Estudios del crecimiento del folículo del pelo con celulas madre de manzana suiza. Prueba con PhytoCellTec Malus Domestica al 0,2%

1. © Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus Domestica
Plant stem cells for skin stem cell protection

2. © Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus Domestica
3 Stories
1. PhytoCellTec Technologie
2. Swiss Apple Story
3. Stem Cell Cosmetics

3. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
The 3 Steps of PhytoCellTec
1. Preparing a sterile plant preparation
2. Wounding the plant to induce callus tissue
3. Growing callus cells in liquid cultures

6. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
2. Swiss Apple Story

8. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
Uttwiler Spätlauber
The importance of the Uttwiler
Spätlauber apple was honored
by the release of a stamp.

11. © Mibelle Biochemistry, Switzerland 2010
Young and Old

12. © Mibelle Biochemistry, Switzerland 2010
The Secret of Storage Ability
• High acid and tannin concentrations
• Longevity of cells
• Undiscovered factors?
This apple can only be used by PCT

13. © Mibelle Biochemistry, Switzerland 2010
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

14. © Mibelle Biochemistry, Switzerland 2010
3. Stem Cell Cosmetics

15. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
Epidermal Stem Cells
Stem cells are located in the basal layer where 2-7% of the cells are stem cells
Differentiating progeny of
one epidermal stem cell
Epidermal
stem cell
Transient
amplifying
cells
Basement
membrane
Dermis
Basal layer
Spinous layer
Granular layer
Stratum corneum

18. © Mibelle Biochemistry, Switzerland 2010
Hair follicle matrix
Epidermal layer
Sebaceous gland
Hair shaft
Inner root sheath
Outer root sheath
Basal layer
Dermal papilla
Hair bulge
stem cells
Infundibulum
Lineages derived from bulge stem cells
Differentiation pathways supplied by bulge stem cells
Hair Bulge Stem Cells

19. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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
+ apple stem cell extract
Incubation
Determination
of CFE

24. © Mibelle Biochemistry, Switzerland 2010
0
20
40
60
80
100
0.01% 0.04%
Concentration of Malus Domestica stem cell extract
IncreaseoftheCFEcomparedtocontrolin%
+ 92%
Result
Number of active and
healthy stem cells is
increased compared to
untreated culturesS-234
Increase of CFE by
PhytoCellTecTM Malus Domestica

25. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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
Day 0 Day 4 Day 7
S-157

27. © Mibelle Biochemistry, Switzerland 2010
Length of Hair Follicle
-70
-60
-50
-40
-30
-20
-10
0
10
20
Day 16 Day 18 Day 20
Changeinlengthcomparedtoday14in%
Control 0.2% Malus Domestica stem cell extract
Senescence of hair
(stem cells) can be delayedS-157

28. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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
After H2O2
+ 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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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 DomesticaS-244

32. © Mibelle Biochemistry, Switzerland 2010
Old cells (P14)
(control)
Old cells (P14)
+ PhytoCellTec MD
No cell growth No cell growth
Day 14 Day 21 Day 28
Young cells (P5)
(control)
S-244

33. © Mibelle Biochemistry, Switzerland 2010
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.
Result

34. © Mibelle Biochemistry, Switzerland 2010
In-Vivo Study: Anti-Wrinkle Effect of
PhytoCellTecTM Malus Domestica
0
4
8
12
16
Day 14 Day 28
Decreaseofwrinkledepthin%
2% PhytoCellTec Malus Domestica
S-186

35. © Mibelle Biochemistry, Switzerland 2010
In-Vivo Study: Anti-Wrinkle Effect of
PhytoCellTecTM Malus Domestica
Day 0
Day 28
2% PCT Malus Domestica
S-186

36. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
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. © Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus Domestica
Plant stem cells for skin stem cell protection
Patent pending