© Mibelle Biochemistry, Switzerland 2010
Plant stem cells to protect skin stem cellsFred 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
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
© 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
© Mibelle Biochemistry, Switzerland 2010
Plant Cell Culture Technology
© Mibelle Biochemistry, Switzerland 2010
Growth of Plant Cell Culture in a Reactor
0
2000
4000
6000
8000
10000
12000
0 5 10 15 20 25
days [d]
tota
l bio
mas
s [g
FS
]
0
5
10
15
20
25
30
35
40
tota
l su
gar
[g
/L],
co
nd
uct
ivit
y [m
S/c
m]
and
pH
-va
lue
Biomass sugar conductivity pH
© 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
+ apple stem cell extract
Incubation
Determination of CFE
© Mibelle Biochemistry, Switzerland 2010
0
20
40
60
80
100
0.01% 0.04%
Concentration of Malus Domestica stem cell extract
Incr
ease
of t
he C
FE c
om
par
ed to
cont
rol i
n %
+ 92%
ResultNumber of active and healthy stem cells is increased compared to untreated culturesS-234
Increase of CFE by PhytoCellTecTM Malus Domestica
© Mibelle Biochemistry, Switzerland 2010
Capacity to Form 3D Epidermis
Study designComparison 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
© 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
© 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
Day 0Day 282% PCT Malus Domestica
S-186
© 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 VitisProtects 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
Epid
erm
isD
erm
is
Rad
iation
RO
S +
toxi
ns
Sun cream with UV-filters
Epidermal stem cell
Basal layer
Basement membrane
Reflection
Absorption
Keratinocyte PhytoCellTecTM Solar Vitis
© 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 VitisClaim 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 RoseCharges skin stem cell resistance
© Mibelle Biochemistry, Switzerland 2010
The Swiss Alp RoseRhododendron 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 cellsProduct: 0.15% Alp rose stem cell extractTreatment: Radiation with UVA+UVB at 450 kJ/m2 Analysis: Determination of colony forming efficacy (CFE)
Alp rose stem cell extract increases the vitality of epidermal stem cells and protects them against UV-induced stress.
S-245
© Mibelle Biochemistry, Switzerland 2010
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)
PhytoCellTecTM Alp Rose Ski Holiday Study
© Mibelle Biochemistry, Switzerland 2010
Ski Holiday Study: Profile Photos (Visioface Quick®) Anti-Wrinkle Effect
D0 D17
S-2
86
© Mibelle Biochemistry, Switzerland 2010
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 characteristics.
Ski Holiday Study: Visible Improvement of the Skin Quality
S-286
© 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
1. Use of plant stem cells to protect skin stem cellsThe 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
PhytoCellTecTM ProductsProvide two Different Modes of Activity
© Mibelle Biochemistry, Switzerland 2010
3 Different PhytoCellTecTM ProductsKey Positioning
LongevityRegeneration
ResistanceBarrier function
UV-ProtectionPhoto-Aging