Cyndy D. Sanberg, Ph.D. President & COO

Stem cells: (U.S. National Institutes of Health 2010)

Can develop into many different cell types during early life and growth within the body.

Located in many tissues, serve as an internal repair system.

Divide without limit to replenish other cells

– When a stem cells divides, each new cell remains a stem cell or becomes another type of cell with specialized function

(i.e. muscle cell, red blood cell, or a brain cell).

What are Stem Cells and Why are They Important?

3 Types of Stem cells: (U.S. National Institutes of Health 2010)

1) Embryonic Stem Cells

2) Adult Stem Cells (Somatic)

3) Induced Pluripotent Stem Cells (iPSCs) – genetically reprogrammed

adult sc that assumes a stem-like state, 2006.

Types of Stem Cells

The Problem:As we age, our adult stem cells decrease and lose their ability to maintain healthy immune function.

are located in most tissues throughout the body. can develop into most cell types.maintain & repair tissue. support a healthy immune system.

Importance of Adult Stem Cells

1) Can we design a formulation from a nutraceutical approach?

2) If we combine potent natural ingredients can we create a synergisticeffect?

Goal - Adult Stem Cell Health

Out of 100 Natural Ingredients Tested at Different Ratios We Discovered

Green Tea

Blueberries

4 Ingredients with Potent Synergy:For adult stem cell proliferationFor protection against oxidationFor memory & neurogenesis

Vitamin D3

L-Carnosine

NT-020 FormulaU.S. Patent #7,442,394

% o

ver

con

tro

l (m

ean

±1

SEM

)

Bickford et al., 2006; Stem Cells & Dev.

NT-020 IncreasesProliferation of Human Stem Cells

Cells were cultured in 96-well plates, and treated with extracts for 72 hrs.

Assay: MTT for cell proliferation

hGM-CSF

Blueberry

Catechin

Carnosine

Green Tea

Vit. D3

Human Bone Marrow Cells

20

0

40

60

80

100Human CD34+ Cells

100

80

60

40

20

0

NT-020 IncreasesProliferation of Human Stem Cells

Bickford et al., 2006; Stem Cells & Dev.

hGM-CSF

Blueberry

Catechin

Carnosine

Green Tea

Vit. D3

40

0

20

60Human CD133+ Cells

% o

ver

con

tro

l (m

ean

±1

SEM

)

NT-020 Increases Neuroprotection

Primary microglial cells were grownin culture and exposed to anoxidative insult (H2O2).

Lactate dehydrogenase (LDH) assaywas used.

Shytle RD, Ehrhart J, Tan J, Vila J, Cole M, Sanberg CD, Sanberg PR, Bickford PC (2007) Oxidative stress of neural, hematopoietic, and stem cells: protection by natural compounds. Rejuvenation Res 10:173-178.

Results: ~90% of the cells in the control groups died (10% survival). However, in the presence of NT-020 cell death was 40% (60% survival).

Primary neuronal cells were grownin culture and exposed to anoxidative insult (H2O2).

Lactate dehydrogenase (LDH) assaywas used.

Shytle RD, Ehrhart J, Tan J, Vila J, Cole M, Sanberg CD, Sanberg PR, Bickford PC (2007) Oxidative stress of neural, hematopoietic, and stem cells: protection by natural compounds. Rejuvenation Res 10:173-178.

Results:~70-80% of the cells in the control groups died (20-30% survival). However, in the presence of NT-020 cell death was 35% (65% survival).

NT-020 Increases Neuroprotection

Animals received NT-020 orvehicle for 2 weeks orally. Bonemarrow stem cells grown inculture, and exposed to anoxidative insult (H2O2).

NT-020Antioxidant Properties

Shytle RD, Ehrhart J, Tan J, Vila J, Cole M, Sanberg CD, Sanberg PR, Bickford PC (2007) Oxidative stress of neural, hematopoietic, and stem cells: protection by natural compounds. Rejuvenation Res 10:173-178.

High dose = 2 caps of NT-020

(13.5mg/kg)

(13.5mg/kg)

(135mg/kg)N = 5 each group

Results:The low dose showed a 20% decrease in cell death; high dose showed a greater than 40% decrease in cell death; compared to control.

NT-020 and Stroke

NT-020 - animals received orally for 14 days

MCAO (stroke) – on day 14

Behavioral testing – occurred prior to stroke and

the 14th day following stroke

Infarction Analysis – tissues collected the 14th day

following stroke after behavioral testing

(Yasuhara et al., 2008; Cell Transplantation)

Bederson Test – neurological

EBST – motor function

(Yasuhara et al., 2008; Cell Transplantation)

Vehicle

NT-020

Vehicle

NT-020

Degree of Damage

NT-020

NT-020 Reduces Damage Post-Stroke

Results: NT-020 decreases the behavioral deficit following a stroke and reduces brain damage.

NT-020

NT-020 (Yasuhara et al., 2008; Cell Transplantation)

NT-020 Increases Stem Cell Proliferation

Results:

NT-020 increases proliferation of stemcells in two brain regions:

1) Subventricular Zone (SVZ) - a stemcell rich zone.

2) Striatum - a target area for themigration of proliferating stem cells.

Stem Cells

Stroke lesion

Control

NT-020

NT-020Enhances Memory

Richard G. Morris (2008), Scholarpedia, 3(8): 6315.

Morris Water Maze4theloveofanimals.com

3 test groups: 1) Young (3 months)2) Old (20 months) 3) Old Treated (NT-020)

NT-020 (135mg/kg/day) was given orally to the NT-020 Old group for 4 weeks. Young and Old Control animals were given water.

Test: Morris Water Maze, spatial learning and memory for 5 days.

NT-020 Enhances Memory

Commons.wikimedia.org/wiki/File:morriswatermaze

Richard G. Morris (2008), Scholarpedia, 3(8): 6315.

Acosta S.A. et al., Society for Neuroscience Abstracts (2009).

NT-020Enhances Memory

Acosta S.A. et al., Society for Neuroscience Abstracts (2009).

Not all of the Old animals showedimpairment. Thus only the oldimpaired groups were compared(top 40% of each group).Cognitively

Impaired

Day 5

Commons.wikimedia.org/wiki/File:morriswatermaze

Results:NT-020 improved the memory of the Older animals.

NT-020Effects Inflammation

Aging increases the expression ofMHC class II receptors on microgliareflecting a rise in inflammation. Old= 21 months

Acosta S.A. et al., Society for Neuroscience Abstracts (2009).

Results:NT-020 significantly reduces MHC Class II expression in the DG, which is responsible for learning and memory.

NT-020 Treatment Group -135mg/kg/day orally for 4 weeksControls – waterYoung – 4 monthsOld – 21 months

NT-020Increases Neurogenesis

Acosta S.A. et al., Society for Neuroscience Abstracts (2009).

Results: A significant age-related decline in proliferating cells (Ki67).

NT-020 increases the number of proliferating cells.

OX6 – SGZ Dentate Gyrus

Young Young

Old Old

OldNT-020

OldNT-020

NT-020 Treatment Group -135mg/kg/day orally for 4 weeksControls - water

Conclusions

A Specific Combination of Natural Ingredients (NT-020):

Promotes the proliferation of adult stem cells.

Has potent antioxidant properties (neuroprotection).

Helps to minimize neural damage in an animal model of stroke.

Increases the migration of adult stem cells to ischemic areas.

Enhances spatial memory.

Reduces MHC Class II expression (reduces inflammation).

Increases neurogenesis.

Acknowledgements

Shinn-Zong Lin, M.D., Ph.D.

Paula C. Bickford, Ph.D.Cesar Borlongan, Ph.D. Doug Shytle, Ph.D.Paul R. Sanberg, M.D., Ph.D., D.Sc.

USF Research Park

3802 Spectrum Blvd., Ste. 142

Tampa, FL 33612

Phone: 813-866-7818

Fax: 813-315-7995

www.naturatherapeutics.com