal of the epider- tions independent of season and other dermis, a stratified epithelium. The epi- mal stem cell is slow, is important, be- environmental restraints. Plant tissue dermis comprises the interfollicular epi- cause they die after a certain number of cultures can be initiated from nearly all dermis, the hair follicles and the seba- divisions and because each division bears plant tissues. The tissue material which ceous glands. Because the terminally dif- the risk of lethal DNA mutations. And is obtained from the plant to culture is ferentiated cells in the epidermis are loss of stem cells is by far more detri- called an explant. As a kind of wound re- shed from the skin, there must be a con- mental for the tissue than when we lose action, new cells are formed on the cut tinuous delivery of newly differentiating differentiated cells. Hair graying for ex- surfaces of the explant. The cells slowly cells. This shows the importance of stem ample could be shown to result from the divide to form a colorless cell mass which cells in the skin. There is strong evidence fact that the melanocyte stem cells in is called callus. These cells have dediffer- that the hair bulge forms a reservoir of the hair follicle have died off (2). entiated into cells that lack the distinc- epidermal stem cells (Fig. 1). From there, tive features of normal plant cells. Cal- stem cells periodically migrate to the lus cells are stem cells comparable to matrix of the hair follicle, the sebaceous Plant Stem Cells and their those in the meristem regions. For high gland and the basal layer in the interfol- Propagation in Culture yield production callus cells can be cul- licular epidermis (shown with dashed tured as individual cells or small cell clus- pink lines) to produce progenitors that Plants also have stem cells. Two popula- ters in a liquid culture. differentiate into hair cells, gland cells or tions of stem cells, one comprising the cells of the upper epidermal layers re- shoot apical meristem and the other the spectively (shown with dashed green root apical meristem, give rise to the Apple Stem Cells of Cultivar lines) (1). plant body. The maintenance of stem with Excellent Storage Properties A fresh apple is a symbol for beautiful skin. Apples with very good storage properties stay fresh over months. These apples must have especially long-living tissue stem Stratum corneum cells. Could we profit from these stem cells? What would be the effect of an ex- Granular layer tract of such long-living stem cells on the skin? But the commercially available apples are not suited for long storage. Spinous layer Epidermal They are selected for intensive cultiva- stem cell tion and for a pleasant sweet flavor. In Transient former times, good storage properties amplifying were an important factor for cultivar se- Basal layer cells lection. Some of these old cultivars sur- vived as isolated trees in areas with less intensive agriculture. The Uttwiler Spät- Dermis Basement lauber is an apple tree that was cultivat- membrane ed especially because of its good storage properties (Fig. 3). The Spätlauber variety Fig. 2 Epidermal stem cell generates transit amplifying cells that differentiate to derives from a seedling that was planted form the stratified layers in the middle of the 18th century. There are still some Uttwiler Spätlauber trees SÖFW-Journal | 134 | 5-2008 31 CO S M ET I C S ST E M C E L L S plant material. Callus induction and sub- Results and Discussion cultivation was carried out according to standard protocols. Incorporation of the Protection of Human Stem Cells dedifferentiated cells in an appropriate Stem cells from umbilical cord blood liquid media, homogenisation of the represent an ethically correct source of cells in suspension and continuous char- human stem cells. An extract of Uttwiler acterisation of the cell suspension was Spätlauber stem cells was tested on cell also carried out according to standard viability of umbilical cord blood stem cells procedures. For up-scaling, 10% of the in two different studies. First, the effect next larger culture volume of a fully of the extract on the proliferative activ- grown cell suspension was used as in- ity of the human stem cells was analyzed. Fig. 3 Isolated Uttwiler Spätlauber tree oculum. Production of biomass was done There was a clear, concentration-depen- found in the Zimmerberg region at in 50 to 100 l cultures with a special dent effect found (Fig. 4). At only 0.1%, the lake of Zürich. bioreactor-system (Wave-Biotech AG, the extract stimulated cell proliferation Tagelswangen, Switzerland). Cultivation by 80%. In a second experiment, the stem was done at 25 °C and an aeration of cells were stressed through irradiation 0.1 vvm. Biomass production was moni- with UV light. Almost 50% of the cells left in certain areas in Switzerland. The tored by analysis of total sugar concen- that were cultured in the growth medi- apples of one of these trees were used to tration, conductivity, pH-value and op- um alone died, whereas cells cultured in obtain tissue explants in order to initiate tical density. Production of secondary presence of an extract of Uttwiler Spät- a plant cell culture. A success full liquid metabolites was followed by HPLC and lauber experienced only a small loss of culture in bioreactors of Uttwiler Spät- UV/VIS analysis. viability (Fig. 5). lauber stem cells could be established. The extract of Uttwiler Spätlauber stem An extract of these cells was tested in a cells was obtained after lysis of the plant series of studies for anti-aging efficacy cells using high pressure homogenisa- Reversion of Senescence Signs in in skin and hair. tion. For preparation of PhytoCellTech™ Fibroblast Cells Malus Domestica, the etract was incor- Senescence is a natural process that, af- porated into lecithin liposomes. The ter about 50 divisions, stops the capaci- Matrials and Methods composition of PhytoCellTech™ Malus ty of the cell to undergo further divi- Domestica is the following (INCI): Malus sions. But senescence can also happen Plant cell cultures Domestica Fruit Cell Culture, Xanthan earlier in a cell’s life, for example as re- Apples of the Uttwiler Spätlauber vari- Gum, Glycerin, Lecithin, Phenoxyethanol sponse to damage to the cellular DNA. ety were used for selection of suitable and Aqua. Premature senescence is especially detri- Fig. 4 Effect of Uttwiler Spätlauber stem cell extract on via- Fig. 5 Uttwiler Spätlauber stem cell extract protects umbilical bility of umbilical cord blood stem cells cord blood stem cells against UV light 32 SÖFW-Journal | 134 | 5-2008 CO S M ET I C S ST E M C E L L S mental when it hits stem cells because they are indispensable for tissue regen- Gene after H2O2 after H2O2 eration. Recently a cellular model for Control + 2% Extract premature senescence was established (% to untreated) (% to untreated) based on fibroblast cells. After a two hour treatment with H2O2, the cells showed cyclin B1: induces proliferation 73 130 the typical signs of senescence. This mod- cyclin E1: controls cell cycle 78 135 el was used to prove an anti-senescence P53: tumor uppressor gene 63 137 effect of an extract of Uttwiler Spät- insulin-like growth factorII: lauber stem cells. After the H2O2 treat- cell prolifertion enhancer 71 117 ment, the fibroblast cells were incu- heme oxigenase 1: antioxidans enzyme 89 211 bated with 2% extract for 144 hours. Then gene expression was analyzed with Table 1 Effect of Uttwiler Spätlauber stem cell extract on gene expression profile a cDNA array system comprising of 150 of fibroblast cells after H2O2 treatment probes related to cellular growth, signal- ing, senescence and aging. Gene expres- sion was compared to that of cells that Fig. 6 shows that follicles kept in pres- ly to the crow’s feet area. Wrinkle depth were kept only in the culture medium af- ence of the Uttwiler Spätlauber stem cell was analyzed with the PRIMOS system ter the H2O2 treatment. In the control extract continued to elongate until day after 2 and 4 weeks. Digital photos of the culture, several genes important for cel- 18, whereas the control follicles started crow’s feet area were taken at the be- lular proliferation and stimulation of to shrink after day 14. ginning and the end of the study. The cellular growth were down regulated af- PhytoCellTecTM Malus Domestica cream ter H2O2 treatment (Table 1). Incubation was found to significantly reduce wrin- with the extract of Uttwiler Spätlauber Anti-Wrinkle Effect on the kle depth after two and after four weeks, stem cells neutralized this down regula- Crow’s Feet Area by 8% and 15% respectively. The effect tion or even induced an up regulation. The anti-wrinkle efficacy of PhytoCell- can be nicely demonstrated by genera- The extract also strongly stimulated the Tec Malus Domestica was demonstrated tion of 3D pictures. Such an example is expression of an important antioxidant in a clinical trial over 4 weeks with 20 shown in Fig. 7. The anti-wrinkle effica- enzyme, heme oxigenase 1, whose expres- subjects. A cream with 2% PhytoCellTec™ cy could also been shown on digital pho- sion was surprisingly not increased in the Malus Domestica was applied twice dai- tos (Fig. 8). control. Retardation of Senescence in Isolated Hair Follicles Human hair follicles can be isolated by microdissection from skin fragments that are left after facelift surgery. Only folli- cles in the anagen phase are collected. Hair follicles are mini organs that repre- sent a natural coculture model of epi- dermal and melanocyte stem cells and differentiated cells. The follicles can be maintained in a growth medium where they elongate until about day 14. Then the follicle cells gradually become senes- cent or undergo apoptosis which is a programmed cell death. Because of the lack of blood circulation, isolated hair follicles cannot be kept alive and grow- ing for a longer period. But isolated hair follicles are test models to screen for ac- tives that can delay the necrosis process. Addition of 0.2% of an extract of Utt- wiler Spätlauber stem cells was found Fig. 6 Uttwiler Spätlauber stem cell extract increases lifespan of isolated human to slightly but clearly postpone senes- hair follicles cence- and apoptosis-induced necrosis. 34 SÖFW-Journal | 134 | 5-2008 CO S M ET I C S ST E M C E L L S resistance against senescence and apop- tosis of human stem cells. In this way, the plant stem cell extract promotes regen- eration of skin and hair and delays the appearance of skin aging signs. The ex- act mechanism is not known. But the Uttwiler Spätlauber apples, known to stay fresh for an extraordinarily long time, might have stem cells with a special epi- genetic profile. It seems that these epi- genetic factors have a positive effect on human stem cells. Fig. 7 3D picture analysis of the crow’s feet area before and after application of a cream with 2% PhytoCellTec™ Malus Domestica References (1) Barthel R. & Aberdam D. (2005) Epidermal stem cells. Journal of European Academy of Derma- tology and Venereology 19:405-13 (2) Nishimura E.K., Granter S.R. & Fisher D.E. (2005) Mechanisms of Hair Graying: Incomplete Me- lanocyte Stem Cell Maintenance in the Niche. Science 307:720-4 (3) Byrne M.E., Kidner C.A. & Martienssen R.A. (2003) Plant stem cells: divergent pathways and common themes in shoots and roots. Cur- rent Opinion in Genetics & Development 13: Before (day 0) After (day 28) 551-7 Fig. 8 Digital photos of the crow’s feet area before and after treatment with a cream with 2% PhytoCellTec™ Malus Domestica * Author’s addresses: Mibelle Biochemistry Bolimattstrasse 1 Conclusions ments. Reduced viability and premature 5033 Buchs senescence or apoptosis of stem cells is Switzerland Stem cells are the most important cells a principal cause for tissue aging. The re- Email: Daniel.Schmid@MIBELLE.CH in the skin. They are the source for con- sults presented in this article show that tinuous regeneration of the epidermis, an extract of Uttwiler Spätlauber stem the formation of new hair and hair pig- cells positively influences viability and SÖFW-Journal | 134 | 5-2008 35
"Plant Stem Cell Extract for Longevity of Skin and Hair"