Post on 16-Jul-2015
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus DomesticaPlant stem cells for skin stem cell protection
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus Domestica3 Stories
1. PhytoCellTec Technologie
2. Swiss Apple Story
3. Stem Cell Cosmetics
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus DomesticaInnovation 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 preparation2. Wounding the plant to induce callus tissue3. Growing callus cells in liquid cultures
© 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
© 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
© 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
The Secret of Storage Ability
• High acid and tannin concentrations • Longevity of cells• Undiscovered factors?
This apple can only be used by PCT
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus Domestica
CompositionMalus Domestica Stem Cells 9.0 %Phospholipids 0.14%Glycerin 0.4 %Phenoxyethanol 1.4 %Keltrol T 1.0 %Deionized water ad.100 %
CTFA/INCIMalus Domestica Fruit Cell Culture Extract (and) Xanthan Gum (and) Glycerin (and) Lecithin (and) Phenoxyethanol (and) Aqua/ Water
© 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
© 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
© 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 membraneDermis
Basal layer
Spinous layer
Granular layer
Stratum corneum
© 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
© 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 Provides two Different Modes of Activity
1. Use of plant stem cells to protect skin stem cellsThe 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 agingThe apple Uttwiler Spätlauber is known to contain ingredients which insure longevity of cells
metabolites
© 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
© 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
co
ntro
l in
%
+ 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
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.
© 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
© Mibelle Biochemistry, Switzerland 2010
Length of Hair Follicle
-70
-60
-50
-40
-30
-20
-10
0
10
20
Day 16 Day 18 Day 20
Cha
nge
in le
ngth
co
mp
ared
to d
ay 1
4 in
%
Control 0.2% Malus Domestica stem cell extract
Senescence of hair (stem cells) can be delayed
S-157
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus DomesticaEffect 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)
© Mibelle Biochemistry, Switzerland 2010
PhytoCellTecTM Malus DomesticaGene 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 H2O2Control
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
© 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
Capacity to Form 3D Epidermis
Study designComparison of 3D epidermis formed by earlypassage (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
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 progenitorcell characteristics. Also cells of late passage are able to proliferate and toundergo the complex process of stratification and differentiation.
Result
© Mibelle Biochemistry, Switzerland 2010
In-Vivo Study: Anti-Wrinkle Effect of PhytoCellTecTM Malus Domestica
0
4
8
12
16
Day 14 Day 28
Dec
reas
e o
f wrin
kle
dep
th in
%
2% PhytoCellTec Malus Domestica
S-186
© 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 Applications
• Stem cell cosmetics• Rejuvenation products for face and
body• Fresh cell treatments• Anti-Aging formulations• Rejuvenation of hair follicles• Delay of hair aging
© 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