Professor Richard Boyd

Group LeaderStem Cells and Immune Regeneration Laboratory,

Dept Anatomy and Developmental Biology

ImmunoGlobalGroup Hong Kong Stem Cell bank

and clinic

MAGELLAN STEM CELLS

“ Stem Cell Medicine” From Placenta to adults – where are we now?Future therapies needs research, commercial partners, Govt

support and public involvement!

Acknowledgements

AsthmaTracy Heng

Sacha KhongTolerance

Tracy HengJess Morison

FRCAnne Fletcher

Peking Union Medical School“Robert” Zhao

Genesis Stem Cells Pty LtdJiansheng (Jason) Meng

Jianqiang YinYang (Claire) Zeng

Thymus regrowth and stem cellsNat Seach, Kahlia Wong, Chew-Li Soh, Ed Stanley, Marco Barsanti, Maree Hammett, Lisa Spyroglou, Jarrod Dudakov,

Gabby Goldberg, Jayne Sutherland (Jade Homan, Jade Barbuto)Neonatal stem cells

Immune RegenerationMSC & AEC in Wound Repair

Standard Operating Procedures for isolation of AECsAdrienne Calder, Anthony Park, Abdulaziz Alsharif

Associate Prof ANN CHIDGEYProfessor ALAN TROUNSON,

Zhicheng XiaoMark “every lab should have one” Malin

Louis Chan, Robert Chin, Anthony Wong, Kenneth Chan

AVSC – Magellan – CMA Dr Dan Bates, David Connell Peter Hansen, Peter Britton, Geoff Barnard

Why the need for stem cells?

• Children develop debilitating diseases that effect them for life

• AND• The older we get, the sicker we get !• Average age has gone from ~30 in 1800’s to now over 80• Living longer but often carrying disease or degenerative

conditions– Quality of life is suffering….

• MAJOR UN-MET CLINICAL NEEDS IN REGENERATIVE MEDICINE

HURDLES – but patients impatient!

• Ethical• Strict Government regulations now in place

• Safety• Must have pre-clinical animal data

• Practical• Which ones to use?• Commercial IP – who owns it?

•Clinical translation• Need care, safety, rigour…. not everything works..

• Funding - public and private co-investment!

Our Goals

To establish:• “State of the Art” (Alpha-type) stem cell treatment

clinics in Australia• prestigious international partners, eg CIRM• rigorous pre-clinical studies “Evidence based medicine”• Isolating, analysing and banking a range of stem cells

– Newborn stem– Adult stem cells

• Innovative, cutting-edge clinical research laboratories

Monash University

Stem Cells Meet Immunology - a new research, therapeutic and commercial platform

Department of Anatomy and Developmental Biology#1 Ranking in Australia

Extensive Research and Development Pipeline in Stem Cells and Immunology

I$ it worth it?

Returns to UK publicly funded cancer-related – (Glover etal BMC Med 2014)

• Total expenditure 1970/2009 - £15 billion• Net monetary benefit of prioritized interventions due to this

research was £124 billion• Internal rate of return for an elapsed time of 15 years – 10%• Compared with previous estimate of 9% for cardiovascular

research

Saving lives and better $ returns than bank interest

Economic Impact of theHuman Genome Project

“How a $3.8 billion investment drove $796 billion in economic impact, created 310,000 jobs and launched the genomic revolution”

Prepared by Battelle Technology Partnership PracticeMay 2011

Stem cells……

• Why are they important?• What can we do with them?

Alan Trounson

Which stem cells to choose..??

Many Types of Stem Cells!

Pluripotent

Induced pluripotential stem cells (iPS)Turning adult mature cells into your own “ES cells”

Adult somatic cells(normal or disease)

“ES” genes(proteins)Oct4, Sox1,2,cMycNanogLin28

Reprogramming

ES cells

Self repair kit; new therapies, diagnostics, disease modeling

ISSUES?? Safety, cancer, autoimmunity, really self??

iPS can be used to create disease in a test tube – diagnosis/drug testing

Are we missing something??

Miracles in motion…

Perinatal Stem Cells

• A source of multipotential/pluripotential stem cells, that are safe, ethical and effective in treating a wide range of diseases

• A 3D membrane suitable for stem cell support and tissue regeneration, treating wounds, ulcers and burns?

• Cord blood, cord tissue, amnion, amnion fluid

Nature’s body repair kit?

…there is a caveat….

Sources of stem cells in new born…

• Umbilical Cord Blood– Haemopoietic stem cells (HSC)– A research, clinical and

commercial gold mine?

www.healthystarttolife.monash.org/

Cord blood

• For transplant: need ~ 25x106 TNC / kg

= 1.25 billion for 50kg patient• Hence need > two cord bags for transplants

– one engrafts but often the other dominates• (MSC can aid engraftment: HSC proliferation

but also improving BM “niche”?)• Is there another source?

Comparison of stem cells derived from Placenta and Cord blood

• Average weight of a placenta = 500g• Average weight of a cord blood unit = 40g (converted from volume).

Whole placenta One Cord blood unit

TNC 2.30 x 10^9 2.12 x10^8

HSC 3.55x 10^7 1.20 x 10^6

MSC 2.60 x 10^8 Not detected

whole placenta one cord blood unit0

500000000

1000000000

1500000000

2000000000

2500000000

TNCs of placenta is 11 times of CB

whole placenta one cord blood unit0

10000000

20000000

30000000

40000000

HSCs of placenta is 30 times of CB

Cord blood (HSC) trials

• 919 registered trials (NIH Clin Trials.gov)• Most haemopoiesis recovery in cancers; very promising • Non cancers:

– BM failure (including MDS)– Inborn errors of metabolism (enzyme storage disease)– birth/prematurity conditions: cerebral palsy; anemia; 

intracranial hemorrhages; retinopathy respiratory distress syndrome;  Bronchopulmonary Dysplasia

– TBI, stroke, autoimmunity

Joanne Kurtzberg (Duke Uni)

Amnion Stem Cells

Readily availablePlentiful supplyNo ethical issues

Towards a cure

Sean Murphy

Made by the baby for the baby.

Chorion Mesenchymal Stem Cells

Amnion membrane Mesenchymal Stem Cells Amnion Epithelial Cells

Placenta: Placenta derived stem cells

Umbilical CordHematopoietic Stem Cells Mesenchymal Stem Cells

Placental tissue Stem CellsAnn Chidgey

ZYGOTE

BLASTOCYST

Hypoblast Epiblast

Mesoderm

WEEK 1

WEEK 2

WEEK 3

Amnion Epithelial Cells

Endoderm Ectoderm

Embryonic Stem Cells

AEC formed from the Epiblast - multipotent stem cells

freshly isolated hAECs

100m

Siva Ilancheran, Ursula Manuelpillai, Sean Murphy, Euan Wallace, Graham Jenkin

muscle

lung brain liver

heart

pancreas

fat

bone

Amnion Epithelial Cell Differentiation

AEC express markers associated with stem cells

• Express markers associated with stem cells

• Risk of tumor formation low compared to ESC/iPS

EpCAM 94%Ck7/8 99%SSEA 3 9%SSEA 4 44%

TRA 1-60 10%TRA 1-81 10%Oct-3/4 5-15% Nanog 5-15% Sox-2 5-15% CD117 20% low CD34 <1%

CD90/105 <1%

Miki et al. 2005, Ilancheran et al. 2007, Murphy et al. 2010

BM MSC ESC hAEC P0 hAEC P5 H Control L Control02468

101214161820

Telo

mer

e Le

ngth

(k

bp)

• Relatively long telomere length

Why Amnion?

• Anti- inflammatory• Immune suppressive• cells secrete appropriate cytokines• Multipotential for differentiation• Antimicrobial • Reduces pain• Reduces scarring• 3D ECM –complex, multilayered• Leads to…..pre-clinical to clinical applications

History of Amniotic Membrane TransplantIn 1910, Davis was the first to introduce the use of

human amniotic membranes (AMs) as a skin graft. In 1913, Sabella used AMs as a permanent coverage

over wounds in burns patients.In 1952, Douglas was the first to report the use of AMs

as a temporary biological dressing in the coverage of burns.

In 1995 Kim and Tseng reintroduced the idea of amniotic membrane for ophthalmic use which has since become increasing more popular.

International Journal of Dermatology 2009, 48, 935-940

Amniotic membrane transplantation for burns and wound healing

Amnion trials

• In addition to burns…..• >160 trials amnion membrane +-/- stem

cells– Corneal - Limbal stem cell insufficiency; data

incomplete– New trial lumbar fusion for degenerative discs;

Jan 2014– Abdominal and skin surgery– Under utilised!

Something to think about…..

The mother is living with a transplant!

Why no rejection?

What actually happens with pregnancy and birth…?

What therapeutic tricks can we learn??

Pregnancy - birth – development

Umbilical cord is MUCH MORE than HSC immune suppression

• Tregulatory lymphocytes (Tregs) • CD4+, CD25+, FoxP3+; release immune suppressing factors

• Mesenchymal Stem Cells• Potent inhibition of immune function; anti-inflammatory

• Combination:• may prevent maternal – fetal rejection;• help “self tolerance” induction?

• MAPC (multipotential “adult” progenitor cells; very low number and variable)

• Cord Blood “ cells” can cross the blood brain barrier

(function of cells or age of recipient?)Joanne Kurtzberg

• 96 trials (May 4th 2014); very few results posted~!• Supplement to HSCT – preventing GvH and

promoting engraftment (HSC expansion/ recovery of niche?) (AuxoCell; Mesoblast)

• HIV – can MSC block immune activation cell death?• Organ transplants (blocking rejection)

• Diabetes (I and II), • Autoimmunity: MS, RA, SLE, • Osteoarthritis

Cord tissue MSC Trialsmaking use of anti-inflammatory/ immune suppressive;

repair induction

What about MSC/ Amnion for asthma?

Key commercial partnership with Mesoblast ARC linkage grant

www.monash.edu.au

35

Lung disease optimal for MSC treatmentTracy Heng

(D. Prockop 2009 Mol Ther)

95% cleared from blood < 5 mins

Half-life = 24hr in lungs

www.monash.edu.au

36

Ovalbumin-induced asthma modelTracy Heng

0

OVA/alum

8 9 10

IntranasalOVA

12

Analysis

11

DAYS

www.monash.edu.au

37

OVA-sensitised mice develop cellular infiltration of the airways

FSC MHC II

CC

R3

CD

3/B

220

CD

11c

SS

C

Mo

Gr

Ly

DCMac

BT

Neu

Eo

OVA SENSITISED

PBS UNSENSITISED

(T.Heng)

*

www.monash.edu.au

38

… MSC reduce eosinophils in asthma lungs

(T.Heng et al 2014)

Multiple Sclerosis: Towards a cure

MSC and AMNION-DERIVED STEM CELLS SUPPRESS THE MULTIPLE SCLEROSIS-LIKE DISEASE IN MICE

McDonald, Jenkin, Bernard

Veterinary applications of MSC’s(Cord and Adipose)

To improve animal health; pre-clinical models for humans• Tendons, ligament injuries, and fractures, • Osteoarthritis • Cancer• Diabetes• Kidney and heart disease• More than 5,000 horses treated since 2003• More than 4,500 dogs treated since 2006• More than 500 dogs, cats, horses in Australia

Commercial partners

Australian Veterinary Stem Cells ,

Vet Stem, San Diego

Veterinary applications - osteoarthritis

Expanded MSC• No serious adverse effects• Strong evidence of pain reduction and return

of capacity • >85% of all animals responded• 100% of younger dogs

(Australian Veterinary Stem Cells , Vet Stem)

To the Clinic - a practical solution?

• Very expensive – who pays?

• In Australia clinical treatments can be conducted under the TGA “Biological Exemption”

• Basically states “ Clinician takes full responsibility for what the patient receives, for one clinical treatment (can be multiple procedures but only for one treatment)”

• Companies and patients can pay for therapy development

• But……

Stem cell therapies: Our code of ethics

We have formed the “Australian Autologous Cell Therapies Group”

Developed a Code of Ethics– Academic, clinical and commercial “clinical groups”– make certain maximal patient safety; clinical rigour, – evidence based treatments, – appropriate thorough patient follow-up; – on-line data management (we are working with

“Clinical Intelligence Pty Ltd. Melb)– Peer group evaluation of data;– International presentations and publications– Appropriate inclusion of commercial partners

Human Chondral Defects, OA Dr DAN BATES

• 12% of the population will suffer chondral lesions

• These progress to osteoarthritis

• >5% of the Australian population suffer Osteoarthritis (3.7 million)

• Costs 23 billion dollars per year to Australian economy

Human Evidence – MSC Safety

Author Patients Disease Injection Follow-up Study Symptom Improvement

Wakitani 2010

41Knee

OsteoarthritisIA 11 years

Safety Study

No Cancer, No infections

Centeno 2010/2011

339Knee, Back,

HipsIA,

Paraspinal2 years

Safety study

No Cancer, No infections

Rodrigeuz 2012

13Rheumatoid

ArthritisIV and IA

13 months

Safety study

One episode of fever & myalgia

Guadalajada 2012

50Perianal fistulas

Local 3 yearsSafety

and efficacy

No Cancer No injection

Left Lateral Femoral Condyle – Pre-treatment

Chondral Defect

12 Months post treatment

Defect Coverage at 12 months (Grey Line)

Osteoarthritis Knee– WOMAC*

pre-treatment 1 month 3months 6months 12 months0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

IH Left PFJ**IH Right PFJ**GH Grade 4TF Grade 3**

WOMAC24 Questions on pain, disability, joint stiffness

Grade 4 OA • Need multiple

injections• Combination cell

therapy

Osteoarthritis Clinical TrialsDrs Julien Freitag / Dan Bates

• Melbourne Stem Cell Centre – Clinic, Box Hill

• “Clean Room” (moving to GMP)– Magellan

• Two Randomised Clinical Trials• Monash/ Latrobe University Ethics• Strong research program

How to maximise MSC effects in OA

• Need to reduce pain• Restore cartilage• Improve strength• But do MSC’s perform this or mediate

through endogenous cells• MSC Short lived!

Need to incorporate support matrices….to improve MSC function and localise to injury site

Tissue Engineering using nanobiotechnology

1. Engineer scaffolds that promote stem cell repair2. Fabricate “smart” scaffolds that can instruct stem cells.

David NisbetJohn ForsytheNick BoydMonash Engineering

Tissue Engineering Scaffolds

Important Features

• Interfiber distance• Surface functionality

Nisbet DR, Forsythe JS, et al. J. Biomater. App. 2009

Factors Controlling Nerve Growth

Interaction of Stem Cells with Nanofibrous Scaffolds

Nisbet DR, Forsythe JS, et al. J. Bio. Sci. Poly. Ed. 2008

Unmodified nanofibrous scaffold

200μm

A

PCL nanofibrous scaffold surface modified

B

3D electrospun/electrosprayed nanocompositessmart surfaces for stem cell therapies Nick Boyd

Nano-HAp suspension

+kV

Collector

PCL + GAA/Pyr+kV

Collector

Step 1: Electrospin PCL Step 2: electrospray nano-HAp

• Use electrospray duration to control surface coverage density of nano-Hap• Then add layer-by-layer growth factors

A

100nm

C

100nm

E

100nm

B

1μm

D

1μm

F

1μm

3D PCL + nano-HAp (low)

3D PCL + nano-HAp (high)

3D PCL

✓

3D electrospun/electrosprayed nanocomposites

Electrospun PCL with varying levels of nano-HAp coating surface density, controlled by duration of electrospraying time. All fibres were electrospun from 15wt% in GAA/pyr(2%), at +12.5kV, -10kV, flow rate: 2.0ml/hour, WD: 13cm, RH: 10-20%. (A, B) no HAp electrosprayed onto the fibres. HAp electrospray from multi-nozzle head using multi-jet spray onto a rotating drum collector. 0.1% (w/v) in methanol, WD: 20cm, R.R: 20mls/hour, +28kV, -20kV was applied to (C,D) for 24 minutes, and (E,F) for 48 minutes.

B

C

D

E

F

100nm 100nm

1μm

C E

F

Stem cell NEW HORIZONS!

Breast milk – natures “jewel” for health

The Value of Maternal Stem Cells at birth

Placental, chorion, decidual tissue• Maternal Mesenchymal Stem Cells for maternal use• Females have higher incidence of autoimmune disease

Breast milk!• Contains ~1 million cells/ml• Immunity- Type II macrophages, T regulatory cells

-> key elements in preventing maternal-fetal rejection also Ab and T cell based immunity

Stem Cells - breast duct -derived pluripotential “epithelial” stem cells; Mesenchymal Stem Cells

• Present as long as lactating; increased with feeding

These “newborn” stem cells are so valuable!- should be banked

• Nature’s whole body repair kit• ProStemCell Pty Ltd Hong Kong• Comprehensive stem cell bank

• Cord blood• Cord tissue (isolated MSC)• Amnion:

• single cells; • cell intact membrane • Acellular intact ECM

• Cord serum/plasma• Breast milk

Hong Kong Stem Cell Bank and clinic

Summary – Perinatal Stem cellsCORD Blood/Tissue• HSC – haemopoiesis• MSC /Tregs – autoimmune disease• “MAPC” tissue repair – cross blood brain barrierPLACENTA• Abundant HSCs, MSCs, AMNION• AECs (100-200 x106 cells per sample• Readily available• Immunosuppressive/ anti-inflammatory• AEC /MSC strong evidence of lung, liver repair-> Can we create Thymus epithelial stem cells??

AMNION MEMBRANE ENHANCES WOUNDS AND BURN REPAIR • Acellular membrane (ECM) for “off the shelf” cell support and tissue

engineering

• Amazing scope for Regenerative Medicine

New Born Stem Cell Banking – a logical choice

Bank it

Donate it

BUT don’t discard it.

It just makes sense!

The BIGGEST PRACTICAL PROBLEM….

How can we overcome this??

• Unless they are your own, stem cell therapies will be considered as foreign transplants and

• will be rejected by the patients immune system!

The thymus

• creates all T cells• deletes or silences autoreactive T cells = self tolerance

Thymus key to long term tolerance of donor therapeutic transplants

Most diseases needing therapy are in the aged…

BUT the thymus degenerates with age?!

Can we use stem cells to restore the old thymus for tolerance inducton?and overcome age-immunodeficiency!

Can we create thymic stem cells?

Derivation of thymus from ES cells:

- GFP targeted “Fox N1” (mouse, human)

Differentiation of FOXN1GFP/w hESCs into hTECS

(Ng et al, 2008, Nature Protocols)

BFGFP

FACS analysis, qPCRs

Chew-Li Soh et al 2014

Inducing tolerance to stem cell therapeutics

Chidgey et al Nature 2008

What are the three worst words a patient can hear?

YOU HAVE CANCER

612 × 358 - en.wikipedia.org

Immunotherapy

Stem cells to boost the immune system to kill cancer

• Combines the power of Ab specificity and flexibility with the killing efficiency of T cells

• Cancer recognition through Ab engineered into T cell membrane

• linked to signal transduction molecules – binding triggers activation

• T cell directly kills cancer cell

Chimeric Antigen Receptor (CAR)

•CAR- T cell Therapy

Stem cell technology enhances immunotherapy!

• CAR- technology can arm millions of patient T cells against their cancer

• Eventually these cells will be “worn out” and severely depleted

• Induced pluripotential stem cell (iPSC) technology can overcome this

Stem cell technology enhances immunotherapy!

• Create iPSC from CAR-T , • effectively immortal, • limitless self renewal, • re-create T cells from these iPSC,• T cells retain the cancer specificity• Can be infused into the patient• OR• Cryopreserved for future relapse

Table 2. Summary of clinical outcomes.Characteristics No. of patients (N = 16) Overall complete response to salvage chemotherapy* 7 44%

Overall complete response to 19-28z CAR T cells 88%In patients with morphologic residual leukemia (n = 9) 7 78%Complete remission (CR) 10 63%Complete remission with incomplete count recovery (CRi) 4 25%Molecular complete remission (CRm)‡ 12† 75%Median time to CR/CRi (days) 24.5Post-CAR T allo-SCT (n = 10 eligible patients)§ 7 70%

“Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia” Davila etal Sci Transl Med 2014

SUMMARY• The world of stem cell therapies is all but on us

– Melbourne Stem Cell Clinic open for business• “Hard yards” of research now gaining clinical traction• The transition from promise to reality is delicately

poised• Funding the key !

– are there commercial partners?– How much should the government pay– Health insurance companies….??– Can/should patients pay for their evolving

therapies?

“Proposition 71 authorizes essential research for new cures intended to save millions of lives” $US3Billion

04/08/2023

Californian Institute for Regenerative Medicine

Alan Trounson

CIRM - Driving Partnerships in Translation and Preclinical Research

Inno

vatio

n an

d so

lutio

ns • Academia• Research

infrastructure• Research

capacity• Innovation• IP

Regu

lato

ry e

xper

ienc

e an

d fo

cus • Industry• Manufacturing• Standards• Focus –

milestones, go-no-go decisions

• Relevance – competition

• IP utilization Del

iver

y of

cel

l the

rapi

es • Clinical

Medicine• Patients• Clinical

infrastructure• Counseling• Monitoring

long term• Clinical trial

capacity

Globalize the Initiatives Alan Trounson

Therapy Progress: Neurological Disorders$94 million

04/08/2023 83

Epilepsy $1.7 million (1)3 million in US

Spinal muscular atrophy $5.6 million (1)

Stroke $20 million (1)140,000 die/year

“There are so many things doctors can do with stem cells that will really help.”- K. Michael Cooper (stroke)

Therapy Progress: Neurological Disorders$94 million

04/08/2023 84

Autism $1.5 million (1)

1 in 110 kids in US

Huntington’s disease$6.6 million (2)30,000 in US

Spinal cord injury$26.5 million (2)200,000 in US

“Turning stem cells into cures”- Roman Reed

Therapy Progress: Neurological Disorders $94 million

04/08/2023 85

Lou Gehrig’s disease(ALS) $10.8 million (1)5,600 diagnosed/year

Alzheimer’s disease$3.6 million (1)

5 Million in the U.S.

Parkinson’s disease$13.9 million (3)

50,000 diagnosed/year

Phase I/II Spinal Cord Injury Trial

• Multi-segmental gains

• ASIA LT changes correlate with quantitative thresholds

• University of Zurich - Balgrist

Hospital, Swissmedic authorised

• Now authorised in Canada and US

• 12 chronic stage patients; T2-T11

injury – enrolling A, B, C