double stemcell plant stem cell extract for longevity of skin and hair sfw 05 08

7/22/2019 Double stemcell Plant Stem Cell Extract for Longevity of Skin and Hair SFW 05 08

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5-2008

English Edition

International Journal for Applied Science

Personal Care Detergents Specialties

D. Schmid, C. Schrch, P. Blum, E. Belser, F. Zlli:

Plant Stem Cell Extract for Longevityof Skin and Hair


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COSMETICSS TE M CE L L S

cells and adult stem cells. Embryonicstem cells are pluripotent and thus candifferentiate into all cell types of thebody. The role of adult stem cells is tomaintain and repair tissues. They aremore restricted and normally able to dif-ferentiate only into the different celltypes of the tissue in which they arefound. But there exist also adult stemcells with a pluripotent differentiationpotential. They are the focus of thenewest research because there are no

ethical concerns with adult stem cells.Each cell, whether stem cell or differen-tiated cell, has the same DNA, meaningthe same genes. So what makes a stemcell a stem cell? Their characteristics de-pend on signals from the microenvirone-

D. Schmid, C. Schrch, P. Blum, E. Belser, F. Zlli*

Plant Stem Cell Extract for Longevityof Skin and Hair

Keywords: stem cells, anti-aging, plant cell culture, senescence, hair follicles

Introduction

What are Stem Cells?Stem cells are unprogrammed cells thatcan differentiate into cells with specificfunctions. Stem cells have therefore abig potential in regenerative medicine.Regenerative therapies are used to stimu-late healing and might be used in the fu-ture to treat Parkinsons, Alzheimers or

diabetes. Regenerative medicine will re-sult in an extended healthy life span. Stemcell research is now a fascinating, hottopic that is already used in cosmetics.Amatokin, a face care product line of VossLaboratories, is claimed to stimulate thestem cells in the skin. And Diors CaptureR60/80 XP range are anti-wrinkle prod-ucts whose mechanism of action is basedon protection of the life force of stemcells.So what exactly are stem cells? The main

principles that define stem cell popula-tions include: (I) the capacity to replen-ish themselves through self-renewal and(II) the ability to generate differentiatedcells. There are two types of stem cells inanimals and humans: embryonic stemS

tem cells of an old apple cul-

tivar with very good storage

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

senescence signs in human fibrob-

last cells and to increase lifespan

of isolated human hair follicles.

PhytoCellTech Malus Domestica,

a cosmetic ingredient based on

encapsulated extract of cultured

apple stem cells, was tested in a

clinical trial over 4 weeks with 20

subjects. This new ingredient was

found to significantly reduce wrin-

kles in the crows feet area.

Summary

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

Epidermal layer

Basal layer

Outer root sheath

Inner root sheath

Hair shaft

Hair follicle matrixDermal papilla

Sebaceous gland

Hair bulgestem cells


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ment, i.e. from the neighbouring cellsthat form a kind of niche. But principal-ly there are signals inside the cell that

control their fate. These signals are calledepigenetic signals. In brief, these are tagson the DNA or the surrounding histoneproteins that regulate the switching onor off of genes.

The Role of Stem Cells in the Skin

The skin is actually the largest organ inthe human body. It is covered by the epi-dermis, a stratified epithelium. The epi-

dermis comprises the interfollicular epi-dermis, the hair follicles and the seba-ceous glands. Because theterminally dif-ferentiated cells in the epidermis areshed from the skin, there must be a con-tinuous delivery of newly differentiatingcells. This shows the importance of stemcells in the skin. There is strong evidencethat the hair bulge forms a reservoir ofepidermal stem cells (Fig. 1). From there,stem cells periodically migrate to thematrix of the hair follicle, the sebaceousgland and the basal layer in the interfol-

licular epidermis (shown with dashedpink lines) to produce progenitors thatdifferentiate into hair cells, gland cells orcells of the upper epidermal layers re-spectively (shown with dashed greenlines) (1).

The basal layer of the epidermis containstwo different types of cell populations(Fig. 2): (I) the slowly dividing epidermal

stem cells and (II) their progeny that arerapidly dividing cells in order to supplynew cells to replace those that get lostby desquamation. After a limited num-ber of divisions, they detach from thebasement membrane and start the dif-ferentiation program leading finally tostratum corneum cells. Newest studiesshowed that the stem cells might com-prise 2 7% of the basal layer cells. Thefact that the self-renewal of the epider-mal stem cell is slow, is important, be-

cause they die after a certain number ofdivisions and because each division bearsthe risk of lethal DNA mutations. Andloss of stem cells is by far more detri-mental for the tissue than when we losedifferentiated cells. Hair graying for ex-ample could be shown to result from thefact that the melanocyte stem cells inthe hair follicle have died off (2).

Plant Stem Cells and theirPropagation in Culture

Plants also have stem cells. Two popula-tions of stem cells, one comprising theshoot apical meristem and the other theroot apical meristem, give rise to theplant body. The maintenance of stem

cells in plants is also dependent uponsignals from the microenvironment andon similar epigenetic control as in mam-

malian stem cells (3). Unlike humans, adultplants contain totipotent stem cells withthe potential to regenerate a whole plant.The plant tissue culture technique isbased on propagation of plant stem cellseither to produce a whole plant, only tis-sue or just single cells in culture to har-vest plant metabolites. This practice al-lows the production of plant materialunder sterile and standardized condi-tions independent of season and otherenvironmental restraints. Plant tissue

cultures can be initiated from nearly allplant tissues. The tissue material whichis obtained from the plant to culture iscalled an explant. As a kind of wound re-action, new cells are formed on the cutsurfaces of the explant. The cells slowlydivide to form a colorless cell mass whichis called callus. These cells have dediffer-entiated into cells that lack the distinc-tive features of normal plant cells. Cal-lus cells are stem cells comparable tothose in the meristem regions. For highyield production callus cells can be cul-

tured as individualcells or small cell clus-ters in a liquid culture.

Apple Stem Cells of Cultivarwith Excellent Storage Properties

A fresh apple is a symbolfor beautiful skin.Apples withvery good storage propertiesstay fresh over months. These apples musthave especially long-living tissue stemcells. Could we profit from these stem

cells? What would be the effect of an ex-tract of such long-living stem cells onthe skin? But the commercially availableapples are not suited for long storage.They are selected for intensive cultiva-tion and for a pleasant sweet flavor. Informer times, good storage propertieswere an important factor for cultivar se-lection. Some of these old cultivars sur-vived as isolated trees in areas with lessintensive agriculture. The Uttwiler Spt-lauber is an apple tree that was cultivat-ed especially because of its good storageproperties(Fig. 3). The Sptlauber varietyderives from a seedling that was plantedin the middle of the 18th century. Thereare still some Uttwiler Sptlauber trees

SFW-Journal| 134 | 5-2008 31


Fig. 2Epidermal stem cell generates transit amplifying cells that differentiate toform the stratified layers

Stratum corneum

Granular layer

Spinous layer

Basal layer

DermisBasement

membrane

Epidermalstem cell

Transientamplifying

cells


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left in certain areas in Switzerland. Theapples of one of these trees were used toobtain tissue explants in order to initiatea plant cell culture. A success full liquidculture in bioreactors of Uttwiler Spt-lauber stem cells could be established.An extract of these cells was tested in aseries of studies for anti-aging efficacyin skin and hair.

Matrials and Methods

Plant cell culturesApples of the Uttwiler Sptlauber vari-ety were used for selection of suitable

plant material. Callus induction and sub-cultivation was carried out according tostandard protocols. Incorporation of the

dedifferentiated cells in an appropriateliquid media, homogenisation of thecells in suspension and continuous char-acterisation of the cell suspension wasalso carried out according to standardprocedures. For up-scaling, 10% of thenext larger culture volume of a fullygrown cell suspension was used as in-oculum. Production of biomass was donein 50 to 100 l cultures with a specialbioreactor-system (Wave-Biotech AG,Tagelswangen, Switzerland). Cultivation

was done at 25 C and an aeration of0.1 vvm. Biomass production was moni-tored by analysis of total sugar concen-tration, conductivity, pH-value and op-tical density. Production of secondarymetabolites was followed by HPLC andUV/VIS analysis.The extract of Uttwiler Sptlauber stemcells was obtained after lysis of the plantcells using high pressure homogenisa-tion. For preparation of PhytoCellTechMalus Domestica, the etract was incor-porated into lecithin liposomes. The

composition of PhytoCellTech MalusDomestica is the following (INCI): MalusDomestica Fruit Cell Culture, XanthanGum, Glycerin, Lecithin, Phenoxyethanoland Aqua.

Results and Discussion

Protection of Human Stem Cells

Stem cells from umbilical cord bloodrepresent an ethically correct source ofhuman stem cells. An extract of UttwilerSptlauber stem cells was tested on cellviability of umbilical cord blood stem cellsin two different studies. First, the effectof the extract on the proliferative activ-ity of thehumanstem cells was analyzed.There was a clear, concentration-depen-dent effect found(Fig. 4). At only 0.1%,the extract stimulated cell proliferationby 80%. In a second experiment, the stem

cells were stressed through irradiationwith UV light. Almost 50% of the cellsthat were cultured in the growth medi-um alone died, whereas cells cultured inpresence of an extract of Uttwiler Spt-lauber experienced only a small loss ofviability(Fig. 5).

Reversion of Senescence Signs inFibroblast CellsSenescence is a natural process that, af-ter about 50 divisions, stops the capaci-

ty of the cell to undergo further divi-sions. But senescence can also happenearlier in a cells life, for example as re-sponse to damage to the cellular DNA.Premature senescence is especially detri-

32 SFW-Journal | 134 | 5-2008


Fig. 3 Isolated Uttwiler Sptlauber treefound in the Zimmerberg region atthe lake of Zrich.

Fig. 4Effect of Uttwiler Sptlauber stem cell extract on via-bilityof umbilical cord blood stem cells

Fig. 5Uttwiler Sptlauber stem cell extract protects umbilicalcord blood stem cells against UV light


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mental when it hits stem cells becausethey are indispensable for tissue regen-eration. Recently a cellular model for

premature senescence was establishedbased on fibroblast cells. After a two hourtreatment with H2O2, the cells showedthe typical signs of senescence. This mod-elwas used to prove an anti-senescenceeffect of an extract of Uttwiler Spt-lauber stem cells. After the H2O2 treat-ment, the fibroblast cells were incu-

bated with 2% extract for 144 hours.

Then gene expression was analyzed witha cDNA array system comprising of 150probes related to cellular growth, signal-

ing, senescence and aging. Gene expres-sion was compared to that of cells thatwere kept only in the culture medium af-ter the H2O2 treatment. In the controlculture, several genes important for cel-lular proliferation and stimulation ofcellular growth were down regulated af-ter H2O2treatment(Table 1). Incubationwith the extract of Uttwiler Sptlauberstem cells neutralized this down regula-tion or even induced an up regulation.The extract also strongly stimulated theexpression of an important antioxidant

enzyme, heme oxigenase 1, whose expres-sion was surprisingly not increased in thecontrol.

Retardation of Senescence inIsolated Hair FolliclesHuman hair follicles can be isolated bymicrodissection from skin fragments thatare 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 anddifferentiated cells. The follicles can bemaintained in a growth medium wherethey elongate until about day 14. Thenthe follicle cells gradually become senes-cent or undergo apoptosis which is aprogrammed cell death. Because of thelack of blood circulation, isolated hairfollicles cannot be kept alive and grow-ing for a longer period. But isolated hairfollicles are test models to screen for ac-tives that can delay the necrosis process.Addition of 0.2% of an extract of Utt-wiler Sptlauber stem cells was found

to slightly but clearly postpone senes-

cence- and apoptosis-induced necrosis.

Fig. 6 shows that follicles kept in pres-ence of the Uttwiler Sptlauber stem cellextract continued to elongate until day18, whereas the control follicles startedto shrink after day 14.

Anti-Wrinkle Effect on theCrows Feet AreaThe anti-wrinkle efficacy of PhytoCell-Tec Malus Domestica was demonstratedin a clinical trial over 4 weeks with 20subjects. A cream with 2% PhytoCellTecMalus Domestica was applied twice dai-

ly to the crows feet area. Wrinkle depthwas analyzed with the PRIMOS systemafter 2 and 4 weeks. Digital photos of thecrows feet area were taken at the be-ginning and the end of the study. ThePhytoCellTecTM Malus Domestica creamwas found to significantly reduce wrin-kle depth after two and after four weeks,by 8% and 15% respectively. The effectcan be nicely demonstrated by genera-tion of 3D pictures. Such an example isshown inFig. 7. The anti-wrinkle effica-

cy could also been shown on digital pho-tos(Fig. 8).

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Gene after H2O2 after H2O2Control + 2% Extract

(% to untreated) (% to untreated)

cyclin B1: induces proliferation 73 130

cyclin E1: controls cell cycle 78 135

P53: tumor uppressor gene 63 137

insulin-like growth factorII:cell prolifertion enhancer 71 117

heme oxigenase 1: antioxidans enzyme 89 211

Table 1Effect of Uttwiler Sptlauber stem cell extract on gene expression profileof fibroblast cells after H2O2treatment

Fig. 6 Uttwiler Sptlauber stem cell extract increases lifespan of isolated humanhair follicles


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Conclusions

Stem cells are the most important cellsin the skin. They are the source for con-tinuous regeneration of the epidermis,the formation of new hair and hair pig-

ments. Reduced viability and prematuresenescence or apoptosis of stem cells isa principal cause for tissue aging. The re-sults presented in this article show thatan extract of Uttwiler Sptlauber stemcells positively influences viability and

resistance against senescence and apop-tosis of human stem cells. In this way, theplant stem cell extract promotes regen-

eration of skin and hair and delays theappearance of skin aging signs. The ex-act mechanism is not known. But theUttwiler Sptlauber apples, known to stayfresh 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 onhuman stem cells.

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., GranterS.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:

551-7

*Authors addresses:

Mibelle BiochemistryBolimattstrasse 1

5033 BuchsSwitzerland

Email: [email protected]

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Fig. 73D picture analysis of the crows feet area before and after application of acream with 2% PhytoCellTec Malus Domestica

Fig. 8Digital photos of the crows feet area before and after treatment with a creamwith 2% PhytoCellTec Malus Domestica

After (day 28)Before (day 0)

double stemcell plant stem cell extract for longevity of skin and hair sfw 05 08

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