stem cells: identification and therapeutic...

Stem Cells: Identification and Therapeutic Use

Prof Andrew C. W. Zannettino Co-Director, Regenerative Medicine Program,

Centre for Stem Cell Research, University of Adelaide

Overview of Presentation

• What are stem cells? • Embryonic stem cells

• Induced Pluripotent Stem Cells

• Stimulus Triggered Acquisition of Pluripotency (STAP) Cells

• Adult Stem Cells – Haemopoietic Stem Cells (HSC) and Mesenchymal Stem Cells (MSC)

• Tissue Engineering Applications of Ex Vivo Expanded MSC:

– i. Orthopaedic

– ii. Cardiac

The Cell

• The basic unit of biological life is the cell

• All biological life is cellular

• Human body contain ~ 210 types of cells, each performing a specific function; e.g. red blood cells carry oxygen; neurones transmit nerve impulses

• All specialized cells in the body are derived from stem cells

• Stem cells have two defining attributes:

– The capacity for self-renewal

– The ability to differentiate into many different cell types

• There are about six classes of stem cells.

• The three most important classes of stem cells:

– Embryonic stem cells

– Induced Pluripotent Stem (iPS) Cells

– Adult stem cells

Stem Cells

EMBRYONIC STEM CELLS

• Derived from the inner cell mass of a blastocyst

• A blastocyst is a hollow ball of cells formed 4-6 days after a human egg is fertilized.

Embryonic Stem (ES) Cells

• In 1964, researchers isolated a single type of cell from a teratocarcinoma, a tumour now known to be derived from a germ cell. These cells replicated and grew in cell culture as a stem cell and are now known as embryonic carcinoma (EC) cells (Kleinsmith LJ and Pierce GB, Cancer Research 1964).

• 1981, embryonic stem cells (ESC) were independently derived from mouse embryos by two groups (Martin Evans and Matthew Kaufman, Nature 1981; and Gail R. Martin, PNAS 1981).

• In 1998, a breakthrough occurred when researchers, led by James Thomson (Thomson et al. Science 1998) at the University of Wisconsin-Madison, developed a technique to isolate and grow human embryonic stem cells in cell culture.

History of ESC Research

Teratoma

Isolate inner cell mass (destroys embryo)

Heart muscle Kidney

Liver

“Special sauce” (feeder cells)

Day 5-6 Blastocyst

Inner cells (forms fetus)

Outer cells (forms placenta)

Heart repaired

Culture cells

Derivation and Use of ES Cells and ES Cell Lines

• Replaceable tissues/organs

• Repair of defective cell types

• Delivery of genetic therapies

• Delivery chemotherapeutic agents

Possible Uses of ES Cell Technology

• Limited number of available Human ES cell lines

• Difficult to cultivate in the absence of feeder cells

• Variation in the potential of different hES cell lines

• Differentiation of ES cells may lead to immune rejection

• Potential transfer of pathogens (HIV, Hepatitis)

• Chromosome abnormalities detected in vitro

• Hard to regulate differentiation: Teratoma (cancer) formation

• Ethical dilemma using embryo-derived tissue

• Human cloning prohibited

Limitations of ES Cell Technology

Teratoma

• The two issues are related or not related based on the answer to the following question:

“Where did the nucleus come from in the fertilized egg used to make the embryonic stem cell.”

How are ES Cells Technically Related to Cloning?

Somatic Cell Nuclear Transfer or “Cloning”

Oocyte Somatic Cell

Pluripotent Stem Cell

• 1998 – Mice cloned

• 1998 – Cows cloned

• 1996 – The first mammal cloned from adult cells was Dolly, the sheep

• 2000 – Pigs cloned

History of Somatic Cell Nuclear Transfer (Cloning)

Dolly

Dolly with her first newborn, Bonnie

• Born in July 1996 at the Roslin Institute in Scotland

• First mammal to be cloned from an adult mammal using the nuclear transfer technique

• 277 attempts were made before the experiment was successful

• Dolly died in February 14, 2003 of progressive lung disease at the age of 6; whereas normal sheep can live up to 12 years of age.

Dolly with her surrogate mother

Mammal Cloning allows propagation of endangered species

January 8, 2001 Noah, a baby bull gaur, became the first clone of an endangered animal.

http://www.google.com.au/imgres?imgurl=http://upload.wikimedia.org/wikipedia/commons/5/5e/MaleGaur_Nagarahole_WLS.jpg&imgrefurl=http://en.wikipedia.org/wiki/File:MaleGaur_Nagarahole_WLS.jpg&h=992&w=1480&sz=458&tbnid=a6tEO7eA7tZ3xM:&tbnh=82&tbnw=123&prev=/search?q=bull+gaur&tbm=isch&tbo=u&zoom=1&q=bull+gaur&usg=__ghXFPkB6k1dwILMjN-lBVyLJT_0=&hl=en-AU&sa=X&ei=eiMbUJ_oKY2WiQfwy4CwAQ&ved=0CDcQ9QEwCg

Comparison of Cloning Success Rates

in Various Animals

Species

Number of oocytes used

Number of live offspring

Notes

Mouse

2468

31 (1.3%)

-

Bovine

440

6 (1.4%)

2 died

Sheep

417

14 (3.4%)

11 died within 6 months

Pig

977

5 (0.5%)

-

Goat

285

3 (1.1%)

-

• Success rates of cloning using mature mammal cells

Yanagimachi, R. 2002. Molecular and Cellular Endocrinology

Birth Defects Related to Cloning

• Cloned offspring often suffer from large offspring syndrome,

where the clone and the placenta that nourished it are

unusually large.

• Cloned offspring often have serious inexplicable respiratory or

circulatory problems, which causes them to die soon after birth.

• Clones tend to have weakened immune systems and sometimes

suffer from total immune system failure.

• Very few clones actually survive to adulthood.

• Clones appear to age faster than normal.

• Clones experience problems associated with old age, such as

arthritis, while they are still young

• Many of these features may relate to the fact that clones have

shorter telomeres

S. Yamanaka, H.M. Blau, Nature 2010

INDUCED PLURIPOTENT STEM (iPS) CELLS

iPS cells are somatic (adult) cells that have been

genetically reprogrammed to an embryonic stem

cell–like state by being forced to express genes and

factors important for maintaining the defining

properties of embryonic stem cells

S. Yamanaka, H.M. Blau, Nature 2010

Induced Pluripotent Stem (iPS) Cells

Tumour Cell Somatic Cell

Pluripotent Stem Cell

c-Myc Klf4

Oct-3/4 Sox2

Apoptosis, Senescence

Immortalization

Takahashi and Yamanaka, Cell, 2006

Induced Pluripotent Stem (iPS) Cells

3) Cells which have been transduced with the four key transcription factors are transferred into culture flasks which contain a layer of mouse embryonic fibroblasts (MEFs)

1) Cells are transduced with the Mouse Slc7a1 lentivirus (Mouse receptor for retroviruses)

2) Slc7a1 transduced fibroblast cells are transduced with Oct4, Sox2, Klf4 and c-Myc retroviruses

4) Cells which have successfully integrated the four key transcription factors will cluster together to form embryonic stem cell-like colonies after 10-30 days in culture

Viral Transduction and iPS Generation

iPS cells have passed the ultimate test: fertile mice in which every cell was from an iPS cell

iPS Cells Can Regenerate and Entire Organism

Lee, Studer Nature Methods 2010

Nature 2009 - “Method of the year”

Basic biology – • differentiation / pluripotency • molecular understanding of diseases

Drug testing –

• Test effectiveness of drug on target human tissue

Tissue engineering – • Patient specific pluripotent stem cells • Ability to generate large quantities of

cells

Gene therapy – • Correction of monogenic disorders

Possible Uses of iPS Cells

• Not hampered by as much ethical, social or political controversy

• Generated from easily accessible tissues

• Disease mechanisms – unattainable sources (brain, heart)

• Genotype specific responses (drug, disease)

Advantages of iPS Cells

• iPS cells are labour intensive • Expensive • Inefficient • Require considerable validation • Late onset disorders? • Cancers?

Teratoma formation

Ectoderm Mesoderm Endoderm

iPS clone

Feeder

Disadvantages of iPS Cells

• Diabetes – generated glucose responsive

pancreatic islet cells from human skin

• Parkinson's – iPS cell-derived neurons

transplanted into rats

• Sickle cell anemia – genetically corrected iPS

cells and differentiated them into

hematopoietic cells, mice

How iPS Cells are Being Utilised

Stimulus Triggered Acquisition of Pluripotency (STAP) Cells

27

• 2014 – Dr Horuko Obokata (Riken Centre for Developmental Biology, Kobe, Japan) described a novel pluripotent cell capable of genrating embryonic (the body) and extra-embryonic tissue (the placenta) – termed STAP cells.

• Obokata hypothesised that stressing cells might make them pluripotent after observing that squeezing adult cells through a capillary tube made them shrink to a size similar to that of stem cells.

• Obokata applied different kinds of stress (heat, starvation and a high-calcium environment etc) and showed that a bacterial toxin that perforates the cell membrane, exposure to low pH (citric acid, pH 5.7) and physical squeezing were each able to coax the cells to show markers of pluripotency.

Obokata H et al, Nature, 505: 641-647, 2014 Obokata H et al, Nature, 505: 676-680, 2014

Stimulus Triggered Acquisition of Pluripotency (STAP) Cells

28

• To show that STAP cells could turn into all cell types (definitive demonstration of pluripotencey), Obokata injected fluorescently tagged cells into a mouse embryo – fluorescently labelled cells observed in every tissue of the resultant mouse.

• Other evidence: Obokata made pluripotent cells by stressing T cells (whose maturity is clear from a rearrangement of the T cell receptor genes)

• Obokata has reprogrammed many different cell types (brain, skin, lung and liver) suggesting that most, if not all, cell types are amenable to STAP cell formation

• On average, 25% of the cells survive the stress and 30% of those convert to pluripotent cells — a higher proportion than the 1% conversion rate of iPS cells

Obokata H et al, Nature, 505: 641-647, 2014 Obokata H et al, Nature, 505: 676-680, 2014

ADULT/SOMATIC STEM CELLS

Adult stem cell (def. Oxford Dictionary)

“An undifferentiated cell found in a differentiated tissue

that can renew itself and (with certain limitations)

differentiate to yield all the specialized cell types of the

tissue from which it originated”

Adult/Somatic Stem Cells

endoderm ectoderm mesoderm

liver gut

pancreas lung

nerve skin hair

pigment cells

blood muscle bone

cartilage

Somatic Lineages Somatic

Stem Cells Germline Stem Cells

Adult/Somatic Stem Cells

• The bone marrow harbours 2 types of adult stem cells:

• Haemopoietic Stem Cells

• Mesenchymal Stem Cells

(Adapted from Kansas Medical Centre, University of Kansas)

• The most well studied are the blood stem cell (hematopoietic stem cell or HSC used in bone marrow transplants) and the neural stem cells

Stem Cells in the Bone Marrow

B lymphocytes

T lymphocytes

Platelets

RBCs

Neutrophils

Macrophages

Pluripotent Haemopoietic

Stem Cell

Lymphocyte Progenitor

Myeloid Progenito

r

Lineage Antigens

Self-Renewal

Bone Marrow-Derived Haemopoietic Stem Cells

HSC are the Gold Standard of Adult Stem Cells

• A single HSC can reconstitute the haematopoietic system of a mouse

Osawa et al, Science, 1996

• Haematological malignancies

- acute leukaemia

- chronic myeloid leukaemia

- multiple myeloma

- Hodgkin’s and Non-Hodgkin’s lymphoma • Aplastic anaemia

• Immunodeficiency syndromes (SCID)

• Inborn errors of metabolism

• Other malignancies

HSC and Bone Marrow Transplantation: Cell Based Therapy for More

than 30 Years

• Stem cells from a given somatic tissue have a

differentiation potential that is limited to the

mature cell population that comprise that

same tissue

• Stem cell differentiation is unidirectional

and irreversible

Adult Stem Cell Dogmas

Lagasse et al, Nat Med 6: 1229, 2000

Haemopoietic Stem Cells Can Differentiate into Hepatocytes in vivo

Orlic et al, Proc Nat Acad Sci 98: 10344, 2001

Mobilised Bone Marrow Cells Repair the Infarcted Heart

• Rare, often difficult to identify and their origins have not been precisely defined

• Usually difficult to propagate in vitro

• Adult stem cells have been derived from tissues that develop from all three embryonic germ layers

• Haemopoietic stem cells from the bone marrow are the most extensively studied and most widely used for clinical applications

• Several additional adult stem cell populations are now being tested in clinical applications

• Some adult stem cells have the apparent ability to differentiate into tissues other than the ones from which they were derived

• Developing effective means to transplant adult stem cells is central to the effective delivery of stem cell therapies

Adult Stem Cells - Summary

Reticular Cell

Smooth Muscle Cell

Adipocyte

Osteoblast

Committed Progenitor

Cell

Self-Renewal

CFU-F

Chondrocyte

Mesenchymal Stem Cell

Lineage Antigens

MESENCHYMAL STEM CELLS

• First identified by Friedenstein et al (1974)

following plating of suspensions of BM:

Colonies resembling spindle-shaped fibroblasts

emerged.

Termed Colony Forming Units-Fibroblasts (CFU-F).

Rare: depending on species, 1-20 CFU-F obtained per

1x105 BM cells plated.

Individual CFU-F have differential capacity for

proliferation & differentiation (ectopic

transplantation beneath the renal capsule of syngeneic

hosts).

BM-derived MSC colony

• Freidenstein et al proposed hierarchy of cellular differentiation supported at its apex by a small compartment of self-renewing, multipotent, stromal stem cells termed MSC.

Mesenchymal Stem Cells (MSC): A Brief History

Reticular Cell

Smooth Muscle Cell

Adipocyte

Osteoblast

Committed Progenitor Cell

Self-Renewal

CFU-F

Chondrocyte

Mesenchymal Stem Cell

Lineage Antigens

Hypothesized Hierarchy of Bone Marrow-Derived Mesenchymal Stem

Cell Differentiation

Classification of Mesenchymal Stem or Stromal Cells

International Society for Cellular Therapy criteria for defining human MSC

• MSC must be plastic-adherent when maintained in standard

culture conditions • MSC must express CD105, CD73 and CD90, and lack expression

of the haematopoietic associated markers, CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules.

• MSC must differentiate into osteoblasts, adipocytes and

chondroblasts in vitro.

Dominici et al. Cytotherapy 2006; Horwitz et al. Cytotherapy 2005

The Monoclonal Antibody STRO-1: A Tool For Isolating MSC

FORWARD LIGHT SCATTER

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BMMNC STRO-1- STRO-1+

• STRO-1 is a murine IgM mAb which identifies an antigen on human stromal elements.

• Does not react with haemopoietic progenitor cells.

BM Cell labelled with Primary Antibody

(STRO-1)

2º Antibody + Biotin

(anti IgM-Biotin)

Streptavidin Microbeads

Negative Fraction Positive Fraction

Magnetic Activated Cell Sorting (MACS) of STRO-1 Positive BM Mononuclear Cells

RE

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LOG FLUORESCENCE (PE)

BM MONONUCLEAR CELLS

STRO-1 NEGATIVE

2%

0.7%

STRO-1bright

STRO-1int

STRO-1lo

STRO-1 POSITIVE

Cloning Efficiency In Vitro

Cell Fraction #CFU-F/105 cells

BMMNC 11 ± 3.2

STRO-1+ 134 ± 19.4

STRO-1- 0

STRO-1lo 0

STRO-1int 62 ± 9.4

STRO-1bright 13,277 ± 517

(~ 1 in 10 cells plated = CFU-F)

Flow Cytometric Analysis of MACS-Sorted STRO-1Bright MSC are Restricted to the

STRO-1Bright Fraction

Anti-CD106 (VCAM-1) bound to STRO-1Pos MACS-selected BM Mononclear Cells FACS Isolation

of CD106+/STRO-

1Pos

MSC

CD106

Cell surface

Adapted from Terese Winslow ©2001

Preparative Flow Cytometric Sorting of STRO-1 MACS Positive BM Mononuclear Cells to Isolate STRO-1BrightCD106+ MSC

CELLS PER WELL

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Gronthos S, Zannettino A, JCS, 2003

STRO-1bright

STRO-1 POSITIVE Poison Distribution Statistics

following limit dilution analysis

Identification and Purification of STRO-1Bright/CD106+ BM Mesenchymal Stem

Cells

B A

C D

E

F

Morphological Characterisation of Purified STRO-1Bright/CD106+ BM

Mesenchymal Stem Cells


Ki-67 FITC

ST

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+ - + - + - + - B

1. CD34+ cells 2. Keratinocytes (+ve control) 3. STRO-1Bright/CD106+ cells 4. Skin Fibroblasts (-ve control)

MACS selected STRO-1+ BM STRO-1Bright/CD106+ cells express telomerase


STRO-1Bright/CD106+ BM MPC Are Quiescent and Express Telomerase In

Vivo: Hallmarks of Stem Cells

CBFA1

OP

ON

BSP

PTH-R

OCN

3 1 2 Bone

PPARg2

H-ALBP

LPL

LEPTIN

3 1 2 Fat

3 1 2

COLL-II

COLL-X

AGGN

Cartilage

COLL-1

1. STRO-1Bright/CD106+ cells 2. Cultured CFU-F at wk 6 3. Tissue

STRO-1Bright/CD106+ BM Mesenchymal Stem Cells Represent an Uncommitted

Stem Cell Population


Bone

Fat Cartilage

Cultured BM MSC

Multipotential Capacity of Ex Vivo Cultured STRO-1Bright/CD106+ BM Mesenchymal Stem Cell In Vitro

2 x 106 Cultured MSC

Hydroxyapatite/Tricalcium Phosphate (HA/TCP) partcles

Histological Analysis of Transplants

Fibrin Glue

In Vivo Bone Formation: Assay to Examine the Osteogenic Potential of

Purified Ex Vivo Expanded MSC

HA/TCP bone

HA/TCP

marrow

BM Mesenchymal Stem Cells Form Human Bone in Immunodeficient Mice

• Do not induce proliferation of allogeneic lymphocytes in vitro. (Le Blanc Exp Heme 2003; Klynshnenkova J Biomed Sci 2005)

• MSC are not lysed by NK cells or cytotoxic T cells. (Le Blanc Bone Marrow Transplant 2003)

• High numbers of MSC are immunosuppressive in vitro. (Le Blanc Scand J Immunol 2003,

2004; Rasmusson Exp Cell Res 2005)

• MSC induce division arrest anergy of activated T cells. (Krampera Blood 2003)

• MSC suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. (Bartholomew et al. Exp Hematol 2002)

• MSC ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. (Zappia et al. Blood 2005)

• Administered MSC protect against ischaemic acute injury through differentiation-independent mechanisms. (Togel et al. Am J Physiol 2005)

• Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. (Le Blanc et al. Lancet 2004)

Immunomodulatory Properties of MSC–Prospect of Allogeneic Use

Immunomodulatory Properties of MSC

Mixed Lymphocyte Reaction

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Colcemid - MSC

Wada et al. J. Cell Physiol. 2009

0.0%

CD40 CD80

0.7% 0.1%

CD86

CD40 CD80 CD86

HLA-ABC

99.7%

HLA-DR

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CD54

HLA-DR HLA-ABC CD54

MSC create a milieu that is immunosuppressive by: • secreting cytokines which down regulate CD40, CD80, CD86 and HL-DR during dendritic cell (DC) maturation-induces tolerance in T cells. • IFN-g induces MSC to express the L-Tryptophan catabolising enzyme Indoleamine 2,3-dioxygenase (IDO). L-tryptophan is an essential amino acid in allogeneic T-cell responses.

MSC lack critical co-stimulatory molecules and evade host immune system

Ryan et al. Mesenchymal stem cells avoid allogeneic rejection. Journal of Inflammation 2005;2(8)

Why do MSC Possess Immunomodulatory Properties ?

Antibody Selection of MSC

• Simmons PJ, Zannettino ACW, Gronthos S. Mesenchymal Stem Cells WO 01/04268A1.

• Gronthos S, Shi S, Zannettino ACW. Perivascular MPC. PCT/AU2004/000416, WO 04/085630.

• Gronthos S, Zannettino ACW. I.HA.PAT.4B - Perivascular MPC Induced Blood Vessel Formation 28-Mar-2003 PCT Application No PCT/AU2004/000417, WO 2004/084921 A1

• Gronthos S, Zannettino ACW. Multipotential expanded Mesenchymal Precursor Cell progeny and uses there off. PCT/AU2005/001445, 24/09/2004, WO 2006/032092.

• Zannettino ACW, Gronthos S, Simmons PJ. Isolation of adult multipotential Cells by tissue non-specific alkaline phosphatase. PCT/AU2006/000494.

• Gronthos S, Zannettino ACW. Method of enhancing proliferation and/or survival of mesenchymal precursor cells. PCT/AU2005/000953, WO 2006/032075 A1.

• Zannettino ACW, Gronthos S. Monoclonal antibody STRO-4 (Provisional; US 61/189,349)

• Development of additional Mesenchymal Stem Cell - specific reagents (STRO-1, STRO-3, STRO-4, CC9)

• The use of MSC for tissue engineering applications - bone, cartilage, cardiac muscle, osteoarthritis, macular degeneration -> MESOBLAST LTD

• Generation of a world wide patent family:

Break Through Technology for Isolation, Expansion and Characterization of

Mesenchymal Stem Cells – IP Portfolio

magnet

MSC

master cell bank

Final product

stem cell-binding antibody

magnetic beads

Bone Marrow + Properties of Prospectively Isolated

Allogeneic Mesenchymal Precursor Cells

• pure initial stem cell pool

• homogeneous population

• efficient large-scale expansion

• lower costs of cell culture process

• batch-to-batch consistency

• stringent release criteria

• greater potency of expanded product

• clinically applicable

Commercial Application of Prospectively Isolated Allogeneic Mesenchymal Stem

Cells (Mesoblast Ltd)

Laboratory Cultivation of Mesenchymal Stem Cells: Therapeutic Product Facility

http://images.google.com/imgres?imgurl=http://www.cun.es/typo3temp/pics/8c54008b30.jpg&imgrefurl=http://www.cun.es/en/the-clinica-universitaria-de-navarra/servicios-medicos/terapia-celular/more-information/gmp-laboratory/&usg=__mtskflkJMqfyfJ9IXOOsVDCFAdw=&h=381&w=254&sz=27&hl=en&start=8&um=1&itbs=1&tbnid=sV0wWEjEL7_nfM:&tbnh=123&tbnw=82&prev=/images?q=GMP+laboratory&um=1&hl=en&sa=N&tbs=isch:1

Mesenchymal Precursor cell

Adipocyte

Cartilage

Tendon & Ligament

Cardiac Muscle

Neural Tissue

Skeletal Muscle

Dermal Cell

Myelosupportive Stroma

Bone

Tissue Engineering Using Adult Bone Marrow Mesenchymal Stem Cells

Structural stability maintained by intramedullary “nail” locked proximally

and distally by transfixing screws

3 cm mid portion of the ovine tibia is surgically resected

Ex vivo expanded ovine MSC

Transplantation of ex vivo cultured ovine MPC + Osteoconductive carrier

(MastergraftTM) into defect site

X-ray assessment of defect at 3, 6 and 12 months

Allogeneic Ovine BM

mAb-based selection of Ovine MSC

Can Allogeneic BM Mesenchymal Stem Cells “Heal” a Critical Sized Tibia Defect ?

Prof John Field, 2007


No MPC 225x106 MPC

X-Ray and mCT @ 6 months

Bone Regeneration Capacity of Allogeneic BM MSC in an Ovine Critical Sized 3cm Tibia

Defect Model

Field JR et al, Comp Orthop Traumatol 2011:24(2):113

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Mastergraft Matrix [HA/TCP/Col I] (n=12)

Mastergraft Matrix [HA/TCP/Col I] + 225m MPC ( n=35)

Allogeneic MSC Combined with MastergraftTM Matrix Induces An Earlier and More Complete Rate of Bone Union in an Ovine Critical Sized

3cm Tibia Defect Model

• In the next decade, the WHO estimates that there will be a 2-3 fold increase in the number of fractures that will require surgical intervention and rehabilitation.

• Mesoblast Ltd - funded Phase I Clinical Trial examining the safety of MSC transplantation into defect site.

• 10 patients with non-union fractures in long bones which have not healed 12 months post injury have been enrolled.

Phase I Clinical Trial Examining the Safety of Human Autologous MSC For Non-Union

Fractures

Aspirate BM from posterior iliac crest

Isolate BM MSC by magnetic separation

Expand MSC ex vivo in GMP laboratory (Cell Therapies,

Melbourne) ~ 4 weeks

Seed MSC onto HA/TCP/Collagen I

Osteoconductive Biomaterial (MastergraftTM)

Transplant MSC/Biomaterial into defect site

Phase I Clinical Trial Examining the Safety of Human Autologous MSC For Non-Union

Fractures

Prof Richard Destiger

X-Ray @ 3 months

X-Ray @ Pre-OP

X-Ray @ 3 months X-Ray @ Pre-OP

• Excellent safety profile of implanted cells, with no adverse events reported.

• 9/10 patients achieved union (5 males and 4 females)

• Time from injury to implant median 10 months (range 8-41 months)

• Number of stem cells received median 111 x 106 (range 89-212 cells)

• Time to union median 18 weeks, range 10-41 weeks

• The need for a second operation to harvest bone from donor site was eliminated following stem cell therapy.

Long Bone Reconstruction

• Mesoblast Ltd received approval for 2 Phase II

bone study using human allogeneic MSC by United States Food and Drug Administration (FDA)

- Spinal fusion

- Repair of long bone defects

FDA Approval for Orthopaedic Clinical Trial

Mesenchymal Precursor cell

Adipocyte

Cartilage

Tendon & Ligament

Cardiac Muscle

Neural Tissue

Skeletal Muscle

Dermal Cell

Myelosupportive Stroma

Bone

Tissue Engineering Using Adult Bone Marrow Mesenchymal Stem Cells

• Coronary heart disease accounts for more hospital admissions than all forms of cancer combined in most developed countries

• Significant medical and budgetary

burden • Over 21% of all deaths in Australia

in 2000 were attributed to coronary or ischaemic heart disease

Coronary Heart Disease and Heart Failure

Myocardial Infarction → Cardiomyocyte cell death → Ventricular remodelling → Heart failure

• With the exception of organ transplant, very few therapies address this cardiomyocyte cell loss → concept of “regenerative cardiology” using stem cells.

Pathway of the Development of Heart Failure: Ventricular Remodelling

Ligation of left anterior descending coronary artery in athymic nude rats

Injection of Human BM MPC to ischemic heart tissue 48 hrs following acute

myocardial infarct

Can BM MSC Regenerate Damaged Cardiac Muscle?-Rodent Model of Acute Myocardial

Infarct

Martens et al. Nature Clin. Pract. Cardio Med 2006

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1x106 Stro- depleted BM-MNC

0.2x106 Stro+ cells

1x106 Stro+ cells

saline

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n=30

n=30

n=30

Injection of Ex Vivo Cultured STRO-1Pos MSC Leads to Improvement of Animal Survival

-20

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Echocardiography of Animals Receiving Myocardial Injection of Ex Vivo Cultured STRO-1Pos MPC Exhibited Global Improvement of Left

Ventricular Function

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(alp

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-SM

A+

/vW

F+

) /

HP

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saline 0.2x106 Stro+ cells

1x106 Stro+ cells

0

5

10

15

20

25

30

35

40

45

saline STRObright STROneg saline STRObright STROneg

injection site distal to injection site

No

. a

rter

iole

s

(alp

ha

-SM

A+

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F+

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Myocardial Injection of Ex Vivo Cultured STRO-1Pos MSC Leads to a Dose-Dependent Increase

in Cardiac Arteriogenesis

MPC

SALINE

MSC Stimulate Endogenous Border Zone Myogenesis

• 6 patients with multi-vessel coronary artery disease and heart muscle damage treated with autologous MSC.

• MSC were injected into damaged heart muscle using the latest generation of myocardial catheters provided by Johnson & Johnson’s companies, Cordis Corporation and Biosense Webster.

• The primary endpoint of safety was achieved and there were no cell-related adverse events.

First in Man Heart Failure Pilot Phase I Trial-John Hunter Hospital, NSW

Aspirate BM from posterior iliac crest

Isolate BM MSC by magnetic separation

Expand MSC ex vivo in GMP laboratory (Cell Therapies,

Melbourne) ~ 4 weeks

Administer MSC into ischemic myocardium using

electromagnetic-guided LV mapping and injection system

Angioblast Systems Inc. - funded Phase I Clinical Trial examining the safety of MSC transplantation

into ischemic myocardium in 6 patients

Dr Suku Thumbar, John Hunter Hospital, Newcastle, NSW

Phase I Clinical Trial Examining the Safety of Human MSC For Left Ventricular Dysfunction

MyostarTM Injection Catheter

NOGA® XP – Injections of MSC

Late Gd. CMR NOGA® XP

Psaltis PJ, Journal of Cardiovascular Translational Research, 2009; 2:48-62

NOGA® XP – Injections of MSC

Kornowski JACC 2000;35:1031

Electrical Mechanical

Hibernating Myocardium

Johnson & Johnson’s companies Cordis Corporation & Biosense Webster

Electromechanical Mapping (NOGA): Transendocardial Delivery of MPC

• The primary endpoint of safety was achieved and there were no cell-related adverse events.

• Heart muscle recovery was seen in all six patients within three months of cell implantation, as defined by either improvement in symptoms of heart failure or heart function.

• All patients demonstrated reduced episodes of chest

pain (angina) and reduced need for anti-anginal medications, suggesting that the stem cell therapy had improved blood flow to the damaged heart muscle.

Heart Failure Pilot Phase I Trial - John Hunter Hospital, NSW

• Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA., Dr. P.C. Gorman, pre-clinical Study of Allogeneic MPC for Myocardial Ischemia (AngioblastSystems Inc.; Mesoblast Ltd.)

• The MI+MSCs group showed (a) significantly higher EF (>40% increase) and cardiac output (CO); (b) significantly decreased LV remodelling (Hamamoto et al. Ann Thorac Surgery 2009)

• IMVS/RAH, ovine model of non-ischemic (Doxorubicin-induced) model of cardiomyopathy

• Eight weeks after Dox treatmment, animals that received MSC showed decrease fibrosis, increased arteriogenesis and increased ejection fraction (Psaltis et al. JACC: Cardiovascular Interventions)

• Conclusion: • Allogeneic MSC are safe and effective at stabilizing heart function

Pre-clinical Efficacy of Allogeneic Ovine MSC to Repair Injured Myocardium

• Phase 2a Congestive Heart Failure (CHF) Clinical Trial • 60 patient multi-center, randomized, controlled trial • Class II-IV CHF with EF < 40% • Randomized 3:1 controls to MPC at 25 M, 75 M, or 150 M cell doses. • Cells injected by JNJ NOGA Myostar™ catheter

• Primary endpoint of safety already met: no adverse events associated • with MPC at any dose Secondary endpoints evaluate effects of MPC on (a) cardiac/heart failure hospitalization events over time (b) cardiac-related mortality over time (c) cardiac functional parameters after patients complete 12 months

Single Intra-Myocardial Injection of Allogeneic MSC for Long-Term Treatment of Congestive

Heart Failure: Phase 2 clinical trial

Over 1.5 Years Study Follow-Up, MPC Treated Patients Had Fewer Cardiac-Related Events, Hospitalizations,

and Deaths Than Controls

*MACE defined as composite of MI,

revascularization, or cardiac death Event

MPC treatment (N=45) No. patients with event

(%)

Controls (N=15) No. patients with event

(%)

p value

Any Serious Adverse Cardiac Event (SAE)

20 (44.4%) 14 (93.3%) 0.001

Repeat SAEs 5 (11.1%) 5 (33.3%) 0.102

Any Hospitalization For Heart Failure

5 (11.1%) 3 (20.0%) 0.4

All Cause Deaths 2 (4.4%) 2 (13.3%) 0.26

Cardiac Deaths 0 (0.0%) 2 (13.3%) 0.059

Any Major Adverse Cardiac Event (MACE*)

3 (6.7%) 6 (40%) 0.005

MACE or Any Hospitalization for

Heart Failure

6 (13.3%) 6 (40%) 0.056

Interim data analysis, after all patients have reached 6 months follow-up *MACE defined as composite of MI, revascularization, or cardiac death

Single treatment with MPC saw a 22% mean increase in EF, whereas controls had an 18% mean decrease in EF at 6 month time period by echocardiogram

Mesenchymal Precursor Cells

Paracrine Effects

Proliferation & Reduced Apoptosis

of Endogenous Cells

Cardiomyocyte

Transdifferentiation

Cardiomyocyte

Regeneration & Repair

Endothelial

Transdifferentiation

Neovascularisation

Cardiac Repair

Psaltis PJ, et al. Int J Cardiovasc Imaging. 2011 Jan;27(1):25-37; Psaltis PJ, et al. J Cardiovasc Transl Res. 2010 Apr;3(2):135-46; Psaltis PJ, et al. Cell Physiol. 2010 May;223(2):530-40; Psaltis PJ, et al. J Card Fail. 2008 Nov;14(9):785-95.

Pre-Clinical and Clinical Trials: Current and Future

Updated: April 2013

Company Location Business Type

Mesoblast Melbourne, Australia Regenerative Medicine

Cardio3 Biosciences Mont-Saint-Guibert, Belgium

Stem Cell Differentiation

Advanced Cell Technology Alameda, CA Stem Cell Technology

DaVinci Biosciences Costa Mesa, CA Cellular Therapies

California Stem Cell Irvine, CA Stem Cell Research Products

Advanced Cell Technology Los Angeles, CA Stem Cell Technology

PCT Cell Therapy (Neostem)

Mountain View, CA Cell Therapy Development Support

SanBio Mountain View, CA Cellular Therapies

StemCells Inc Palo Alto, CA Biologics / Stem Cells

OncoMed Pharmaceuticals Redwood City, CA Cancer Stem Cells

Cytori Therapeutics San Diego, CA Medical Devices / Biotechnology

Histogen San Diego, CA Regenerative Medicine

ViaCyte San Diego, CA Stem Cell Therapies

iperian South SF, CA Induced Pluripotent Stem Cells

Vistagen South SF, CA Stem Cell Technology

Bioheart Sunrise, FL Cell Therapies

Vivalis Saint-Herblain, France Stem Cell Lines

ViaCyte Athens, GA Stem Cell Therapies

ProBioGen Berlin, Germany Contract Cell-Line Work

Tissue Genesis Honolulu, HI Adipose Cell Isolation

NewLink Genetics Ames, IA Cell Therapy / Small Molecules

Cytori Therapeutics Florence, Italy Medical Devices / Biotechnology

MolMed Milan, Italy Biologics, Small Molecules, Cellular Therapy

Okairos Rome, Italy T-Cell Based Vaccines

DNAVEC Ibaraki, Japan Viral Vectors & Gene Therapy

Cytori Therapeutics Tokyo, Japan Medical Devices / Biotechnology

NuPotential Baton Rouge, LA Cell Line Production

Gingko Bioworks Boston, MA Engineered Organisms

OvaScience Boston, MA Fertility, Mitochondria Transplantation

ViaCord (PerkinElmer) Boston, MA Cord Blood Banking

NeoStem Cambridge, MA Adult Stem Cell Storage

Advanced Cell Technology

Worcester, MA Stem Cell Technology

Cognate Bioservices Baltimore, MD Cell Therapy Services

Osiris Therapeutics Baltimore, MD Stem Cell Technology

Osiris Therapeutics Columbia, MD Stem Cell Technology


52 Cell Therapy Companies Worldwide

http://www.mesoblast.com/careers/http://www.c3bs.com/en/careers/job-opportunities.htmlhttp://www.advancedcell.com/company/careers/http://www.dvbiosciences.com/careers.phphttp://www.californiastemcell.com/contact/jobs-at-csc/http://www.advancedcell.com/company/careers/http://pctcelltherapy.com/company/careers/http://pctcelltherapy.com/company/careers/http://www.san-bio.com/careers/http://www.stemcellsinc.com/opportunities.htmlhttp://www.oncomed.com/Careers.htmlhttp://www.cytori.com/Company/Careers/CareerOpportunities.aspxhttp://www.histogen.com/aboutus/careers.htmhttp://viacyte.com/careers/http://www.ipierian.com/company/careers/open-positions/http://www.vistagen.com/About-Us/careers/default.aspxhttp://www.bioheartinc.com/Careershttp://www.vivalis.com/http://viacyte.com/careers/http://www.probiogen.de/company/careers.htmlhttp://www.tissuegenesis.com/careers.htmlhttp://www.linkp.com/careers/index.htmlhttp://www.cytori.com/Company/Careers/CareerOpportunities.aspxhttp://www.molmed.com/node/1467http://www.okairos.com/e/inners?m=00080http://www.dnavec.co.jp/en/company/employment.htmlhttp://www.cytori.com/Company/Careers/CareerOpportunities.aspxhttp://www.nupotentialinc.com/Contact/contact2.htmlhttp://ginkgobioworks.com/careers.htmlhttp://ovascience.com/contact/careers.aspxhttp://www.viacord.com/career-opportunities.htmhttp://www.neostem.com/careers.htmlhttp://www.advancedcell.com/company/careers/http://www.advancedcell.com/company/careers/http://www.cognatebioservices.com/index.php?option=com_content&view=article&id=65&Itemid=72http://www.osiristx.com/career.phphttp://www.osiristx.com/career.php

Aastrom Ann Arbor, MI Cellular Products

PCT Cell Therapy (Neostem) Allendale, NJ Cell Therapy Development Support

Proteonomix Mountainside, NJ

Cellular Therapies

ChromoCell N. Brunswick, NJ

Drug Discovery / Cellular Therapy

Mesoblast New York , NY Regenerative Medicine

Orgenesis White Plains, NY

Autologous Cellular Conversion

Fibrocell Science Exton, PA Personalized Skin Therapy

Cellartis Goteborg, Sweden

Stem Cell Technology

Cognate Bioservices Memphis, TN Cell Therapy Services

Bellicum Pharmaceuticals Houston, TX Cellular Therapy

Opexa Therapeutics The Woodlands, TX

Cell Therapy

Cellartis Dundee, UK Stem Cell Technology

Intercytex Manchester, UK Cell-Based Products

Roslin Cells Roslin, UK Stem Cells

American Type Culture Collection (ATCC)

Manassas, VA Biologic / Cell Line Management

Cell Line Genetics Madison, WI Pharmaceutical Services

Cellular Dynamics Madison, WI Induced Pluripotent Stem Cells

Stratatech Madison, WI Cell Therapy / Tissue Engineering


52 Cell Therapy Companies Worldwide

http://investors.aastrom.com/JobSearch.cfmhttp://pctcelltherapy.com/company/careers/http://www.proteonomix.com/careers.htmhttp://www.chromocell.com/careers.htmlhttp://www.mesoblast.com/careers/http://www.orgenesis.com/contact/careershttps://www.fibrocellscience.com/working-with-us/careers/http://www.cellartis.com/the-company/career-at-cellartishttp://www.cognatebioservices.com/index.php?option=com_content&view=article&id=65&Itemid=72http://www.bellicum.com/about-us/careershttp://www.opexatherapeutics.com/index4614.html?page=positions&section=careershttp://www.cellartis.com/the-company/career-at-cellartishttp://www.intercytex.com/index.php?option=com_content&view=article&id=10&Itemid=12http://roslincells.com/careers/http://www.atcc.org/About/CareerOpportunities/tabid/144/Default.aspxhttp://www.atcc.org/About/CareerOpportunities/tabid/144/Default.aspxhttp://www.clgenetics.com/pages/careershttp://www.cellulardynamics.com/about/careers.htmlhttp://www.stratatechcorp.com/about/careers.php

• Purification of human MSC to near homogeneity using mAbs to the STRO-1/CD106 cell surface molecules.

• Ex vivo expanded, prospectively isolated human and ovine MSC exhibit multi-differentiation potential, induce angiogenesis and immunosupress activated lymphocytes.

• Ex vivo expanded, human/ovine MSC exhibit contribution to skeletal and cardiac repair

• MSC may act by – (a) secreting factors that attract endogenous cells to the area of injury, (b) contributing directly to specific tissue following differentiation, (c) increasing the vascular supply to the injured area.

• Successful pre-clinical and phase I/IIb studies have paved the way for Phase III skeletal and cardiac regeneration trials.

Summary

School of Medical Sciences, University of Adelaide S Gronthos

Comparative Orthopaedic Research, Flinders University, South Australia

J Field

Royal Melbourne Hospital R Destiger

University of Melbourne Angioblast Systems Inc/Mesoblast Ltd.

S Itescu M Schuster

Cardiovascular Research Centre, Royal Adelaide Hospital

Stephen Worthley Peter Psaltis

National Health and Medical Research Council of Australia

John Hunter Hospital, Newcastle S Thumbar

Acknowledgements

stem cells: identification and therapeutic...

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