© Mibelle Biochemistry, Switzerland 2011
Stem Cell Cosmetics The anti-aging potential of plant derived stem cells
In-Cosmetics 2011, Milan Beata Hurst, Mibelle Biochemistry
© Mibelle Biochemistry, Switzerland 2011
The Anti-Aging Potential of plant derived
stem cells
1. Stem cells
– Stem cells in men and plants
– PhytoCellTecTM, a novel biotechnology to cultivate plant stem cells
2. Stem cells cosmetics
– Properties and the role of epidermal stem cell
– Skin stem cells and aging
– Protection of longevity and efficiency of skin stem stells with
PhytoCellTecTM actives
3. Next target – dermal stem cells
– Identification and characteristics
– PhytoCellTecTM Argan – next generation of stem cell cosmetics
© Mibelle Biochemistry, Switzerland 2011
Stem Cells
What is Special about these Cells?
Stem cells have two properties:
• 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
Beside the embryonic stem cells,
adults have so called adult stem cells
in every organ to replenish dying cells
and regenerate damaged tissues
© Mibelle Biochemistry, Switzerland 2011
Human: Embryonic Stem Cells (Totipotent)
• 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 2011
Adult Stem Cells in Human, Multi-Potent
• Stem cells are the only cells in adults
that can divide and regenerate tissues
• We have only a limited number of
these cells
• Stem cells lose vitality with age,
replication cycles are slower and even
can stop
Stem cells are the most precious cells
of our body and need to be protected
and supported to maintain their
vitality
© Mibelle Biochemistry, Switzerland 2011
Stem Cells in Plants
• In contrast to human, every plant
cell has the potency to
regenerate new organs (leaves,
stem, flowers, and seeds) or even
the whole plant throughout their
whole life
• Plant cells can dedifferentiate
under certain conditions and
become a stem cells
© Mibelle Biochemistry, Switzerland 2011
PhytoCellTecTM
A selected part of the
plant is cut in to small
pieces;
it is wounded to
induce the formation of a
callus tissue
A novel technology to dedifferentiate plant
cells and produce stem cell actives
© Mibelle Biochemistry, Switzerland 2011
Plant Cell Cultures
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
© Mibelle Biochemistry, Switzerland 2011
Production of PhytoCellTecTM Stem Cells
• After complete dedifferentiation of the cells and generation of a
homogenous culture, the cells are brought into suspension with liquid
media
• The suspension culture grows continuously (doubling time 4 - 10 days)
© Mibelle Biochemistry, Switzerland 2011
PhytoCellTecTM
A Challenging and sensitive Process
• Preparation of sterile plants 1 – 2 months
• Callus induction 3 – 12 months
• Isolation of a stable stem cell line from callus 3 – 6 months
• Transformation into liquid media 1 – 3 months
• Transformation into bioreactors 1 – 3 months
© Mibelle Biochemistry, Switzerland 2011
Sustainable Sourcing for raw material
Further benefits:
• Free of pollutants or other impurities
• Independency regarding seasons
• Not limited to availability on the market
• Consistent quality
PhytoCellTecTM makes it now possible to cultivate cells of endangered
and rare species helps to preserve rare or limited plant stock
© Mibelle Biochemistry, Switzerland 2011
The Anti-Aging Potential of plant derived
stem cells
1. Stem cells
– Stem cells in men and plants
– PhytoCellTecTM, a novel biotechnology to cultivate plant stem cells
2. Stem cells cosmetics
– Properties and the role of epidermal stem cell
– Skin stem cells and aging
– Protection of longevity and efficiency of skin stem stells with
PhytoCellTecTM actives
3. Next target – dermal stem cells
– Identification and characteristics
– PhytoCellTecTM Argan – next generation of stem cell cosmetics
© Mibelle Biochemistry, Switzerland 2011
Skin Stem CellsProperties
Differentiation - the capacity to differentiate
into other cell types of the same tissue
Self-renewal - the ability to go through
numerous cycles and cell divisions while
maintaining the undifferentiated state
Primary roles - replenish dying cells and
regenerate damaged tissues
© Mibelle Biochemistry, Switzerland 2011
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
membraneDermis
Basal layer
Spinous layer
Granular layer
Stratum corneum
© Mibelle Biochemistry, Switzerland 2011
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 2011
Decrease of Colony Forming Efficiency
(CFE)
Stem cell (progenitor cell)
Keratinocyte
Young stem cell efficiency Aging/stressed stem cell efficiency Aging/stressed stem cell
which lost its «stemness »
© Mibelle Biochemistry, Switzerland 2011
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 2011
0
20
40
60
80
100
0.01% 0.04%
Concentration of Malus Domestica stem cell extract
Incr
ease
of th
e C
FE
co
mp
are
d to
co
ntr
ol i
n %
+ 92%
Result
The vitality and activity
of epidermal stem cells
is increased compared
to untreated culturesS-234
Increase of Colony Forming Efficiency by
PhytoCellTecTM Malus Domestica
© Mibelle Biochemistry, Switzerland 2011
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
© Mibelle Biochemistry, Switzerland 2011
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 2011
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
PhytoCellTecTM Products
Provides two Different Modes of Activity
© Mibelle Biochemistry, Switzerland 2011
3 Different PhytoCellTecTM Products
Key Applications
Malus Domestica
• Longevity
• Regeneration
Alp Rose
• Resistance
• Barrier function
Solar Vitis
• UV-Protection
• Photo-Aging
© Mibelle Biochemistry, Switzerland 2011
PhytoCellTecTM Malus Domestica
USP
Key benefits:
• Longevity, delay of senescence
• Regeneration
Specific study:
• 3 D epidermis (maintenance of skin
stem cells)
• Senescence of fibroblasts
• Lifespan of hair follicles
Plant story:
• Longliving, rare Swiss apple
© Mibelle Biochemistry, Switzerland 2011
Uttwiler Spätlauber
The importance of the Uttwiler
Spätlauber apple was honored by
the release of a stamp.
© Mibelle Biochemistry, Switzerland 2011
PhytoCellTecTM Malus Domestica Claims
• Delays senescence of essential
cells
• Protects longevity of skin stem cells
• Protection of skin stem cells
• Rejuvenation of skin
• Combats chronological aging
© Mibelle Biochemistry, Switzerland 2011
PhytoCellTec™ Solar Vitis
USP
Key benefits:
• Skin stem cell protection against UV-
stress
• Photo-aging
• UV-tolerance
Specific study:
• Protection of stem cells against UV-
stress
• In-vivo: Increase of UV-tolerance
Plant:
• Extremely UV-resistant Burgundy grape
© Mibelle Biochemistry, Switzerland 2011
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 2011
PhytoCellTecTM Alp Rose
USP
Key benefits:
• Skin stem cell resistance
• Barrier function
• Environmental stress protection
Specific study:
• In-vivo half side test during ski
holiday
• TEWL
• Wrinkle, Complexion
Plant:
• Extremely resistant, rare alpine
flower
© Mibelle Biochemistry, Switzerland 2011
The Anti-Aging Potential of plant derived
stem cells
1. Stem cells
– Stem cells in men and plants
– PhytoCellTecTM, a novel biotechnology to cultivate plant stem cells
2. Stem cells cosmetics
– Properties and the role of epidermal stem cell
– Skin stem cells and aging
– Protection of longevity and efficiency of skin stem stells with
PhytoCellTecTM actives
3. Next target – dermal stem cells
– Identification and characteristics
– PhytoCellTecTM Argan – next generation of stem cell cosmetics
© Mibelle Biochemistry, Switzerland 2011
PhytoCellTecTM Argan
Vitalizing dermal stem cells for
fundamental anti-aging effectiveness
Dermal stem cells – our next target
© Mibelle Biochemistry, Switzerland 2011
Next target: dermal stem cells
• Epidermis: epidermal stem cells in the
basal layer continuously regenerate
the epidermal layer
• Dermis: dermal stem cells confer
firmness and elasticity to the skin and
are involved in wound healing
processes
But: the stem cell reservoir in the dermis
remained a secret for a long time
© Mibelle Biochemistry, Switzerland 2011
Dermal Papilla Stem Cells and their Role in
Skin
self renewal
differentiation into
inter-folicular
fibroblasts
sphere
formation
wound
healing
End of 2009 a research
group from the
University of Toronto
(Freda Miller et al.,
Hospital for Sick
Children, Toronto, CA)
discovered that the
dermal papilla is a
niche for dermal
progenitor/stem cells.
© Mibelle Biochemistry, Switzerland 2011
Characterization of “Dermal Stem Cells”
Sphere-forming properties
Human dermal papilla cells grow in three
dimensional spheres.
Sox2 expression
The expression of the specific Sox2 marker
gene is required for maintaining the
pluripotency in stem cells.
© Mibelle Biochemistry, Switzerland 2011
1. Establishment of a Cell Line with Human
Dermal Papilla Cells
Sphere of dermal papilla cells
(ca. 3000 cells/ 10 μl drop)
Isolation of dermal papilla
cells from a hair bulb
Hanging drop culture in 96 well plate
Formation of primary spheres
Hanging drops after Higgins et al., 2003
Passaging
+/- active
substance
© Mibelle Biochemistry, Switzerland 2011
Staining of Spheres
Nuclear staining Sox2 stainingHistological analysis of primary
spheres
Real dermal stem cells!
© Mibelle Biochemistry, Switzerland 2011
2. Preparation of Secondary Spheres
Spontaneous formation of secondary
spheres (10’000 cells/0.5 ml)
Hanging drop culture in 96 well plate
Formation of primary spheres
Test system for
novel cosmetic
actives!
Spheres are
trypsinised
and
solubilised
Passaging
+/- active
substance
© Mibelle Biochemistry, Switzerland 2011
1. Effect of PhytoCellTecTM Argan
on Sox2 ExpressionControl + PhytoCellTecTM Argan
DP passage 9,
16 days
Sox2 expression is enhanced
“stemness” of the dermal stem cells is improved
© Mibelle Biochemistry, Switzerland 2011
2. Effect of PhytoCellTecTM Argan on
Sphere Formation
• Secondary spheres of human
dermal stem cells
• Microscopic counting of
sphere number after 3 weeks
Increase of sphere
number with
PhytoCellTecTM ArganS-330
© Mibelle Biochemistry, Switzerland 2011
And the Absortion into the Dermis?
In PhytoCellTecTM Argan the oil- and
water-soluble ingredients are
vectorized in liposomes
Hair follicles represent a highly
relevant and efficient penetration
pathway and reservoir for topically
applied substances
dermal papilla, where the dermal
stem cell pool is localized, can be
reached over follicular penetration
© Mibelle Biochemistry, Switzerland 2011
Innovation: Next Generation of Stem Cell
Cosmetics
1. The identification of dermal stem cells opens the door to the next
generation of stem cell cosmetics: Protection and vitalization of human
dermal stem cells for a deep-seated rejuvenation of the skin, resulting in
restoration of firmness and wrinkle reduction.
2. We have established a specific test system: cell cultures of human dermal
stem cells that.
3. Screening for a cosmetic active that has the potential to enhance the
vitality of dermal stem cells.
Plant stem cells obtained with our PhytoCellTecTM technology from a very
resistant and rare plant – the argan tree.
© Mibelle Biochemistry, Switzerland 2011
Claim Ideas with PhytoCellTecTM Argan
• Deep-seated rejuvenation of the skin
• Vitalizes dermal stem cells, which
are responsible for collagen and
elastin production
• Accelerates skin's natural repair
process
• Combats chronological aging
• Fights wrinkle and loss of firmness