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
PE
RP
EN
DIC
UL
AR
LIG
HT
SC
AT
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R
RE
LA
TIV
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EL
L
CO
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T
STRO-1 (FITC)
6.5% ± 0.8, n=20
0
100
50
1 10 100 1000 10000
CF
U-F
PE
R 1
x10
5
CE
LL
S
0
10
20
30
40
50
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
LA
TIV
E
CE
LL
N
UM
BE
R
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
NE
GA
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EL
LS
(%
TO
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L)
1
10
100
0 1 2 3 4 5 6 7 8 9 10
y=93.708 x 10-0.196x
STRO-1
VC
AM
-1 (
CD
106
)
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
Gronthos S, Zannettino A, JCS, 2003
Ki-67 FITC
ST
RO
-1
PE
A
1 2 4 3
+ - + - + - + - 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
Gronthos S, Zannettino A, JCS, 2003
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
Gronthos S, Zannettino A, JCS, 2003
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
3H
-th
ym
idin
e In
corp
ora
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n (
CP
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x10
3)
0
10
20
30
40
50
60
70
80
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
0.7% 3.6%
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
Can Allogeneic BM Mesenchymal Stem Cells “Heal” a Critical Sized Tibia Defect ?
Can Allogeneic BM Mesenchymal Stem Cells “Heal” a Critical Sized Tibia Defect ?
Can Allogeneic BM Mesenchymal Stem Cells “Heal” a Critical Sized Tibia Defect ?
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
10
0
20
30
40
50
60
70
80
90
Per
cen
tag
e U
nio
n i
n C
riti
cal
Siz
ed
3 c
m T
ibia
Def
ect
Mo
de
l
2 months 6 months
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
% S
urv
iva
l a
t 6
wee
ks
po
st-M
I
0
10
20
30
40
50
60
70
80
90
100
1x106 Stro- depleted BM-MNC
0.2x106 Stro+ cells
1x106 Stro+ cells
saline
n=30
n=30
n=30
n=30
Injection of Ex Vivo Cultured STRO-1Pos MSC Leads to Improvement of Animal Survival
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
% C
ha
ng
e E
ject
ion
Fra
ctio
n
6 w
ks
po
st-M
I
Martens et al. Nature Clin. Pract. Cardio Med 2006
1x106 Stro- depleted BM-MNC
0.2x106 Stro+ cells
1x106 Stro+ cells
saline
Echocardiography of Animals Receiving Myocardial Injection of Ex Vivo Cultured STRO-1Pos MPC Exhibited Global Improvement of Left
Ventricular Function
alp
ha
-SM
A S
tain
ing
Saline
1x106 Stro-1+ cells
0
2
4
6
8
10
12
14
No
. a
rter
iole
s
(alp
ha
-SM
A+
/vW
F+
) /
HP
F
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+
/vW
F+
) /
HP
F
Martens et al. Nature Clin. Pract. Cardio Med 2006
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
Company Location Business Type
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
Company Location Business Type
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§ion=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