1. PhytoCellTecTM Malus Domestica
Plant stem cells for skin stem cell protection

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

3. 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

4. 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)

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

6. 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

7. 2. Swiss Apple Story

8. 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

9. 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

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

11. Young and Old

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

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

14. 3. Stem Cell Cosmetics

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

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

25. 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.

26. 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

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

28. 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)

29. 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

30. 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

31. 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

32. 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

33. 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.

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

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

36. 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

37. 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

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