P Phyto Tell Pec Calus Tomestica (New Version)

PhytoCellTecTM Malus Domestica
Plant stem cells for skin stem cell protection
© Mibelle Biochemistry, Switzerland 2009

3 Stories

1. PCT Technologie

2. Swiss Apple Story

3. Stem Cell Cosmetics


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

1. Preparing a sterile plant preparation
2. Wounding the plant to induce callus tissue
3. 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



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

Epithelial skin stem cells Basal layer of the epidermis

Hair bulge stem cells Hair follicle


Epithelial Stem Cells

Stratum corneum

Granular layer

Spinous layer
Epidermal
stem cell

Transient
Basal layer amplifying
cells

Basement
Dermis
Differentiating progeny of membrane
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
expection
• 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


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

Protection of Stem Cells against
UV Damage – Study Design

Cell type
Umbilical cord blood stem cells

Study design
Incubation for 24h with PhytoCellTec Malus
Domestica Extract, UV radiation of cell cultures Control

Determination of living cells
MTS assay 48h after UV radiation

+ 0.1% PhytoCellTecTM Extract


Protection of Stem Cells against
UV Damage – Results

PhytoCellTec Malus
Domestica improves the
vitality of stem cells

Increased protection
S-142 against UV stress


Novel Progenitor Cell Targeting
Technology

• Cells: human epidermal stem cells (primary human keratinocytes) from a
biopsie 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 keratinocyte 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
+ apple stem cell extract of CFE

Incubation


Increase of CFE by
100 + 92%
Increase of the CFE compared to control in %

80

60

40 Result
Number of active and
20 healthy stem cells is
increased compared to
S-234 untreated cultures
0
0.01% 0.04%
Concentration of Malus Domestica stem cell extract


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

Control 0.2% Malus Domestica stem cell extract

20

10
Change in length compared to day 14 in %

0

-10

-20

-30

-40

-50

-60 Senescence of hair
S-157
(stem cells) can be delayed
-70
Day 16 Day 18 Day 20


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)


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 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

• Analysis of wrinkle depth in the crow‘s feet area
• 20 volunteers
• 28 days
• Application twice daily
• Against inital conditions


2% PhytoCellTec Malus Domestica

16

12
Decrease of wrinkle depth in %

8

4

S-186
0
Day 14 Day 28



S-186

Day 28
Day 0 2% PCT 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


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

Plant stem cells for skin stem cell protection

Patent pending


P Phyto Tell Pec Calus Tomestica (New Version)

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