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Dr R V S N Sarma MD MSc

Consultant Physician &

Chest Specialist

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Stem Cell Research

Advancements & Applications

Special focus on Cord Blood

Basics of Cloning and GE

Stem Cell Research

Advancements & Applications

Special focus on Cord Blood

Basics of Cloning and GE

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The Outline of Discussion

What are Stem Cells ? Why Stem Cells ? What are the sources of Stem Cells

(SC) ? What are the methods of SC

Production ? How Cord Blood SCs are harvested ? What are the applications of Stem

Cells ? Where are we now ? Where do we go from here ? Cord Blood Banking resources –

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Cell Therapy – Regenerative Medicine

Autologous (from the same individual) Allogeneic (from a different

individual) Xenogeneic (from a different species)

1. Differentiated – specialized cells of same or other tissue type, such as heart, muscle, blood cells etc.

2. Undifferentiated or unspecialized or uncommitted cells such as stem cells.

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Different Types of Stem Cells

Totipotent (totally undifferentiated – potential to become any cell type including whole organism)

Example: The inner cell mass of embryo

Pluripotent (undifferentiated – potential to be any cell type but not an entire organism)

Emb Stem Cells (hESC), Emb Germ Cells (hEGC)

Multipotent (differentiated and yet undifferentiated)

The UCSC, BMSC, MSC – limited in potential

Use of the stem cells raises ethical / legal concerns

Wide variation in national policies for stem cell use

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Hierarchy of Stem Cells

Multipotent

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What is special about Stem Cells ?

1. Capability of dividing and renewing themselves for long periods - almost immortal

2. Stem cells are unspecialized “uncommitted” cells.

3. Stem cells can give rise to specialized cells. Remain “uncommitted” until they receive signals from their environment to develop into specialized cells.

4. Stem Cells have Plasticity - Stem Cells from one tissue are able to give rise to cells of a completely different type of tissue

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Hectic Competition

The ultimate in love

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Week 1 of Gestation

FertilizationDay 1

2 cell stage Day 2

4 cell stage Late Day 2

MorulaDay 3

BlastocystDay 5

ImplantationDay 6 - 7

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The Blastocyst – Day 5

The Blastocyst consists of

Inner Cell Mass

(embryoblast)

Trophoblast

Blastocele

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Week 2 of Gestation - Day 7.5

Blastocystic Cavity

Cytotrophoblast

Inner Cell Mass

Syncytiotrophoblast

Uterine Epithelium

Uterine Artery

Uterine Gland

Syncitio-trophoblsat

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Week 2: Early Development

Cytotrophoblast

Inner Cell Mass :

Epiblast

Hypoblast

Amniotic Cavity

Amnioblasts

Primary Yolk Sac

Exocoelomic Membrane

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Embryonic Cell Types – at Gastrulation

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GENES

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Sexual Reproduction

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Cloning or Asexual Reproduction

SCNT

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Parthenogenesis

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Embryonic Stem Cell Cultures

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Human Genetic Engineering

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Germline Engineering

Combines the application of

1. Stem Cell use

2. Genetic Engineering

3. Embryo Cloning and

To Produce Designer Babies

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In Vivo – Pluripotent Stem Cells

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Different Types of Stem Cells

Embryonic cells – by In Vitro Fertilization (IVF)

ESC and Embryonic Germ Cells (EGC)

Somatic cell nuclear transfer (SCNT) - Cloning

Used to clone ‘Dolly’

Adult Stem Cells (ASC) – Best eg. BMT, MSC

Umbilical cord blood stem cells (UCSC)

Already differentiated to some degree ?

Can they be made to become pluripotent ?

Will they rapidly lose capacity to differentiate ?

Amniotic Fluid Stem Cells – AFSC – 9th Jan 2007

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ESC versus ASC

Embryonic Stem Cell (ESC)

High malleability Potential for undesired

development (teratomas)

Infinite lifespan,

unlimited supply High ethical burden Uncertain legal status

Adult Stem Cells (ASC) Limited developmental

potential Better behaved, easier

to manage Lose their ability to

proliferate or differentiate after a time in culture

Less moral ambiguity Less legal controversy

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Methods of Stem Cell Generation

SCNT

IVF

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Stem Cell Maturation in Bone Marrow

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Alternatives to Embryonic Stem Cells from Living Human Embryos

From dead embryos Less than 8 wks MTPs Non-destructive biopsy Bioengineered cells (genetically

altered) Reprogrammed adult somatic cells

[but] Live human embryos hold most promise

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Stem Cell Programming Stem Cell Differentiation

‘RECIPE’

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Therapeutic Applications of Stem Cells

Regenerative medicine – permanent repair of failing organs - e.g.Cardiomyocytes for heart diseaseAngiogenesis in CAD – ‘Auto bypass’ Islet cells for diabetesNeural cells for Parkinson’sBlood cells for cancerChondrocytes for osteoporosisKeratinocytes for burns

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Stem Cell Applications

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The Promise of Stem Cell Research

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Innovations of Cell Therapies

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Cell Therapies – Global Over View

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Estimated Market for CT versus IT

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Electron Micrographs of Stem Cells

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Programming of

Stem Cells – ex vivo

EMG of Stem Cell

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Stem Cells in CAD

Cardiomyogenesis Coronary Angiogenesis

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The Enormous Power of the Umbilical Cord

Let us Understand Let us Understand

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Umbilical Cord Blood Banking

Collection, Processing, Cryo-preservation of blood in the placenta and umbilical cord after the separation of baby

In-Utero collection, Ex-Utero collection

45 disease are now treatable with Cord blood Stem Cells

These are not ‘experimental’ but ‘mainstream’ therapies

The stem cell concentration is 10 times higher than BM

For BMT overall only 25% get a suitable HLA match

Reduced GVH-Disease in the recipient

Painless and easy to collect, Immediately available

BMT costs 3-4 lakhs – Cord blood has lower cost

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UCSC Rx. for which diseases ?

Acute and Chronic Leukemias Myelodystrophic syndromes Stem Cell disorders – FA, PNHU, Aplastic Anemia Myelo and Lympho proloferative Disorders Liposomal storage Diseases, Plasma cell

disorders Phagocyte Disorders Histiocytic Disorders, Platelet abnormalities Other Malignancies – BC, Ewings, NB, RCC RBC Abnormalities – Sickle cell, Beta

Thalassemia SCID – Congenital immune system disorders

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Promising Applications of UCSC

Parkinsonism Alzheimer's Disease Type 1 Diabetes Mellitus Cardio-myopathies Coronary revascularization for CAD Retinoblastoma – Retinopathy Burns and skin regeneration Osteo and other degenerative

arthritides Cirrhosis, Hepatitis Osteoporosis

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The Placental Barrier

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Why Use Cord Blood Stem Cells ?

Absolutely non-controversial Absolutely simple to collect More potent than Adult stem cells Non-contaminated Readily available Both for vaginal and c-section

deliveries Low rates of rejection for transplants Most importantly very high chances of

HLA matching

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Stem Cell therapy centres in India

Tata Memorial Hospital, Mumbai Adyar Cancer Centre, Madras Apollo Specialty Hospital, Madras, Apollo Hospital, Global Hospitals, NIMS,

Hyderabad Christian Medical College, Vellore Narayana Hruduyalaya , Bangalore R&R Army Hospital, New Delhi AIIMS , New Delhi Inlaks Hospital, Pune Armed Forces Medical College, Pune Sanjay Gandhi PGIMS, Lucknow

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Storage of Umbilical Bloodfor Stem Cells

Commercial enterprise (Cordbank) stores umbilical stem cells from birth for future autologous use.

“Saving your baby’s umbilical cord stem cells could save your baby’s life”

Others advocate for a public cord blood bank

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Cord Blood Collection

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Thawing Method

Based upon COBLT (Cord Blood Transplantation Study) Method

Used for thawing cord blood products received from COBLT Banks frozen in MedSep (80/20) bags

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Cord Blood Preservation

Under liquid N2 at – 192o C

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Preparation

2 Hours prior to thaw: Review product paperwork

Gather supplies and

reagents

Prepare wash media

(Dextran 40 and human

serum albumin).

Prepare wash bag setup:

MedSep Transplant Set+

300 mL plastic transfer

bag.

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Preparation

Bag 1 Transfer 250 mL Dextran 40 into 300 mL transfer bag. Add 50 mL 25% Albumin. Final conc. of albumin is 4.1%

Connect the Dextran/Albumin bag to a MedSep Transplant Set (Bag 2 & 3) using sterile connecting device. Bag 1 Bag 3Bag 2

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Preparation of Wash Bags

Transfer 125 mL

of the

Dextran/Albumin

to Bag 2 and chill

bags for

approximately

one hour in

refrigerator.

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Retrieval

Remove from

storage

Verify all label

information

with 2nd

technologist

Detach segment

and store

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Thawing

Place the

product inside

a Ziploc bag,

seal tightly,

and submerge

in the 37C

waterbath until

slushy.

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Connect to Wash Bags

Clean scissors and CBU bag

with alcohol. Cut port and

rewipe with alcohol

Connect CBU

With all clamps closed

spike both ports of the

CBU bag with MedSep

transplant set. Open

clamps for CBU and Bag

2 (125mL Dex/Alb

solution)

Bag 2Bag 2

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Diluting Cells

Dilute Cells

Gradually (over 4-

5 minutes) mix the

cells in the

cryobag with the

Dextran/Albumin

solution (Bag 2)

by raising and

lowering bags

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Diluting Cells

Transfer cells to Bag 2

(125mL wash media)

Rinse the CBU bag twice

with an additional 25 ml

of the Dextran/Albumin

solution from Bag 1.

Drain into Bag 2

The total volume in the

Bag 2 should be

approximately 200 mL

Bag 2

Bag 1

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First Centrifugation

Heat seal and remove

the CBU and Bag 1

(Dextran/Alb)

Centrifuge cells and

remaining bag @ 880

x g for 20 minutes at

4C.

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Expressing Supernatant

Express the

supernatant into the

attached transfer bag

Leave approx. 25 mL

of cell suspension in

bag.

Heat seal and

disconnect CBU from

supernatant bag

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Spinning the Supernatant

Connect a 300 mL transfer

bag to the supernatant

bag.

Centrifuge the supernatant

bag and attached transfer

bag to 880 x g for 15

minutes at 4C.

Express the supernatant

leaving approximately 10-

20 mL of cell suspension in

the original bag.

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Final Pooling

Combine cells

recovered from the 1st

and 2nd spins into 1

bag.

Add additional

Dextran/Albumin

solution so that final

product is 50mL (peds)

and 100mL (adults)

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QC Testing

QC Samples

Nucleated Cell

Hematocrit

Viability

ABO/Rh

CFU

CD34

Sterility

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Checking the Cord Blood for Disease

Syphilis

HIV Ag and Ab

Hepatitis B and C

ALT

Malaria

Leptospirosis

HTLV 1 and 2, CMV, EB Virus

HLA typing is not done routinely while banking. It is done only before Stem Cell transplant

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Final Product

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Potential Safety Issues

Older stem cells can turn cancerous, e.g.Human adipose stem cells in animalsUnexpected in adult stem cellsProblem is a function of age (number

of divisions outside body)Cell cultures should be no more than

60 generations old Need for pre-clinical research prior to

therapeutic trials

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International regulations regarding embryonic stem cell research A: Prohibition B: Utilisation C: Utilisation and

extraction D: Utilisation, extraction and creation

Austria Brazil Costa Rica Denmark Equador Ireland Peru Poland

Germany USA

Australia Canada Greece Japan Spain Sweden The Netherlands France

Singapore UK Israel China

[Taken from Towns C.R. Embryonic Stem Cell Research: Understanding and Analysis at the Interfaces of Science, Medicine and Ethics. PhD Thesis, University of Otago, 2004]

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American Association for Advancement of Science

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To learn more about theStem Cell Research visit

www.stemcells.nih.gov

National Marrow Donor Program (NMDP) www.marrow.org

This presentation is on the our website

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International Stem Cell Forum

http://stemcellforum.org

Currently representatives from the following countries:

• Australia

• Canada

• Czech Republic

• Finland

• France

• Germany

• Israel

• Japan

• Singapore

• Sweden

• Switzerland

• The Netherlands

• UK

• USA (NIH)

JDRF is the only non-government partner

JDRF funds outside national borders

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Cord Blood Banking Facilities

Public Cord Blood BankingReliance Life Science (RLS), Bombay

2001– RLS has net work all over India 2005– ReliCord-S and ReliCord-A

Histostem ( of Korea) bank in Mumbai,– Additional centres for Delhi, Chennai

and Kolkata. – Govt of India has 10% equity stake

Private Cord Blood Banking – 2004 byLifeCell of ACCPL (Asia Cryo Cell P ltd)

– Affiliate of Cryo-Cell International (USA)

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