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Organised by:

Malaysian Healthy Ageing Society

Co-Sponsored:

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Overview of Stem Cell Therapy

Chin Sze Piaw MBBS MRCP FNHAM

Consultant Cardiologist, Mawar Medical Centre

Visiting Cardiologist, PPUKM

Clinical and Research Advisor, Cytopeutics

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Stem Cell Potential

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Defining Stem Cells

Stem cells are cells that can self-replicate and differentiate

into other specialized cells

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What is True Stem Cell?

Embryonic stem cell in first 14 days of life

Fetal stem cell divides along 3 germ layers

Unethical

Banned

Causes Cancer

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Embryonic Stem Cells Can Cause Cancer

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Stem cells exist in adults to elicit daily repair

The human body recycles itself every 20 years

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Adult stem cells reside in bone marrow Bone marrow transplant established for 30 years

Bone marrow transplant is essentially haematopoeitic stem

cell (HSC) treatment

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BMSC for non-haematopoeitic diseases?

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Mesenchymal Stem/Stromal Cell (MSC)

Differentiates naturally into:

• Adipose Cells

• Osteoblasts

• Cartilage

• Cardiac Muscle

• Smooth Muscle

• Skeletal Muscle

Transdifferentiates into:

•Hepatocytes

• Neurons

• Insulin Producing Cells

• Renal Mesenchyme Cells

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Differing actions of MSC and PBSC/HSC

HSC/PBSC destroys bone; MSC builds

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MSC is present in minute numbers but may be rapidly expanded

- 13 - - 13 - Choong, Cheong et al, Cytotherapy 2007

MSC Regenerates New Joint Cartilage

MSC-derived cartilage cells staining for Type II Collagen and still

retaining capacity for replication (arrows)

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Lee et al, Stem Cells 2007

MSC-HA Saline HA Only

6 wk

12 wk

MSC Regenerates New Joint Cartilage

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MSC Regenerates Hyaline Cartilage

MSC-HA Saline HA Only

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MSC can be changed into osteoblast

Wong et al 2008, Pathology

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MSC for Cardiac Muscle

MSC engrafts and fuses with

damaged heart muscle cells

MSC fuses with Damaged Heart Muscle

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MSC Repairs Damaged Heart Muscle

MSC replaces disorganized scar tissue in the heart with new cardiomyocyte. Laflamme 2007

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MSC Forms New Blood Vessels

MSC differentiates into vascular smooth muscle cells for arteriolar formation (“Not just capillary proliferation”)

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MSC Forms New Blood Vessels

MSC differentiates into vascular smooth muscle cells for artieriolar formation

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MSC forms the building blocks for musculoskeletal and supporting structures

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M S C Transdifferentiation - Neuron Induction

Choong, Mok, Cheong, Cytotherapy 2010

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Functional neurons with neurotransmitter expression

Choong, Mok, Cheong, Cytotherapy 2010

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IV M S C - Mechanisms of Actions in Stroke

Angiogenesis

Neuron cell regeneration Reduced Scarring

Engraftment

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MSC hastens recovery from acute renal injury in a mouse model

Wong, Cheong Pathology 2008

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MSC Cell clusters stain crimson red with dilthizone (for insulin expression)

Wong, Chin 2010

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TEM confirms increasing secretory granules in induced MSC

Wong, Chin 2010

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Induced MSC produces insulin in response to different concentrations of glucose stimulation

Insulin Production by induced IPC upon various

glucose stimulation

2.1 2.67 2.83.43

13.63

48.2

0

10

20

30

40

50

60

5.56 16.7 25

Glucose Stimulation (mMol)

Insu

lin C

on

cen

trat

ion

(m

icro

IU/m

L)

Uninduced MSC Induced IPC

M S C For Diabetes

Wong, Chin 2010

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Proven that our MSC can be differentiated into specialized cell types

MSC

Osteoblast

Chondro-blast

(& NP Cell)

Adipocyte

Arteriole Cardiac

Muscle

Neuron

Insulin (beta islet) cell

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We Have Successfully Produced True MSC

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3 days 6 days

12 days (MSC reached confluence) 9 days

Using Proprietary, Animal-free Medium for rapid expansion,…

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Safe for Clinical Applications

We are able to culture 500 million high quality MSC from just 10-

20 ml BMA within a month for clinical treatment

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We ensure all cells retain identical characteristics as original

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All processing in a GMP compliant lab Large clean room 100 facility

Large clean room class 100 for (future expansion)

Laminar air flow

Individualized incubators

Layout (Goods flow and Human flow) according to GMP

ISO 15189 for hematology testing

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Product Traceability and Safety during storage and transportation

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On-site Final Preparation

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ISO 15189, CLEAN ROOM CLASS 100, GMP COMPLIANCE BY BPFK

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Potential mesenchymal stem cell applications

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Bone Marrow Cells Are Proven Safe

Autologous Bone-Marrow Derived Stem Cells Are Safe

Meta-analysis of 999 heart failure patients

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From Atherosclerosis to MI and Heart Failure

Smoking Diabetes HPT Lipid

Inflammation

Endothelial Cell Damage & EPC

Dysfunction

Atherosclerosis & Atherothrombosis

MI/Heart Attack

Scarring

Dilatation & Electrical instability

Heart Failure & Cardiac Arrest

Death

Endothelial Progenitor

Cell (EPC) also

dysfunctional in

cardiovascular patient.

Therefore unable to

repair endothelial cell

damage, leading to

atherosclerosis

Scar tissue is easily

stretched, does not

return to shape. Gives

rise to progressive

heart dilatation and

also electrical

arrhythmias

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Methodology

(20) Symptomatic (NYHA II-IV)

Severe Dilated Cardiomyopathy (LVEF < 35%)

Suitable for/ likely to benefit from revascularization alone

Suitable for bivent pacing

(10) No.

Assignment

(5) Multivessel disease

Intramyocardial MSC with concurrent CABG

(5) Patent vessels (2) /Non-ischemic (3)

Intracoronary MSC injection alone

(10) Yes.

Plan for revasc (8) or bivent pacing (2) first

Phase IIb Trial. (NMRR Research ID No: 582) 20 patients screened between 2008-2009

•8 patients had significant viable myocardium: sent for revascularization initially

•2 with BBB: sent for consideration of bivent pacing

•5 with ischemic cardiomyopathy

deemed unlikely to benefit from

revascularization alone

•2 had previous and patent vessels

•3 non-ischemic cardiomopathy

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LV Ejection Fraction

Baseline 6 weeks 3 months 6 months 12 months

Total 26.5 + 6.7 38.6 + 12.8^ 44.5 + 12.3* 47.4 + 7.2* 58.7 + 12.6^

Intracoronary 26.9 + 6.2 33.9 + 6.7 39.6 + 6.3^ 41.7 + 4.1* 47.7 + 2.8^

Intramyocardial 26.1 + 7.8 43.4 + 16.3^ 49.5 + 7.9^ 53.1 + 4.4^ 69.6 + 5.7^

P values when compared to baseline

^ P less than 0.05

* P less than 0.01

Accepted Eur Heart J 2010

Early rapid recovery – Paracrine effect

Late sustained recovery - Regeneration

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MRI viability study – Intramyocardial injection

Baseline 12 months

Gadolinium delayed enhanced constrast

MRI shows concentric and full thickness

scarring (white area)

Resolution of scar and increased

myocardial thickness with new

myocardium (grey and black areas)

Accepted Eur Heart J 2010

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Findings corroborated by independent research group

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REPAIR-AMI: 1 year follow-up

0

5

10

15

20

25

30

35

Death MI HF Hosp PCI Stroke Arrhythmias/Syncope

Placebo BMC

Schachinger et al 2006

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Patient Population

• 15 patients (Male 4; Female 11)

• Mean age 68 + 12.7 years

• Severe OA

• Significant varus/valgus deformity

• Pain and reduced mobility on NSAIDs or COX2

• Failed multiple hylauronic injections

• No change or worsening OKQ * in previous 6 months

* Oxford Knee Qustionaire

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Chondrocell in a 3D biodegradable scaffold

MSC as a Single Injection in Severe refractory

osteoarthritis

2-3 mLs injected into knee capsule containing

1-2 million cells per kg body weight

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Change in Functional Score from baseline

-5 -2 -1 -1 -1

-10 -13

-3 -5

-3 -4

6 3 2

6

6 10

10

-15

-10

-5

0

5

10

15

Prior to Tx 6 mths 12 mths

Major Improvement

Mild Improvement

No Change

Worse

Comparing OKQ scores to baseline

-4 or less: Worse

-3 to 3: No change

4 to 9: mild improvement

10 or more: major improvement

R L

R R L

L

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MRI independantly reported

• Increased in cartilage

thickness by 40% (noted

in 40%)

• Resolution of

subchondral cysts (noted

in further 10%)

• Reduction in effusion

(noted in further 10%)

Das Gupta, et al (Accepted Int J Rheuamtic Dis 2010)

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Randomized, prospective, double blind study at

7 leading sports centre in the US,

Single injection

LD 50 million MSC

In biodegradable matrix

55 Patients with moderate-severe OA and torn meniscus

1 WEEK

MSC Effective for OA in a FDA

Multi-Centre Clinical Trial

Method:

Single injection

HD 150 million MSC

In biodegradable matrix

Multiple injection

HA

To assess the safety of an injection of stem cells into the joint capsule and to gain

preliminary efficacy data on the ability of MSC to impact symptomatic improvement, tissue

regeneration and the development of osteoarthritis

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Superior Pain Reduction by MSC

Normal pain

reduction by

multiple HA inj

is 9-23 pts

only

48

56

28

19

0

10

20

30

40

50

60

All OA Severe OA Patients only

Vis

ua

l A

na

log

ue

Sc

ore

1 Year Magnitude of Pain Reduction by VAS

MSC HA

p=0.05 p=0.004

Effects were dose-dependant and pain scores improved from 6 months to 1 year

implying that an Active Biological Process

MSC group were 3.5 times less likely to experience degenerative bone changes as

compared to multiple HA injections

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Positive Dose-Response Effect

Severe OA patients only 56

26

19

0

10

20

30

40

50

60

High Dose (150 million) Low dose (50 million) HA

Vis

uA

L A

na

log

ue

Sc

ore

Improvement of Pain from Baseline

No. of Cells Matter

Moderate OA: Min. 50 million cells

Severe OA: Min. 100-150 million cells

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Bone Marrow Cells (BMC) better than Peripheral Blood Cell (PBC) for Cartilage Repair

Reference: Fei Chang et al, J Orthopaedic Reseach Jan 2008.

Bone Marrow Stem Cells are Superior

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Comparing regeneration of normal hyaline cartilage with growth factor induced and non-induced MSC

Reference: Gelse 2007

MSC non-induced MSC induced Untreated

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MSC EPC combination for occlusive PAD

• 62 year old Male

• Diabetes

• Moderate Kidney Failure

• Heart Failure

• Severe Peripheral Artery Disease

• Not suitable for Bypass or angioplasty

• Ulcer on right heel not healing after 2 months

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* Intensive treatment with topical gels, hyperbaric oxygen and oral medicines and supplements

First presentation

After 1 month intensive

treatment *

3 weeks after intramuscular

Dermacell 1st injection

After 3 months Dermacell

Injection

After 6 months Dermacell

Injection

Deep ischemic non-healing ulcer –

no improvement after 1 month

Ulcer starting to become

smaller, shallow with

healthy bleeding tissue and

overriding fibrin plaque –

derived from new blood

vessel and flow to ulcer

Healed ulcer with no

scarring

MSC EPC combination for occlusive PAD

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M S C - Nucleus Pulposus Cell Induction

Uninduced MSC

Induction with TGF B

2 wks 4 wks

Collagen II after 4 wks

Proteoglycan Production

evident by metachromatic

staining with Safranin-O

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L5/S1 IVD Disease 33 year old man

Hyaluron (white appearance) inside disc present at 6 months

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Clinical Trial of IV MSC for Stroke

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IV MSC appears feasible and safe and aid recovery after recent stroke

• Barthel Index at baseline, 3,6 and 12 months

• Significant functional recovery (early and sustained).

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MSC reduces autoimmune reactions THE PARACRINE EFFECT

MSC inhibits T-cell proliferation and activity

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Role of T-Cells in Autoimmune disease

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M S C For Autoimmune Disease

M S C from patients with active Rheumatoid Arthritis inhibiting autologous T-cell proliferation and activity

Das Gupta, Wong CY, Chin et al (accepted Int J Rheumatic Dis 2010)

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M S C Use in Autoimmune Disease

• MSC is Immunoprivileged

• Dampens inflammation and immune reactions

• Demonstrated against GvHD, Crohn’s disease

• Early benefits against MS, Lupus nephritis, T1DM.

• Likely immune-mediated effects in cardiomyopathy, arthritis

Wong, Cheong Cytotherapy 2008

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NK-DC Cells – not stem cells – are used in cancer

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World Active Stem Cell Sites

• 200 Stem cell research and therapy centres in 2009

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Future Directions

• Alternative autologous source (induced pluripotent cell from

skin fibroblast)

• Cell therapy versus tissue engineering

• Best source for off-the-shelf use?

• Wellness and Healthy Aging

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Conclusions

• Potential in stem cell applications progressively and

consistently demonstrated for MSC

• Safety issues determined by:

• Source

• Ex vivo expansion

• Storage and transport procedures

• Conduct in context of a study with proper informed

consent, patient selection and follow-up

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Thank You

Chin.sze.piaw@gmail.com

• Prof Dr Cheong Soon Keng

• Prof Dr S Fadilah Wahab

• A/Prof Dr Leong Chooi Fun

• Dr Then Kong Yong

• Dr Lian QiZhou

• Wong Chee Ying

• Ooi Ghee Hian

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Who is considered a healthy donor?

• Cancer risk increases with age

• Biotech advances have failed to reduce rate of cancer incidence and mortality

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Most cancers are due to mutations in adulthoood caused by environmental factors or diet

• Inherited from birth

• Acquired Mutations

• Virus infections

• Radiation exposure

• Contaminants

• Diet

• Smoking

• UV/Sunlight

• Occupational factors

• Alcohol

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Therefore the healthiest stem cells must be the youngest possible donor - the umbilical cord blood

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Wharton’s jelly MSC Resilient to hypoxia and tridifferentiation ability

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Highest proliferative potential seen with UC-MSC compared to adipose tissue and bone marrow

• Note: BMA obtained from 18 patients aged 64-84; adipose tissue

liposuction obtained from 18 patients aged 26-57

• Therefore the differences in proliferative capacity may be due to age

Kern 2006 Stem Cells

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UC derived MSC is best source for allogeneic stem cell treatment

Highest proliferative capacity

Free from cancer caused by environmental factors

Easy to trace family tree and family medical history

Balance of regenerative potential and paracrine effect

Easy to obtain, expand and store

Same efficacy or better in latest trials

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Current Status

• Pioneering research on stem cell transdifferentiation (neuron cells, insulin producing cells, immune-modulating cells)

• Developmental work and clinical trials with promising results (safe and efficacious in end-stage heart failure, severe OA,

peripheral arterial occlusive disease)

• Pilot studies for autoimmune disease, neuro diseases

• Bone marrow MSC has best evidence across disciplines

• Autologous source accepted for clinical studies

• Allogeneic MSC - - USE UMBILICAL CORD-DERIVED

• Embryonic and fetal source strictly experimental (major

safety concerns)

• Animal source prohibited

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Acknowledgements

• Prof Dr Cheong Soon Keng

• Prof Dr S Fadilah Wahab

• A/Prof Leong Chooi Fun

• Dr Then Kong Yong

• Dr Lian QiZhou

• Dr Rebecca Wong Shin Yee

• Wong Chee Yin

• Ng Mei Theng

• Dan Siew Peng

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Exercise caution!

• Promising use of stem cells for therapies in future

• Understand the disease process

• Understand the rationale for treatment

• Determine the best route for administration

• Part of adjunctive treatment of established options – not an alternative at this stage

• Aware of complications

• To be treated by specialists only in setting of clinical trial

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Differing actions of PBSC/HSC and MSC

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• Fracture Repair from cancellous bone chips, demoniralized bone matrix and autogenic bone grafts

• (but not possible with PBSC)

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Osteogenic potential

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Differing actions of MSC and PBSC/HSC

HSC/PBSC cause inflammation and healing by fibrosis

MSC regulates inflammatory process and healing through repair

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MSC from different sources are not the same

Genomic study comparing MSC from bone marrow (BM) with

umbilical cord blood (UCB1 and UCB2):

A. Number of gene sequences shared

B. Concentration of gene expression

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Using a stranger’s cells has many concerns

RISK OF VIRAL

TRANSMISSION

Some viruses remain latent for up to 10 years. Many new viral diseases

being discovered which is not routinely tested during blood donation

RISK OF

CARCINOGENICITY

Many cancers have familial of genetic link. Therefore potential risk of

genetic transfer by live nucleated cells. A young man may donate stem

cells now and develop cancer 20 years later

RISK OF GENETIC

ABNORMALITY

Few centres also routinely screen for genetic abnormalities which may

manifest later in life

ETHICAL CONCERN

WITH DONOR

Was donation voluntary or was patient coerced? Did donor know cells

used for research or treatment of other patients

LAW VIOLATION No money or inducement can be given to donor.

Donated tissue cannot be bought or sold

QUALITY Cells that has been stored for a long time – is it safe? Do we know what

is expiry date? How many passages of cell multiplication?

COMPATIBILITY How do we check if we are compatible? Not as simple as ABO.

LACK OF EVIDENCE Scarce clinical data on efficacy and safety unlike autologous treatment

Allogeneic BMT associated with 20-30% GvHD

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MSC is not 100% immunoprivileged