A scientific overview and rationale

Development of autologous Adipose Derived Mesenchymal Stem Cell conditioned saline as an injectable cell-free therapy and stem cell banking services

Introduction to the Project

Adult derived mesenchymal stem cells were first isolated

in 1982 and have generated an extensive literature as an

alternative source of stem cells in regenerative medicine.

Mesenchymal stem cells can be isolated from human

bone marrow or from fat tissue of a patient in need of

treatment and thus provide a source of stem cells which

are autologous for the specific patient and so provide a

safer and ethically more acceptable population of stem cells

compared to embryonic stem cells. Despite the 28 years

of extensive research in both animals and humans, there

has been limited progress in translating this research to

therapy. Recently, our traditional view of stem cell therapy

mediating reparative effects in degenerative disease by

direct replacement of damaged cells by healthy cells has

been challenged. Many laboratories including our own have

found that stem cells secrete various factors which rescue

damaged tissue and stimulate regeneration in a variety of

disease models. These data permit an alternative source of

therapy rather than direct stem cell transplantation, namely

the harvesting of such secretory factors in physiological

saline from in-vitro cultured autologous stem cells and using

such saline (conditioned media) as an injectable agent to

ameliorate disease. It is to this approach that this proposal

is addressed. We present examples of our own pre-clinical

animal data for a typical degenerative condition (Dementia),

review the published supporting literature and propose

clinical protocols for assaying such effects in human patients

on a named patient compassionate use basis in a small

private clinic.

Natural Biosciences SA: Company Background.

Natural Biosciences SA. Is a Swiss company, established in

2009, to bring to clinical practice its intellectual property and

expertise in the fields of stem cell derived macromolecules

and regenerative medicine. Our clinical procedures focus

on the use of autologous cell-free injectable lysates and

conditioned media, derived from regulatory authority

compliant adipose derived mesenchymal stem cells, as a

safer (autologous) form

of cell therapy.

A review of the pre-clinical and clinical literature shows an

extensive body of published literature reporting evidence

for cell-free stem cell derived extracts as a safe, minimally

invasive and efficacious therapeutic. Further, many

laboratories including our own, now show such cell free

injections to show the same efficacy as intact stem cell

therapy with less risk to the patient as no intact cells are

transplanted. The approach has been widely used and

stimulates repair and regeneration in a wide range of soft

and hard tissue organs.

The company is focused on two immediate term services,

the facility to offer adults and children the opportunity to

store their own stem cells for future use in the event of

accident or disease. Secondly, to offer clinical therapy using

autologous ADMSC generated cell free therapeutics at our

clinic in Switzerland.

Part 1: Brief overview

Part 1: Overview of therapy and banking services page 2 to 4

Part 2: Scientific rationale for therapy page 5 to 11

Part 3: Resume of Chief Scientist page 12 to 15

2

3

1) Autologous stem cell banking services

There are several well established commercial stem

cell banks offering the opportunity for new parents to store

umbilical cord blood derived stem cells for the child’s use in

later life if the child develops an illness. However, currently

there are no provisions for adult clients to similarly bank

their own stem cells for either future or immediate use in

case of disease or injury. Natural Biosciences offers this

service within the current stem cell regulatory framework to

an international client base, with storage services for intact

stem cell populations suitable for transplantation, and cell

free molecular fractions for immediate use by our consulting

physicians.

Advantages of banking stem cells

• Many acute medical crises e.g. heart attack, stroke

or trauma occur with little or no warning, similarly the

treatment window to optimize repair and regeneration

is brief. To maximize the benefits of regenerative

medicine, pre-stored stem cell product for immediate

use is the only option as isolation, separation,

expansion and therapy after the event would have

significantly less efficacy or none at all.

• Stem cell quality and quantity do deteriorate with age.

Most chronic degenerative conditions e.g. dementias,

Parkinson’s disease, lung diseases, liver and kidney

problems, vascular diseases occur in older individuals

with sub optimal stem cell populations. Logically it is

better to harvest an optimal population when the

client is in good health for future use to exploit the

many repair and regenerative effects of the various

forms of stem cell therapy offered by this company,

and others, such effects relevant to our therapies are

reviewed briefly below.

• Regenerative and anti-ageing medicine has become

one of the fastest growing areas of medical sciences

with new developments announced in the scientific

and popular literature announced daily. Adult stem

cell banking offers the client the opportunity to take

full advantage of current and future developments in

stem cell medicine with their own optimized stem

cells ready for immediate use.

2) Autologous ADMSC generated cell-free therapeutics

Natural Biosciences SA has developed a portfolio of

intellectual property based on the paracrine effects of adult

stem cells. Following an extensive period of pre-clinical

testing in our (UK Government regulated) laboratories, we

are at a phase of development to translate this research to

therapeutic application on a Named Patient Use basis in

Switzerland.

Our approach uses the secretory factors from autologous

stem cells as a therapeutic product. These are generated

from the patients own ADMSCs collected by liposuction,

separated from fat and expanded as clinical grade cells.

These cells are then used to condition sterile physiological

saline. Such conditioned saline may then be condensed

for injection or further fractionated for specific injectables.

One such fractionation is the centrifugal separation of

microvesicles from the conditioned saline. Microvesicles

are becoming appreciated as an important communication

system between damaged tissue and stem cells both

in-vivo and in-vitro. They, like whole stem cell conditioned

saline, can mediate numerous tissue regenerative effects

following non-invasive systemic injection. Such cell free

approaches to therapy, offers many safety advantages over

intact stem cell transplantation minimising the potential risk

of tumour genesis from transplanted cells, immunological

issues or inappropriate stem cell differentiation. The

technique is minimally invasive involving either intravenous

or local injection and is used to support healing following

conventional repair of tissue or as a stand alone therapy to

induce tissue regeneration.

The therapy requires minor aspiration of fat. This is

processed by one of our European compliant laboratories

to produce clinical grade autologous ADMSCs. Following

stringent testing, these cells are used to generate the

autologous stem cell generated cell free injectable products.

These therapies are designed to be used to stimulate

endogenous repair and regeneration mechanisms in the

body and so lend themselves well to support other surgical

and medical interventional programmes.

Brief overview (Cont.)

A further advantage is that the patients’ stem cells may

also be banked cryogenically, and future therapies prepared

immediately from the same sample.

The production of such clinical cell free autologous

products is summarised in figure 1

Fig. 1

The company currently holds a unique market advantage

of owning intellectual property and ‘know how’ for

immediate application in adult stem cell banking

conforming to all regulatory bodies, and is positioned

to offer clients immediate access to autologous cell free

therapy. The cell free autologous injectables will only be

used on a named patient basis in our own clinics and will

not be manufactured or supplied to any third party by

Natural Biosciences SA. We are not seeking to produce a

licensed medicine, only to operate as a private clinic.

4

Autologous ADMSC generated cell free therapeutics

Natural Biosciences SA has developed a portfolio of

intellectual property based on the paracrine effects of adult

stem cells. Following an extensive period of pre-clinical

testing in our (UK Government regulated) laboratories, we

are at a phase of development to translate this research

to therapeutic application on a Named Patient Use /

Compassionate trial basis in Switzerland.

Our approach uses the secretory factors from autologous

stem cells as a therapeutic product. These are generated

from the patient’s own ADMSCs collected by liposuction,

separated from fat and expanded as clinical grade cells.

These cells are then used to condition sterile physiological

saline. Such conditioned saline may then be condensed

for injection or further fractionated for specific injectables.

One such fractionation is the centrifugal separation of

microvesicles from the conditioned saline. Microvesicles

are becoming appreciated as an important communication

system between damaged tissue and stem cells both in-vivo

and in-vitro. They, like whole stem cell conditioned saline,

can mediate numerous tissue regenerative effects following

non-invasive systemic injection. Such cell free approaches

to therapy, offers many safety advantages over intact stem

cell transplantation minimising the potential risk of tumour

genesis from transplanted cells, immunological issues or

inappropriate stem cell differentiation.

Advantages of ADMSCs

They are easily and safely obtained from lipoaspirates using

clinically approved commercial kits. Housman (2002) reports

a study by the American Society for Dermatological Surgery

of outpatient cosmetic liposuction performed between 1994

and 2000 showed zero deaths on 66570 procedures and a

serious adverse event rate of 0.68 per 1000 cases (1).

ADMSC are also easy to isolate from lipoaspirates using a

digestion buffer containing 0.1%collagenase (type 1, Sigma)

and 0.25% trypsin (sigma) dissolved in Hank’s buffer

then gravity separation and centrifugation (1500 rpm for

10 minutes) (2). This separation method avoids the use of

magnetic bead or antibody mediated positive or negative

selection of stem cell populations. Purity of cells can also

easily be assessed in ADMSCs by high levels of stem cell

related antigens (CD13, CD29, CD44, CD105, and CD166).

Moreover, the clinical utility is further enhanced compared

to other stem cell types by positive expression of Nanog,

Oct4, Sox-2 and Rex-1, genes normally associated with

embryonic stem cells (2).

The most important feature of ADMSCs is that they may be

obtained in very large, clinically significant, numbers from a

single liposuction procedure without the need to expand the

cells. Fraser et al (2006) report that the frequency of BMSCs

in skeletally mature adults is approximately between 1 in

50,000 and 1 in 100,000 cells in bone marrow aspirates

compared to ADMSCs in lipoaspirates with a frequency of 1

in 100 cells some 500 fold more than found in marrow (3).

Another important advantage of ADMSCs is that they are

easily expanded in tissue culture (2, 3). ADMSCs may be

expanded through at least 25 passages without lose of

stem cell characteristics. Thus, large volumes of ADMSCs

may be obtained.

ADMSCs are also known to have excellent potential to

differentiate in to a wide variety of tissue types an essential

feature for stem cell therapy. Illustrative of this ability,

ADMSCs have been differentiated into Neural tissue (4,5,6),

Cardiac tissue (7,8,9), Skeletal muscle (10,11,12), Cartilage (13,14,15),

Hepatocytes (16), Hematopoietic cells (17), endothelial cells

(18), and Bone (19,20). Thus an expansive literature shows

ADMSCs to be one of the most versatile adult derived stem

cell capable of a wide range of regenerative effects in both

humans and animals.

In conclusion, it is clearly evident from the published

scientific literature and the clinical literature, that autologous

derived ADMSCs offer an attractive source of cells for a

variety of applications in regenerative medicine.

Paracrine effects

Adult derived stem cell transplantation has now been

Part 2: Scientific Rationale for the therapy

5

studied and used clinically for well over 26 years; however

it is only in the last five years that scientists and clinicians

have begun to realise that stem cells have more than one

therapeutic benefit. Previously it was thought that injected

stem cells home to tissue damage and replace lost cell

populations, hence the popular rational for transplanting

whole cells. Recently, many authors report impressive

regenerative effects in both animal and human studies which

are mediated by paracrine actions of the stem cells i.e.

secretory molecules which have a rescue/stimulatory effect

on tissue damage in a variety of pathologies.

There are now several seminal papers illustrative of the

clinical potential of using autologous stem cell extracts

or media/saline conditioned by such cells as a safer,

less invasive clinical protocol compared with intact cell

transplantation. Amongst the first papers to report such

effects was the report by Togel et al. (2005) entitled

“Administered mesenchymal stem cells protect against

ischemic acute renal failure through differentiation-

independent mechanisms” (21). These authors conclude

that the profound recovers observed was mediated by

secretory factors produced by the cells and that it was

these growth factors which ameliorated renal damage.

Similar conclusions have now been reported in many

other pathologies including, but not limited to, recovery

of ischemic limb disease by growth factors secreted by

adipose tissue derived stem cells (22). Increasingly the

factors responsible for such effects are being elucidated

in a variety of pathologies including cardiac protection

and functional recovery (23), liver injury repair (24), brain and

spinal tissue damage (25) and many others beyond the

scope of this article; indeed the entire secretion proteome

of mesenchymal stem cells has now been mapped and

reported (26). An ever expanding literature on this topic has

led many groups to investigate direct injection of stem cell

derived lysates to ameliorate tissue damage following spinal

cord injury (27), wound healing (28), tendon repair (29) amongst

many other pathologies. Further, Yeghiazarians et al. (30)

show that cell extract injection into infarcted hearts results

in functional improvement comparable to intact cell therapy.

Similarly, Shabbir et al. report that heart failure therapy can

be mediated by the trophic activities of mesenchymal stem

cells following injection of stem cell conditioned media or

stem cells intra muscularly into the hamstring muscle (31).

They report attenuating heart tissue injury, inhibiting fibrotic

remodelling, and promoting angiogenesis, stimulating

recruitment and proliferation of endogenous tissue stem

cells and reducing inflammatory oxidative stress as some of

the multiple trophic factor effects of MSCs.

Stem cell conditioned media results have been confirmed

by other laboratories around the world in both human and

animal studies including – stem cell conditioned media

induced neuroprotective effects in a model of Huntington’s

disease (32), other laboratories report excellent stem cell

conditioned media effects on injured spinal cords (33), stroke

(34), cardiac damage (35, 36), diabetes (37) and age related

damage (38, 39).

In summary, conditioned media provides a filtered, cell

free, autologous protein and RNA rich saline suitable for

injection to stimulate regeneration. Such conditioned media

is also known to contain microvesicles small membrane

bound structures (20nm – 1µm in diameter) known to be

powerful intercellular communicators with profound ability

to differentiate, proliferate and integrate endogenous and

exogenous stem cells (40,41,42,43,44) and stimulate tissue

repair by a variety of mechanisms (45,46,49). The clinical use of

differentiated or undifferentiated stem cell conditioned media

again provides an attractive, less invasive and cell free

alternative to cell transplantation.

Human use of mesenchymal stem cells

The potential uses of stem cells for tissue repair and

regeneration is beginning to be realised with extensive

clinical trials already completed for adult derived stem

cells and FDA approval for commencement of human

trials for embryo stem cells. An indication of the number of

clinical trials involving stem cells is best illustrated in fig 1.

which shows the number of human stem cell clinical trials

registered with ClinicalTrial.gov. per annum

The 70,000 records are from 165 countries and are

registered federal or privately funded trials. Further, there

are many clinics internationally offering both autologous and non-autologous stem cell therapy

Scientific Rationale for the therapy (Cont.)

6

(e.g. www.xcell-centre.com, www.cellmedicine.com,

www.medistem.com, www.emcell.com,

www.tissustemcell.com) these are well established clinical

practices with impeccable safety records using

a variety of stem cell types including umbilical cord stem

cells, bone marrow mesenchymal stem cells or adipose

derived stem cells, by a variety of different injection routes

including intravenous administration and intraspinal injection,

all use intact cells as the injectable material.

Adipose derived mesenchymal stem cells are also currently

used extensively in clinical medicine as intact cell transplants

in breast reconstruction, ligament and tendon repair, bone

defect repair and cosmetic surgery (e.g. www.cytoritx.com,

www.theamarclinic.com, www.facesplus.com,

www.stemnow.com) and also as cell free injectable material

e.g. Sloane Clinic (www.sloaneclinic.com). There are also

extensive publication lists available at www.stemcelldocs.

org reviewing pre-clinical and clinical data.

Similarly, in the peer reviewed literature, mesenchymal stem

cells have been reported to have safe therapeutic potential

following transplantation in humans with osteogenic

imperfecta (78), bone fracture (79), traumatic brain injury (80),

stroke (81), amyotrophic lateral sclerosis (82), graft versus host

disease (83,84), myocardial infarction (85,86). No significant side

effects have been reported.

Sourced from: http://thestemcellblog.com/2009/03/21/

7

References cited in document:

1) Houseman, T.S. (2002)

The safety of liposuction: results of a national survey.

Dermatological Surg. 28. 971 – 978.

2) Zhu, Y. et al. (2008)

Adipose-derived stem cells: a better stem cell than BMSC.

Cell Biochemistry and Function. 26. 664 – 675.

3) Fraser, J.K. et al. (2006)

Fat tissue: an unappreciated source of stem cells

for biotechnology.

Trends in Biochemistry. 24(4). 150 – 154.

4) Ashjian, P.H. et al. (2003)

In vitro differentiation of human processed lipoaspirate cells

into early neural progenitors.

Plast. Reconstructive. Surg. 111. 1922 – 1931.

5) Kang, S.K. et al. (2003)

Improvement of neurological deficits by intracerebral

transplantation of human adipose tissue-derived stromal cells

after cerebral ischemia in rats.

Exp. Neurol. 183. 355 – 366.

6) Safford, K.M. et al. (2002)

Neurogenic differentiation of murine and human

adipose-derived stromal cells.

Biochem. Biophys. Research Communications. 294. 371 – 379.

7) Rangappa, S. et al. (2003)

Transformation of adult mesenchymal stem cells isolated from

fatty tissue into cardiomyocytes.

Ann. Thorac. Surg. 75. 775 -779.

8) Gausted, K.G. et al. (2004)

Differentiation of human adipose tissue stem cells using extracts

of rat cardiomyocytes.

Biochem. Biophys. Research Communications. 314. 420 – 427.

9) Planat-Bernard, V. et al (2004)

Spontaneous cardiomyocytes differentiation from adipose tissue

stroma cells.

Circ. Res. 94. 223 – 229.

10) Rodriguez, A.M. et al. (2005)

Transplantation of a multipotent cell population from

human adipose tissue induces dystrophin expression in the

immunocompetent mdx mouse.

J. Exp. Med. 201. 1397 -1405.

11) Mizuno, H. et al. (2002)

Myogenic differentiation by human processed lipoaspirate cells.

Plast. Reconstr. Surg. 109. 199 – 209.

12) Bacou, F. et al. (2004)

Transplantation of adipose tissue derived stromal cells increases

mass and functional capacity of damaged skeletal muscle.

Cell Transplant. 13. 103 – 111.

13) Winter, A. et al. (2003)

Cartilage-like gene expression in differentiated human stem cell

spheroids: a comparison of bone marrow-derived and adipose

tissue-derived stromal cells.

Arthritis Rheum. 48. 418 – 429.

14) Erickson, G.R. et al. (2002)

Chondrogenic potential of adipose tissue-derived stromal cells in

vitro and in vivo.

Biochem. Biophys. Res. Commun. 209. 763-769.

15) Dragoo, J.L. et al. (2003)

Tissue engineered cartilage and bone using stem cells from

human infrapatellar fat pads.

J. Bone Joint Surg. Br. 85. 740-747.

16) Seo, M.J. et al. (2005)

Differentiation of human adipose stromal cells into hepatic lineage

in vitro and in vivo.

Biochem. Biophys. Res. Commun. 328. 258-264.

17) Cousin, B. et al. (2003)

Reconstitution of lethally irradiated mice by cells isolated from

adipose tissue.

Biochem. Biophys. Res. Commun. 301. 1016 – 1022.

18) Miranville, A. et al. (2004)

Improvement of postnatal neovascularisation by human adipose

tissue-derived stem cells.

Circulation. 110. 349 – 355.

19) Zuk, P.A. et al (2001)

Multilineage cells from human adipose tissue: implications foe

cell-based therapies.

Tissue Eng. 7. 211 – 228.

20) Hicok, K.C. et al. (2004)

Human adipose-derived adult stem cells produce osteoid in-vivo.

Tissue Eng. 10. 371 – 380.

21) Togel, F. et al. (2005)

Administred mesenchymal stem cells protect against

ischemic acute renal failure through differentiation

-independent mechanisms.

Am. J. Physiol – Renal Physiol. 289. 31 – 42.

22) Nakagami, H. et al. (2005)

Novel autologous cell therapy in ischemic limb disease through

growth factor secretion by cultured adipose tissue-derived stem cells.

Arterioscler. Thromb. Vasc. Biol. 25. 2542 – 2547.

23) Gneechi, M. et al. (2006)

Evidence supporting paracrine hypothesis for Akt- modified

mesenchymal stem cell-mediated cardiac protection and

functional improvement.

FASEB J. 20. 661 – 669.

24) Lin, N. et al. (2008)

Hedgehog-mediated paracrine interactions between hepatic

stellate cells and marrow derived mesenchymal stem cells.

Biochem. Biophys Res. Comm. 372. 260 – 265.

8

25) Todas. H. et al. (2003)

Stem cell-derived neural stem/progenitor cell supporting factor

for adult neural stem/progenitor cells.

J. Biological Chemistry. 278 (37). 35491 – 35500.

26) Kwan Sze. S. et al. (2007)

Elucidating the secretion proteome of human embryonic

stem cell-derived mesenchymal stem cells.

Molecular & Cellular Proteomics. 6. 1680 – 1689.

27) Kang. S.K. et al. (2007)

Cytoplasmic extracts from adipose tissue stromal cells alleviates

secondary damage by modulating apoptosis and promotes

functional recovery following spinal cord injury.

Brain Pathol. 17. 263 -275.

28) Fu, X. et al. (2007)

Adipose tissue extract enhances skin wound healing.

Wound Repair and Regeneration. 15. 540 – 548.

29) Moon, Y. L. et al. (2008)

Autologous bone marrow plasma injection after arthroscopic

debridement for elbow tendinosis.

Ann. Acad. Med. Singapore. 37. 559 -563.

30) Yeghiazarians, Y. et al. (2009)

Injection of bone marrow cell extract into infracted hearts results

in functional improvement compared to intact cell therapy.

Molecular Therapy. Advanced on line doi:10.1038.mt.2009.85.

31) Shabbir, A. et al. (2009)

Heart failure therapy mediated by trophic activities of bone marrow

mesenchymal stem cells: a non invasive therapeutic regimen.

Am. J. Physiol. Heart. Circ. Physiol. 296. H1888 – H1897.

32) Lim. H.C. et al (2008)

Neuroprotective effect of neural stem cell-conditioned media in in

vitro model of Huntingdon’s disease.

Neuroscience Letters. 435. 175 – 180.

33) Peng, L. et al (2006)

Human neural stem cells promote corticospinal axons regenerating

and synapse reformation in injured spinal cord of rats.

Chinese Medical Journal. 119 (16). 1331 – 1338.

34) Fatar, M. et al. (2008)

Lipoaspirate-derived adult mesenchymal stem cells improve

functional outcome during intracerebral hemorrage by

proliferation of endogenous progenitor cells stem cells in

intracerebral hemorrages.

Neuroscience Letters. 443. 174-178.

35) Doyle. B. et al. (2008)

Progenitor cell therapy in a porcine acute myocardial infarction

model induces cardiac hypertrophy, mediated by paracrine

secretion of cardiotrophic factors including TGFbeta1.

Stem cells and development. 17(5). 941-951.

36) Crisostomo, P.R. et al. (2007)

Stem cell mechanisms and paracrine effects: potential in

cardiac surgery.

Shock. 28(4). 375-383.

37) Moriscot, C. et al. (2005)

Human bone marrow mesenchymal stem cells can express insulin

and key transcription factors of the endocrine pancreas

developmental pathway upon genetic and/or microenvironment

manipulation in vitro.

Stem Cells. 23. 594 – 604.

38) Kim, W.S. et al (2009)

Antiwrinkle effects of adipose-derived stem cells: activation of

dermal fibroblasts by secretory factors.

J. Dermatological Science. 53. 96 – 102.

39) Kim. W.S. et al. (2008)

Evidence supporting antioxidant action of adipose-derived stem

cells: protection of human dermal fibroblasts from oxidative stress.

J. Dermatological Science. 49. 133 – 142.

40) Ratajczak, J. et al. (2006)

Embryonic stem cell-derived microvesicles reprogram

hematopoietic progenitors: evidence for horizontal transfer of

mRNA and protein delivery.

Leukemia. 20. 847-856.

41) Ratajczak, J. et al. (2006b)

Membrane derived microvesicles: important and underappreciated

mediators of cell-to-cell communication.

Leukemia. 20. 1487 – 1495.

42) Yuan, A. et al (2009)

Transfer of microRNAs by embryonic stem cell microvesicles.

PLoS One. 4(3).e4722.

43) Aliotta, J.M. et al (2007)

Alteration of marrow cell gene expression, protein production,

and engraftment into lung by lung-derived microvesicles: a novel

mechanism for phenotype modulation.

Stem Cells. 25(9). 2245 – 2256.

44) Ray.S. (2008)

Patent application Microvesicles. PCT/GB2009/000004

45) Bruno, S. et al. (2009)

Mesenchymal stem cell derived microvesicles protect against

acute tubular injury.

J. Am. Soc. Nephrol. 20. 1053 – 1067.

46) Spees, J.L. et al. (2006)

Mitochondrial transfer between cells can rescue

aerobic respiration.

Proc. Nat. Acad. Sci USA. 103(5). 1283 – 1288.

47) Van Poll, D. et al. (2008)

Mesenchymal stem cell – derived molecules directly modulate

hepatocellular death and regeneration in-vitro and in-vivo.

Hepatology. 47 (5). 1634 – 1643.

48) Hagan, M. et al. (2003) Neuroprotection by human neural

progenitor cells after experimental contusion in rats.

Neuroscience Letters. 351. 149 – 152.

9

49) Hyun Ok Kim1,2, Scong-Mi Choi1, and Han-Soo Kim. (2013)

Mesenchymal Stem Cell-Derived Secretome and Microvesicles as a

Cell-Free Therapeutics for Neurodegenerative Disorders.

Tissue Engineering and Regenerative Medicine. Vol. 10,

No. 3, pp 93-101DOI 10.1007/s13770-013-0010-7

50) Doyle, B. et al (2008)

Progenitor cell therapy in a porcine acute myocardial infarction

model induces cardiac hypertrophy, mediated by paracrine

secretion of cardiotrophic factors including TGFbeta1.

Stem cells and development. 17. 941 – 51.

51) Lanza, C. et al. (2009)

Neuroprotective mesenchymal stem cells are endowed with

a potent antioxidant effect in – vivo.

J. of Neurochemistry. 110. 1674 – 1684.

52) Kyung Kang, S. et al. (2007)

Cytoplasmic extracts from adipose tissue stromal cells alleviates

secondary damage by modulating apoptosis and promotes

functional recovery following spinal cord injury.

Brain Pathology. 17. 263 – 275.

53) Burchfield, J.S. & Dimmeler, S. (2008)

Role of paracrine factors in stem and projenitor cell mediated

cardiac repair and tissue fibrosis.

Fibrosis & Tissue repair. 1:4. 1 – 11.

54) Rehman, J. et al. (2004)

Secretion of angiogenic and antiapoptotic factors by human

adipose stromal cells.

Circulation. 109. 1292 – 1298.

55) Kang, Y et al. (2009)

Proteomic characterization of the conditioned media produced

by the visceral endoderm-like cell line HepG2 and END2: towards

a defined medium for the Osteogenic/chondrogenic differentiation

of embryonic stem cells.

Stem cells and development. 18(1). 77 - 82.

56) Vaca, P. et al. (2006)

Induction of differentiation of embryonic stem cells into

insulin-secreting cells by fetal soluble factors.

Stem Cells. 24. 258 – 265.

57) Kale, V.P. & Limaye. L.S. (1999)

Stimulation of adult human bone marrow by factors secreted

by fetal liver hematopoietic cells: In vitro evaluation using

semisolid clonal assay system,

Stem Cells. 17. 107 – 116.

58) Aliotta, J.M. et al. (2008)

Tissue-specific gene expression of marrow cells co-cultured with

various murine organs. Abstract presented to the American

Society of Hematology 50th meeting. San Francisco.

59) Bentz, K. et al. (2007)

Embryonic stem cells produce neurotrophins in response

to cerebral tissue extract: Cell line-dependent differences.

J. Neuroscience Research. 85. 1057 – 1064.

60) Liu, Y. et al. (2009)

Cell extract from fetal liver promotes hematopoietic differentiation

of human embryonic stem cells.

Cloning and stem cells. 11(1). 51 – 60.

61) Xu, Y.X. (2009) Mesenchymal stem cells treated with rat pancreatic

extract secrete cytokines that improve the glycometabolism of

diabetic rats.

Stem Cell Biology. 41. 1878 – 1884.

62) Di Santo, S. et al. (2009)

Novel cell-free strategy for therapeutic angiogenesis:

in-vitro generated conditioned medium can replace progenitor

cell transplantation.

PLoS ONE 4(5):e5643

63) Planat-Benard, V. et al. (2004)

Spontaneous cardiomyocytes differentiation from adipose tissue

stromal cells.

Circ. Research. 94. 223 – 229

64) Song, Y.H. et al. (2007)

VEGF is critical for spontaneous differentiation of stem cells

into cardiomyocytes.

Biochem. Biophy. Res. Commun. 354. 999 – 1003.

65) Safford, K.M. et al. (2002)

Neurogenic differentiation of murine and human adipose

derived stromal cells.

Biochem. Biophy. Res. Comm. 294. 371 – 379.

66) Safford, K.M. et al. (2004)

Characterization of neuronal/glia differentiation of murine adipose

derived adult stromal cells.

Exp. Neurol. 187. 319 – 328.

67) Ashjian, P.H. et al. (2003)

In-vitro differentiation of human processed lipoaspirate into early

neural progenitors.

Plast. Reconstr. Surg. 111. 1922 – 1931.

68) Zuk, P.A. et al. (2001)

Multilineage cells from human adipose tissue: implications for

cell based therapies.

Tissue Eng. 7. 211 – 228.

69) Mizuno, H. et al. (2002)

Myogenic differentiation by human processed lipoaspirate cells.

Plast. Reconstr. Surg. 109. 199 – 209.

70) Miranville, A. et al (2004)

Improvement of postnatal neovascularisation by human adipose

tissue-derived stem cells.

Circulation. 110. 349 – 355.

71) Planat-Bernard, V. et al. (2004)

Plasticity of human adipose lineage cells toward endothelial cells:

physiological and therapeutic perspectives.

Circulation. 109. 656 – 663.

10

72) Rehman, T. et al. (2004)

Secretion of angiogenic and antiapoptotic factors by human

adipose stromal cells.

Circulation. 109. 1292 – 1298.

73) Halvorsen, Y.D. et al. (2001)

Extracellular matrix mineralization ans osteoblast gene

expression by human adipose tissue derived stromal cells.

Tissue Eng. 7. 729 – 741.

74) Hicok, K.C. et al. (2004)

Human adipose derived stem cells produce osteoids in vivo.

Tissue Eng. 10. 371 – 380.

75) Stewart C A and Morris R G M (1993).

The watermaze. In “Behavioural Neuroscience. A Practical

Approach. Volume I”. Ed A Saghal, IRL Press at Oxford University

press, Oxford, New York, Tokyo, pp107-122.

76) Kuen-Jer, T. et al. (2007)

G-CSF rescues the memory impairment of animal models

of Alzheimer’s disease. J. Expt Med.

On-line www.jem.org/cgi/doi/10.1084/jem20062481.

77) Smith, D.L. et al. (2009)

Reversal of long term dendritic spine alterations in Alzheimer’s

disease models.

Proc. Natl. Acad. Sci. USA. 106. (39) 16877 – 16882.

78) Horwitz, E.M. et al. (2002)

Isolated allogenic bone marrow derived mesenchymal cells

engraft and stimulate growth in children with osteogenesis

imperfecta : implications for cell therapy of bone.

Proc. Natl. Acad.Sci. USA. 99. 8932 – 8937.

79) Bajada, S. Et al. (2007)

Successful treatment of refractory tibial non-union using calcium

sulphate and bone marrow stromal cell implantation.

J. Bone Joint Surg. Br. 89. 1382 – 1386.

80) Zhang, Z.X. et al. (2008)

A combined procedure to deliver autologous mesenchymal

stromal cells to patients with traumatic brain injury.

Cytotherapy. 10. 134 – 139.

81) Bang, O.Y. et al. (2005)

Autologous mesenchymal stem cell transplantation in

stroke patients.

Ann. Neurol. 57. 874 – 882.

82) Ferrero, I. Et al. (2008)

Bone marrow mesenchymal stem cells from healthy donors and

sporadic amyotrphic lateral sclerosis patients.

Cell Transplant. 17. 255 – 266.

83) Ringden, O. et al. (2006)

Mesenchymal stem cells for treatment of therapy resistant graft

versus host disease.

Transplantation. 81. 1390 – 1397.

84) Muller, I. et al. (2008)

Mesenchymal stem cell therapy for degenerative

inflammatory disorders.

Current opinion in Organ Transplantation. 13. 638 – 644.

85) Chen, S.L. et al. (2004)

Effect on left ventricular function of intracoronary transplantation

of autologous bone marrow mesenchymal stem cells in patients

with acute myocardial infarction.

Am. J. Cardiol. 94. 92 – 95.

86) Katritsis, D.G. et al. (2005)

Transcoronary transplantation of autologous mesenchymal stem

cells and endothelial progenitors into infracted human myocardium.

Catheter. Cardiovasc. Interv. 65. 321 – 329.

11

Occupation: Chief Scientist Natural Biosciences SA

Senior Lecturer in Clinical Physiology,

Oxford Brookes University UK

Academic qualifications:

BSc (Hons.) Psychology

Plymouth Polytechnic -1984

PhD Neuroscience

Plymouth Polytechnic-1988

Stephen Ray BSc PhD

Resume

Employment:

2008: Present Chief Scientist Natural Biosciences SA.

2005 – 2008: Chief Scientist Systems Biology

Laboratory Ltd.

2003 – 2005: Chief Scientist Ribostem Ltd.

1993-2003: Senior lecturer in Clinical Physiology School

of Biological & Molecular Sciences, Oxford Brookes

University

Research interests: The biological basis of learning and memory, neural

tissue transplantation, stem cell transplantation, cell and

cell extract based therapy, physiological measurement

of stress.

12

Selected research publications

Henry Collins-Hooper, Roberta Sartori, Raymond

Macharia, Komtip Visanuvimol, Keith Foster, Antonios

Matsakas, Hannah Flasskamp, Steve Ray, Philip

R Dash, Marco Sandri, and Ketan Patel (2013).

Propeptide-Mediated Inhibition of Myostatin Increases

Muscle Mass Through Inhibiting Proteolytic Pathways in

Aged Mice, Journal of Gerontology A Biol Sci Med Sci.

1 - 11.

Henry Collins-Hooper, Graham Luke, Mark Cranfield,

William R. Otto, Steve Ray, and Ketan Patel. (2011)

Efficient myogenic reprogramming of adult white fat

stem cells and bone marrow stem cells by freshly

isolated skeletal muscle fibers. Translational Research.

Vol. 158 (6) 334 - 343

Walthall H, and Ray S. (2002)

Do perioperative variables have an effect on extubation

timing following coronary artery bypass grafts? Heart

and Lung. Vol. 31.

Walthall H, Robson D, and Ray, S. (2001)

Do any preoperative variables affect extubation time

after coronary artery bypass grafts? Abstract selected

by Dannemiller Memorial Educational Foundation

for AnesthesiaFile.

Walthall, H., Robson, D., & Ray, S. (2001)

Do any preoperative variables affect extubation time

after coronary artery bypass graft surgery?

Heart & Lung. Vol. 30. (3). 216-224.

Walthall H, Robson D, and Ray S. (2001)

Does extubation cause haemodynamic instability

in patients following coronary artery bypass grafts?

Journal of Intensive and Critical Care Nursing.

Vol. 17. 286 – 293.

Ray, S. & Bagnall, L. (2001)

Gender differences in rat learning assessed on

spatial navigation, taste aversion and visual

discrimination tasks.

Animal Technology. Vol. 52. (3). 219 – 226.

13

Martin, M. & Ray, S. (2001)

A comparison of learning ability between water or food

deprived and non deprived rats on

visual discrimination and conditioned taste

aversion paradigms.

Animal Technology. Vol. 52. (3) 211- 217.

Butcher, O. & Ray, S. (2000)

Olfactory learning in the neonate rat: a comparison

of aversive and appetitive conditioning.

Animal Technology. Vol. 51. (3). 151 – 160.

Butcher, O. & Ray, S. (2000)

Development of a suitable in-vivo technique to

explore the effects of Brain derived extracts on neonate

rat maturation.

Animal Technology. Vol. 51 (2). 91- 100.

Ferneyhough, B.M. & Ray, S. (2000)

Long term captivity and its effects on olfactory learning

in the Honeybee Apis mellifera.

Journal of Apicultural Research.

Ferneyhough, B.M. & Ray, S. (1999)

The Honey bee as a laboratory animal in the study of

the biological basis of behaviour.

Animal Technology. Vol. 50. (3). 145 – 154.

Bagnall, L. & Ray, S. (1999)

Rat strain differences on performance in the

Morris water maze.

Animal Technology. Vol. 50 (2). 69 - 77.

Ray, S. & Ferneyhough, B.M. (1999)

Behavioural development and olfactory learning in the

Honeybee (Apis mellifera).

Developmental Psychobiology. Vol. 34. 21 - 27.

Ray, S. (1999)

Survival of Olfactory memory through metamorphosis in

the fly Musca domestica.

Neuroscience letters. Vol. 259. 37 - 40.

Ray, S. (1998)

An alternative to water deprivation techniques in animal

learning studies.

Animal Technology. Vol. 49 (3). 113 – 120.

Ray, S. (1998)

Learning in the land snail Helix aspersa.

Animal Technology. Vol. 49 (3). 135 – 143.

Schofield, L., Ferguson, J.C. & Ray, S. (1997)

Physiological measurement of the response to and

recovery from stress during a selection procedure.

Proceedings of the British Psychological Society.

Vol. 5 (2). 114.

Ray, S. & Ferneyhough, B.M. (1997)

Seasonal variation of proboscis extension reflex

conditioning in the honey bee (Apis mellifera).

Journal of Apicultural Research. Vol. 36 (2). 108 – 110.

Ray, S. & Ferneyhough, B.M. (1997)

The effects of age on olfactory learning in the honey bee

Apis mellifera.

Neuroreport. Vol 8. 789 – 793.

Schofield, L., Ferguson, J.C. & Ray, S. (1997)

Physiological measurement of the response to and

recovery from stress during a selection procedure.

Proceedings of the British Psychological Society. Vol.

5 (2). 114.

Ray, S. (1993)

Recovery from stress measured by Salivary IgA. British

Psychophysiology Society Newsletter. Vol 20. 33.

Resume (Cont.)

14

Selected conference papers

Keeton, S.J., Ray,S. & Dash, P.R. (2013) Plasticity of cell migration st transition zones between different dimensions and substrates. Invadosomes Conference Nijmegen.

Ray, S. (2012) Microvesicles and Tissue repair. NeuroRehab*

Ray, S. (2011) Therapeutic cell banking applications of adipose derived Mesenchymal stem cells in anti-aging or regenerative medicine. Institute of South African Plastic Surgeons National Meeting. *

Ray, S. (2011) Stem cells and neurological repair. NeuroRehab Conf. Berkshire.*

Ray, S. (2009) Paracrine stem cell mediated repair of tissue Damage. Muscular Dystrophy UK.

Ray, S. (2007) Stem cell Repair in Neuromuscular Diseases. Ataxia Society.

Ray, S. (2007) Stem Cell Therapy in Degenerative Diseases. World Anti-aging Medicine Conference . Athens.

Ray, S. (2004) Cell Therapy: promises, promises, promises! Multiple Sclerosis Society Annual Conference.

Ray, S. (2004) Potential applications of cell therapy for neurodegenerative diseases. MS Therapy Centre Annual Lecture. Berkshire.*

Ray, S. (2003) Stem cells and the future of regenerative medicine. Multiple Sclerosis Society. Henley on Thames.

Ray, S. (2002) RNA, are memories made of this? Michaelmas Lecture. Oxford Graduate Society. Oxford

Ray, S. (2002) Potential applications of cell therapy for neurodegenerative diseases. Motor Neurone Disease Association. Nottingham.

Ray. S. (2002) Stem cell therapy in neurodegenerative diseases: an update. Motor Neurone Disease Association Annual Conference, Birmingham.*

Ray, S. (2002) Generation of glia from Bone Marrow stem cells in – vitro. Applications in the treatment of Multiple Sclerosis. Multiple Sclerosis Society Regional Research Conference.

Ray, S. (2001) Macromolecular theories of memory. Institute of Biologists. Reading.

Ray, S. (2001) Stem cell therapy: the use of Bone Marrow Stromal stem cells for global cell replacement in neurodegenerative disease. Multiple Sclerosis Society Research Conference. Manchester.*

Ray, S. (2001) The potential application of stem cell therapy in Motor Neurone Disease. Motor Neurone Disease Association Annual Conference. Birmingham.*

Ray, S. & Freeman-May, A. (2001) Stress profiling of emergency medical staff: A physiological perspective. Ambulance Research & Development, University of Hertford.

Ferneyhough, B.M. & Ray. S. (2001) The effects of behavioural development on olfactory learning and memory in the honeybee (Apis mellifera). Experimental Analysis of Behaviour Conference, University of London.

Martin, M. & Ray. S. (2001) Taste aversion learning in the rat in the absence of food/ water deprivation. Institute of Animal Technology Congress. Jersey.

Ray, S. (2000) The Anatomy of the creative human brain. YPSL European Quality Conference. Thames Valley University.

Ray, S. (1998) Physiological profiling of the stress response in occupational medicine. IPD Conference. Harrogate.*

15

Bagnall, L. & Ray, S. (1998) Rat strain differences in spatial learning assessed on the Morris water maze. Institute of Animal Technology Congress. Jersey.

Ray, S., Howells, K.F., Abbas, S., Butcher, O.L. & Bagnall, L. (1998) The effects of brain derived protein fractions on recipient brain anatomy and behaviour following systemic injection in the rat. Joint meeting of the European Neuropeptide Club and the Summer Neuropeptide Conference, University of Gent, Belgium.

Ray, S. & Ferneyhough, B. (1998) The involvement of neuropeptides in the survival of memory through insect metamorphosis. Joint meeting of the European Neuropeptide Club and the Summer Neuropeptide Conference, University of Gent, Belgium.

Ferneyhough, B. & Ray, S. (1998) Neuropeptides and the ontogeny of learning in the honeybee. Joint meeting of the European Neuropeptide Club and the Summer Neuropeptide Conference, University of Gent, Belgium.

Butcher, O.L. & Ray, S. (1998) The effects of exogenous neuropeptides on the development of motor co-ordination in the neonate rat. Joint meeting of the European Neuropeptide Club and the Summer Neuropeptide Conference, University of Gent, Belgium.

Abbas, S. & Ray, S. (1998) An in-vitro assay for neuropeptide induced plasticity in neuronal and glial transplantation. Joint meeting of the European Neuropeptide Club and the Summer Neuropeptide Conference, University of Gent, Belgium.

Schofield, L.A., Ferguson, J.C. & Ray, S. (1997) Physiological measurement of the response to and recovery from stress during selection procedures. British Psychological Society Occupational Psychology Conference.

Ray, S. (1997) The Physiology of Stress. IPD Annual Conference. Harrogate.

Ray, S., & Blythe J. (1997) Survival of memory through metamorphosis, Experimental Analysis of Behaviour Conference, University of London.

Ray, S. (1997) Neural transplantation and learned time signals, Experimental Analysis of Behaviour Conference, University of London.

Patents

1. BBSRC industrial Studentship Professor Ketan Patel,

Dr Steve Ray Stem cell generated paracrine Therapy

2. BBSRC industrial Studentship Professor Ketan Patel

(University of Reading), Dr Dionne Tannetta

(University of Oxford & Dr Steve Ray (Natural

Biosciences Ltd) Characterising the role of

microvesicles as anti-aging reagents through their

ability to clear protein aggregates

3. BBSRC industrial Studentship Dr Phil Dash &

Dr Steve Ray Microvesicle shedding in cell migration

4. Myostis Support Group Research Grant

Dr Steve Ray, Stem cell microvesicles and

muscle repair

5. BBSRC Studentship Professor Ketan Patel,

Dr Steve Ray, Dr Henry Collins-Hooper, The role of

RNA in stem cell differentiation abd tissue

regeneration

Ray, S. (2003) GB0316089.2 Method of altering cell properties by Administering RNA.

Ray, S. (2004) PCT.GB2004/00298. Method of altering cell properties by Administering RNA.

Ray, S. & Fischer, M. (2006) WO2006077409. Method of genotypic modification by administration of RNA.

Ray, S. (2008) GB0804932.2 Microvesicles

Ray, S. (2009) WO/2009/087361 Microvesicles

* Key Note Speaker

Natural Biosciences SA155A Seestrasse,Kilchberg 8802ZurichSwitzerlandT +41 (0) 44 716 4828 natural-biosciences.com