PhytoCellTecTM Malus Domestica Plant stem cells for skin stem cell protection

PhytoCellTecTM Malus Domestica 3 Stories 1. PhytoCellTec Technologie 2. Swiss Apple Story 3. Stem Cell Cosmetics

PhytoCellTecTM Malus Domestica Innovation Price BSB Innovation Price for most innovative natural active Received during incosmetics 2008 Patent pending in CH, EU, USA and Korea

1. Plant Cell Cultures New cell culture technology makes it now possible to cultivate cells of endangered and rare species Selected plant material (leave, fruit) is wounded to induce the formation of a callus tissue The callus of a plant consists of dedifferentiated, totipotent cells (plant stem cells)

The 3 Steps of PhytoCellTec Preparing a sterile plant preparation Wounding the plant to induce callus tissue Growing callus cells in liquid cultures

Plant Cellculture Technology (PhytoCellTecTM =PCT) Suspension cultures from plant stem cells are grown in bioreactors Medium consists of sucrose, trace elements and plant hormones (cytokinins and auxins) Cultures grow with oxygen and without light (chlorophyll free) Batch is harvested after all sugar has been metabolized The cells are washed and homogenized to release the secondary metabolites Oil- and water-soluble ingredients are extracted by liposomal structures

2. Swiss Apple Story

Storage Ability of Apples and Skin Aging Apples with very good storage abilities stay fresh over months This was an important factor for cultivar selection Modern apple cultivars are selected for intensive cultivation and for a sweet flavour since storage is no longer a problem Therefore cultivars with excellent storage abilities disappeared from the market

Uttwiler Spätlauber An old cultivar is still alive in Switzerland but no longer cultivated The Spätlauber variety derives from the middle of the 18th century It was famous for its tremendous storability without shrinking Today this is a very rare and endangered apple variety with only a few trees left in the world

Uttwiler Spätlauber The importance of the Uttwiler Spätlauber apple was honored by the release of a stamp.

Young and Old

The Secret of Storage Ability High acid and tannin concentrations Longevity of cells Undiscovered factors? This apple can only be used by PCT

PhytoCellTecTM Malus Domestica Composition Malus Domestica Stem Cells 9.0 % Phospholipids 0.14% Glycerin 0.4 % Phenoxyethanol 1.4 % Keltrol T 1.0 % Deionized water ad.100 % CTFA/INCI Malus Domestica Fruit Cell Culture Extract (and) Xanthan Gum (and) Glycerin (and) Lecithin (and) Phenoxyethanol (and) Aqua/ Water

3. Stem Cell Cosmetics

What are Skin Stem Cells? Properties of skin stem cells: Self-renewal - the ability to go through numerous cycles and cell divisions while maintaining the undifferentiated state Differentiation - the capacity to differentiate into other cell types of the same tissue Primary roles - replenish dying cells and regenerate damaged tissues

Adult Stem Cells in Skin In skin, different types of stem cells have been found: Type Place Epidermal skin stem cells Basal layer of the epidermis Hair bulge stem cells Hair follicle

Epidermal Stem Cells Stratum corneum Granular layer Spinous layer Epidermal stem cell Transient amplifying cells Basal layer Basement membrane Dermis Differentiating progeny of one epidermal stem cell Stem cells are located in the basal layer where 2-7% of the cells are stem cells

Hair Bulge Stem Cells Epidermal layer Basal layer Infundibulum Sebaceous gland Outer root sheath Inner root sheath Hair shaft Hair bulge stem cells Hair follicle matrix Dermal papilla Lineages derived from bulge stem cells Differentiation pathways supplied by bulge stem cells

Stem Cells and Aging Stem cells have a limited life expectation Environmental and intrinsic stress factors effect stem cells In aged skin the number and activity of stem cells is reduced Skin stem cells have to be protected and supported to delay preliminary aging

Epigenetic Factors Composition of Plant Stem Cells Plant stem cells contain similar epigenetic factors as human stem cells Plant stem cell extracts provide important nutrients to human stem cells  Plant stem cell nutrients improve the viability and healthiness of human stem cells

PhytoCellTecTM Malus Domestica Provides two Different Modes of Activity 1. Use of plant stem cells to protect skin stem cells The components of plant stem cells such as Malus domestica are rich in specific factors relevant to protect and maintain the function of skin stem cells  epigenetic factors 2. Use of longevity cell extract to delay skin aging The apple Uttwiler Spätlauber is known to contain ingredients which insure longevity of cells  metabolites

Novel Progenitor Cell Targeting Technology Biopsy: human epidermal cells  ca. 6% of cells are stem cells (optically not different from normal basal cells) Medium: specifically designed, fully defined medium that mimics the physiological environment of the in vivo stem cell niche  enrichment of so called progenitor cells (stem cells) 3x

Activity Assay for Epidermal Stem Cells Stimulation of growth or over-expression of markers are not good parameters for the activity of stem cells Colony forming efficiency (CFE) Cell cultures are seeded at low density. The number of colonies formed is a value for the concentration and activity of progenitor/stem cells Determination of CFE + apple stem cell extract Incubation

Increase of CFE by PhytoCellTecTM Malus Domestica + 92% Result Number of active and healthy stem cells is increased compared to untreated cultures S-234

Result PhytoCellTec apple stem cell extract enhanced CFE up to 92% compared to the control. Maintenance of the stem cell vitality of epidermal stem cells was improved.

Growth of Hair Follicle Hair follicles From human skin fragments (from micro dissection) Test substance PhytoCellTecTM Malus Domestica 0.2% Design Length of hair follicle increases until day 14 → after day 14 induction of senescence → length of hair follicle decreases S-157 Day 0 Day 4 Day 7

Length of Hair Follicle Senescence of hair (stem cells) can be delayed S-157

PhytoCellTecTM Malus Domestica Effect on Gene Expression in Senescent Dermal Fibroblasts Cell line Normal Human Dermal Fibroblasts Design  H2O2 induced senescence (2h) followed by a treatment with 2% PhytoCellTecTM Malus Domestica or not (144 h) Parameters  Gene profile (microarray)

PhytoCellTecTM Malus Domestica Gene Profile (Microarray) ↓ Gen, that is down-regulated by senescence induction (see control) ↑ Gen, that is up-regulated or normalized in senescent cells by PhytoCellTec Malus Domestica Gen After H2O2 Control + 2% Malus Domestica stem cell extract cyclin B1: induces proliferation 73 ↓ 130 ↑ cyclin E1: cell cycle regulator 78 ↓ 135 ↑ p53: tumor suppressor gene 63 ↓ 137 ↑ insulin-like growth factor II: cell proliferation enhancer 71 ↓ 117 ↑ heme oxigenase 1: antioxidans enzyme 89 ↓ 211 ↑ S-194

In-Vivo Study: Anti-Wrinkle Effect of PhytoCellTecTM Malus Domestica Analysis of wrinkle depth in the crow‘s feet area 20 volunteers 28 days Application twice daily Against inital conditions

Capacity to Form 3D Epidermis Study design Comparison of 3D epidermis formed by early passage (young) and by late passage (old) epidermal progenitor cells. Test substance without test substance (control) 0.01% PhytoCellTecTM Malus Domestica S-244

Day 14 Day 21 Day 28 Young cells (P5) (control) Old cells (P14) No cell growth No cell growth Old cells (P14) + PhytoCellTec MD S-244

Result Epidermal progenitor (stem) cells of late passage (P14) loose the capacity to form a 3D epidermis. But when these cells are cultured with PhytoCellTecTM Malus Domestica, they retain the capacity to form a 3D epidermis similar to early passage progenitor cells. PhytoCellTecTM Malus Domestica clearly helps to conserve the progenitor cell characteristics. Also cells of late passage are able to proliferate and to undergo the complex process of stratification and differentiation.

In-Vivo Study: Anti-Wrinkle Effect of PhytoCellTecTM Malus Domestica

In-Vivo Study: Anti-Wrinkle Effect of PhytoCellTecTM Malus Domestica Day 28 2% PCT Malus Domestica Day 0

PhytoCellTecTM Malus Domestica Applications Stem cell cosmetics Rejuvenation products for face and body Fresh cell treatments Anti-Aging formulations Rejuvenation of hair follicles Delay of hair aging

PhytoCellTecTM Malus Domestica Claims Delays senescence of essential cells Protects longevity of skin stem cells Protection of skin stem cells Rejuvenation of skin Maintains the power of regeneration Decrease in wrinkles Combats chronological aging With plant stem cells to maintain the power of regeneration With plant stem cells to delay senescence of essential skin cells

PhytoCellTecTM Malus Domestica Plant stem cells for skin stem cell protection Patent pending