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Stem Cell Research Advancements & Applications Special focus on Cord Blood Basics of Cloning and GE. Dr R V S N Sarma MD MSc Consultant Physician & Chest Specialist. www.drsarma.in. The Outline of Discussion. What are Stem Cells ? Why Stem Cells ? What are the sources of Stem Cells (SC) ?

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Stem cell research advancements applications special focus on cord blood basics of cloning and ge

Stem Cell Research Advancements & ApplicationsSpecial focus on Cord BloodBasics of Cloning and GE

Dr R V S N Sarma MD MSc

Consultant Physician &

Chest Specialist

www.drsarma.in

www.drsarma.in


The outline of discussion
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
Cell Therapy – Regenerative Medicine

  • Autologous (from the same individual)

  • Allogeneic (from a different individual)

  • Xenogeneic (from a different species)

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

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

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Different types of stem cells
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
Hierarchy of Stem Cells

Multipotent

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What is special about stem cells
What is special about Stem Cells ?

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

  • Stem cells are unspecialized “uncommitted” cells.

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

  • 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
Hectic Competition

The ultimate in love

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Week 1 of gestation

4 cell stage

Late Day 2

Morula

Day 3

2 cell stage

Day 2

Blastocyst

Day 5

Implantation

Day 6 - 7

Fertilization

Day 1

Week 1 of Gestation

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The blastocyst day 5
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
Week 2 of Gestation - Day 7.5

Uterine Artery

Uterine Gland

Syncitio-trophoblsat

Blastocystic

Cavity

Uterine Epithelium

Inner Cell Mass

Cytotrophoblast

Syncytiotrophoblast

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Week 2 early development
Week 2: Early Development

Cytotrophoblast

Amniotic Cavity

Amnioblasts

Inner Cell Mass :

Epiblast

Hypoblast

Primary Yolk Sac

Exocoelomic

Membrane

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Genes
GENES

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Sexual reproduction
Sexual Reproduction

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Cloning or asexual reproduction
Cloning or Asexual Reproduction

SCNT

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Parthenogenesis
Parthenogenesis

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Germline engineering
Germline Engineering

  • Combines the application of

  • Stem Cell use

  • Genetic Engineering

  • Embryo Cloning and

  • To Produce Designer Babies

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Different types of stem cells1
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

ESC versus ASC

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Methods of stem cell generation
Methods of Stem Cell Generation

IVF

SCNT

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Alternatives to embryonic stem cells from living human embryos
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 Embryos

Stem Cell Programming

‘RECIPE’

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Therapeutic applications of stem cells
Therapeutic Applications of Stem Cells Embryos

  • Regenerative medicine –

    permanent repair of failing organs - e.g.

    • Cardiomyocytes for heart disease

    • Angiogenesis in CAD – ‘Auto bypass’

    • Islet cells for diabetes

    • Neural cells for Parkinson’s

    • Blood cells for cancer

    • Chondrocytes for osteoporosis

    • Keratinocytes for burns

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Stem cell applications
Stem Cell Applications Embryos

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The promise of stem cell research
The Promise of Stem Cell Research Embryos

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Innovations of cell therapies
Innovations of Cell Therapies Embryos

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Cell therapies global over view
Cell Therapies – Global Over View Embryos

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Estimated market for ct versus it
Estimated Market for CT versus IT Embryos

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Electron micrographs of stem cells
Electron Micrographs of Stem Cells Embryos

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Programming of stem cells ex vivo
Programming of Stem Cells – ex vivo Embryos

EMG of Stem Cell

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Stem cells in cad
Stem Cells in CAD Embryos

Cardiomyogenesis

Coronary Angiogenesis

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The enormous power of the umbilical cord

Let us Understand Embryos

The Enormous Power of the Umbilical Cord

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Umbilical cord blood banking
Umbilical Cord Blood Banking Embryos

  • 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
UCSC Rx. for which diseases ? Embryos

  • 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
Promising Applications of UCSC Embryos

  • 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
The Placental Barrier Embryos

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Why use cord blood stem cells
Why Use Cord Blood Stem Cells ? Embryos

  • 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
Stem Cell therapy centres in India Embryos

  • 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 blood for stem cells
Storage of Umbilical Blood Embryosfor 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
Cord Blood Collection Embryos

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Thawing method
Thawing Method Embryos

  • 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
Cord Blood Preservation Embryos

Under liquid N2 at – 192o C

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Preparation
Preparation Embryos

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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Preparation1
Preparation Embryos

  • 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 2

Bag 3

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Preparation of wash bags
Preparation of Wash Bags Embryos

  • Transfer 125 mL of the Dextran/Albumin to Bag 2 and chill bags for approximately one hour in refrigerator.

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Retrieval
Retrieval Embryos

  • Remove from storage

    • Verify all label information with 2nd technologist

    • Detach segment and store

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Thawing
Thawing Embryos

  • Place the product inside a Ziploc bag, seal tightly, and submerge in the 37C waterbath until slushy.

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Connect to wash bags
Connect to Wash Bags Embryos

  • 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 2

Bag 2

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Diluting cells
Diluting Cells Embryos

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 cells1
Diluting Cells Embryos

  • 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 1

Bag 2

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First centrifugation
First Centrifugation Embryos

  • Heat seal and remove the CBU and Bag 1 (Dextran/Alb)

  • Centrifuge cells and remaining bag @ 880 x g for 20 minutes at 4C.

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Expressing supernatant
Expressing Supernatant Embryos

  • 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
Spinning the Supernatant Embryos

  • 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 4C.

  • Express the supernatant leaving approximately 10-20 mL of cell suspension in the original bag.

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Final pooling
Final Pooling Embryos

  • 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 Testing Embryos

  • QC Samples

    • Nucleated Cell

    • Hematocrit

    • Viability

    • ABO/Rh

    • CFU

    • CD34

    • Sterility

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Checking the cord blood for disease
Checking the Cord Blood for Disease Embryos

  • 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
Final Product Embryos

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Potential safety issues
Potential Safety Issues Embryos

  • Older stem cells can turn cancerous, e.g.

    • Human adipose stem cells in animals

    • Unexpected in adult stem cells

    • Problem 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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To learn more about the Embryos

Stem Cell Research visit

www.stemcells.nih.gov

National Marrow Donor Program

(NMDP) www.marrow.org

This presentation is

on the our website

www.drsarma.in

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International Stem Cell Forum Embryos

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
Cord Blood Banking Facilities Embryos

  • Public Cord Blood Banking

    • Reliance 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 by

    • LifeCell of ACCPL (Asia Cryo Cell P ltd)

      • Affiliate of Cryo-Cell International (USA)

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