Plant stem cells to protect skin stem cells

Fred Zülli - Mibelle Biochemistry Switzerland

© Mibelle Biochemistry, Switzerland 2010
Agenda

1. Introduction “aging”
2. Stem cell cosmetics
3. Activity of apple stem cells
4. New PhytoCellTec products based on grapes and alpine rose

© Mibelle Biochemistry, Switzerland 2010

1. Anti-Aging Cosmetics

Skin aging is based on

• Intrinsic aging
(chronological ageing, ROS,
reduced cell renewal)
• Extrinsic aging
(environmental influences:
UV, toxines, life style)

© Mibelle Biochemistry, Switzerland 2010

Theory of Evolution and Aging

General Considerations
Natural selection (Darwin) favours surviving
and reproduction in younger years versus
vitality and beauty in older ages.

Many children vs. beauty at older age

Jeanne Calment, 20 /122 y

© Mibelle Biochemistry, Switzerland 2010
Theory of Evolution and Aging

Bad news:
Nature / evolution does not support today’s
beauty trends

Good news:
Prevention and treatments with cosmetic
products can counteract aging due to:
– High technology / science
– Consumer spend money for the products

Mimi Weddell, 93
© Mibelle Biochemistry, Switzerland 2010
Aging and Longevity in Different Countries

Genetic Control ?
Environment?
Life Style?

© Mibelle Biochemistry, Switzerland 2010

Why Can‘t we Live Forever?

• Mouse 4 y (90% dye<1y)

• Dog 29 y
• Chimpanzee 59 y
• Elefant 86 y
• Men 122 y
• Turtle 190 y
• Whale 211 y
• Bristlecone Pine 4838 y

© Mibelle Biochemistry, Switzerland 2010

Aging Postulate

• Limited cell divisions (stem cells)

telomer shortening

• Oxidative stress (caloric restriction)

fuel + oxygen  energy to move a car
food + oxygen  energy to move our body
oxygen  ROS  cell death

© Mibelle Biochemistry, Switzerland 2010

What are Stem Cells?

• Stem cells have two properties:

Self-renewal - the ability to go through

numerous cycles of cell divisions in an
undifferentiated state
Unlimited potency - the capability to
differentiate into other cell types

• Toti-, pluri-, multi-, unipotent stem cells

© Mibelle Biochemistry, Switzerland 2010

Human: Embryonic Stem Cells (Pluripotent)

• Cells derived from the inner cell

mass of an early stage embryo
• Blastocyst, 4-5 days post
fertilization, 50-150 cells
• Pluripotent: Differentiate into one of
the more than 220 different cell
types of an adult body
• The use of embryonic stem cells in
medicin is discussed contradictorily

© Mibelle Biochemistry, Switzerland 2010

Human: Adult Stem Cells (Multipotent)

• Undifferentiated cells among

differentiated cells in a tissue or
organ
• Generate all the cell types of the
organ from which they originate —
possibility to regenerate the entire
organ from a few cells
• Function: Replenish dying cells and
regenerate damaged tissues

© Mibelle Biochemistry, Switzerland 2010

2. Stem Cell Cosmetics

© Mibelle Biochemistry, Switzerland 2010

Epidermal 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

© 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

© 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

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTecTM Malus Domestica
3 Stories

1. PhytoCellTec Technologie

2. Swiss Apple Story

3. Stem Cell Cosmetics

© 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

© 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)

© 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

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Plant Cell Culture Technology

© Mibelle Biochemistry, Switzerland 2010

 Growth of Plant Cell Culture in a Reactor
Biomass sugar conductivity pH

12000 40

35
10000

30

8000
25
total biomass [g FS]

6000 20

15
4000

10

2000
5

0 0
0 5 10 15 20 25
days [d] © Mibelle Biochemistry, Switzerland 2010
PhytoCellTec
A Difficult and Tedious Process

• Preparation of sterile plants 2 months

• Callus induction 12 months
• Isolation of a stable stem cell line from callus 6 months
• Transfer into liquid media 3 months
• Transfer into bioreactors 3 months

© Mibelle Biochemistry, Switzerland 2010

2. Swiss Apple Story

© 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
© Mibelle Biochemistry, Switzerland 2010
Uttwiler Spätlauber

The importance of the Uttwiler

Spätlauber apple was honored
by the release of a stamp.

© Mibelle Biochemistry, Switzerland 2010

Young and Old

© 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

Determination
+ apple stem cell extract of CFE

Incubation

© Mibelle Biochemistry, Switzerland 2010

 Increase of CFE by
PhytoCellTecTM Malus Domestica
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

© 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)
S-244
• 0.01% PhytoCellTecTM Malus Domestica

© Mibelle Biochemistry, Switzerland 2010

Day 14 Day 21 Day 28

Young cells (P5)

(control)

No cell growth No cell growth Old cells (P14)

(control)

Old cells (P14)

+ PhytoCellTec MD

S-244

© 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

© Mibelle Biochemistry, Switzerland 2010

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

S-186

Day 28
Day 0 2% PCT Malus Domestica
© 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

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTecTM Solar Vitis
Protects epidermal stem cells against UV damage
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM - A new Technology to
Produce Stem Cell Extracts

Stem cell culture from grapes

 A part of the flesh is selected
and cut

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTecTM : Cell Culture Technology
with Plant Stem Cells
Preparation of plant stem cells
• A part of a grape is selected and sterilized
• Induction of callus formation by cutting the plant
• Isolation of a stable stem cell line from the callus
• Incubation and proliferation on agar plates

© Mibelle Biochemistry, Switzerland 2010

 UV Radiation and Skin Stem Cells

Reflection
n R
d
a
ito

Sun cream with UV-filters

Absorption
rm
sid
ep
E

in+
O
R
S
to
x

Keratinocyte
s

Epidermal stem cell

PhytoCellTecTM Solar Vitis
Basal layer

Basement membrane
rm
s
iD
e

© Mibelle Biochemistry, Switzerland 2010

Increase of CFE with PCT Solar Vitis

S-261/©Mibelle Biochemistry 2009

© Mibelle Biochemistry, Switzerland 2010

Protection of Epidermal Stem Cells against
UV Radiation

• Stimulation of CFE by 86% with

PCT Solar Vitis
• 58% decrease of CFE after
exposure to UV
• With PCT Solar Vitis stem cells
were fully protected against
negative effects of UV

S-245/©Mibelle Biochemistry 2009

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTec™ Solar Vitis Protects against
UV-Stress

Study design
• Determination of the individual MED in 20
volunteers (19 to 63 y).
• Application: cream with SPF 10 with and
without 0.4% PhytoCellTec™ Solar Vitis.
• Irradiation with UV doses whose
intensities were in the range of the MED
multiplied by the SPF.
• 24 hours later, the redness of the skin
was evaluated again.

PhytoCellTec™ Solar Vitis significantly

increased the MED (MED+25% UV ~
SPF increase from 10 to 13).
S-268

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTec™ Solar Vitis
Claim Ideas

• Protects skin stem cells

against UV-stress
• Increases the skin‘s UV
tolerance
• Fights photo-aging
• For a vital and healthy-looking
skin

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTecTM Alp Rose
Charges skin stem cell resistance
© Mibelle Biochemistry, Switzerland 2010
The Swiss Alp Rose
Rhododendron ferrugineum L.

• In the Swiss Alps, the alpine rose is one

of the most typical and prominent
flowers
• Rhododendron (rhodon = rose, dendron
= tree, ferrugo = rust)
• Ericaceae
• 1500 – 2800 m
• 1 m high, winter-green shrub, highly
branched
• Pink, bell-shaped flowers
• Egg-shaped, lanceolate leaves,
underneath with rust-colored scales

© Mibelle Biochemistry, Switzerland 2010

Production of PhytoCellTecTM Alp Rose

• Leaf of alpine rose is wounded to induce callus formation

• Callus cells are transferred to agar plates
• The developed callus with its stem cells is harvested and further cultivated
on agar plates until complete dedifferentiation of the cells and generation of a
homogenous culture

© Mibelle Biochemistry, Switzerland 2010

Viscous Circle Impairs Skin Barrier

© Mibelle Biochemistry, Switzerland 2010

Protection/Improvement of Skin Stem Cell
Vitality with PhytoCellTecTM Alp Rose

Cell line: Epidermal stem cells

Product: 0.15% Alp rose stem cell
extract
Treatment: Radiation with UVA+UVB at
450 kJ/m2
Analysis: Determination of colony
forming efficacy (CFE)

Alp rose stem cell extract increases the

S-245
vitality of epidermal stem cells and
protects them against UV-induced
stress.
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Alp Rose
Ski Holiday Study

Test products
• Sun cream SPF 30
+ 0.4% PhytoCellTecTM Alp Rose
• Placebo (sun cream SPF 30 without active)
Volunteers: 22 (20 – 52 y)
Application: 3 times daily (morning, noon, evening)
Test area: Face, half side test (blind, randomized)

© Mibelle Biochemistry, Switzerland 2010

Ski Holiday Study:
Profile Photos (Visioface Quick®)
Anti-Wrinkle Effect

S-286
D0 D17

© Mibelle Biochemistry, Switzerland 2010

Ski Holiday Study:
Visible Improvement of the Skin Quality

Scoring:
Visual scoring by dermatologists on
day 0 and day 17

The skin side treated with

PhytoCellTec™ Alp Rose showed
an improvement of all 3
S-286
characteristics.

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTecTM Alp Rose
Claim Ideas

• Increases skin stem cell vitality

• Boosts epidermal regeneration
• Improves the skin barrier function
• Helps the skin to cope with climate
changes

© Mibelle Biochemistry, Switzerland 2010

3 Different PhytoCellTecTM Products:
Positioning

© Mibelle Biochemistry, Switzerland 2010

PhytoCellTecTM Products
Provide two Different Modes of Activity

1. Use of plant stem cells to protect skin stem cells

The components of plant stem cells such as from the apple, the grape and the
alpine rose are rich in specific factors relevant to protect and maintain the function
of skin stem cells
 epigenetic factors

2. Use of plant specific factors for target activity

 “Uttwiler Spätlauber” apple > longevity
 “Gamay Teinturier Fréaux” grape > UV protection
 Swiss alpine roses > resistance

 metabolites
© Mibelle Biochemistry, Switzerland 2010
3 Different PhytoCellTecTM Products
Key Positioning

Longevity Resistance UV-Protection

Regeneration Barrier function Photo-Aging

© Mibelle Biochemistry, Switzerland 2010