The anti-aging potential of plant derived stem cells...The Anti-Aging Potential of plant derived...

© Mibelle Biochemistry, Switzerland 2011

Stem Cell Cosmetics The anti-aging potential of plant derived stem cells

In-Cosmetics 2011, Milan Beata Hurst, Mibelle Biochemistry


Fountain of youth – a dream?


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


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


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


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


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


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


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


From Callus to Plants


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)


5-Wave Bioreactor


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


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



stem cells

1. Stem cells












Stem Cell Cosmetics


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


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


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


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 »


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


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


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


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


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


3 Different PhytoCellTecTM Products

Key Applications

Malus Domestica

• Longevity

• Regeneration

Alp Rose

• Resistance

• Barrier function

Solar Vitis

• UV-Protection

• Photo-Aging


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


Uttwiler Spätlauber

The importance of the Uttwiler

Spätlauber apple was honored by

the release of a stamp.


http://www.sxc.hu/browse.phtml?f=download&id=217661


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


PPR with PhytoCellTecTM Malus Domestica


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


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


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



stem cells

1. Stem cells












PhytoCellTecTM Argan

Vitalizing dermal stem cells for

fundamental anti-aging effectiveness

Dermal stem cells – our next target


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


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.


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.


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


Staining of Spheres

Nuclear staining Sox2 stainingHistological analysis of primary

spheres

Real dermal stem cells!


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


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


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


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


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.


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


Thank you

for

your interest

[email protected]

Booth E70

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