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Stem Cell Research by Dr Sarma

Stem Cell Research by Dr Sarma

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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
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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 ? www.drsarma.in  Where are we now ?

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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 www.drsarma.in or uncommitted cells such as stem
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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)
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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 www.drsarma.in
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Hectic Competition

The ultimate in love
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Week 1 of Gestation
2 cell stage Day 2 4 cell stage Late Day 2 Morula Day 3 Blastocyst Day 5

Fertilization Day 1

Implantation Day 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

Uterine Artery Uterine Gland Syncitiotrophoblsat Blastocystic Cavity Uterine Epithelium
www.drsarma.in Cytotrophoblast

Inner Cell Mass

Syncytiotrophoblast

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Week 2: Early Development
Cytotrophoblast Amniotic Cavity Amnioblasts Inner Cell Mass : Epiblast Hypoblast 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 ?
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 Can they be made to become pluripotent

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ESC versus ASC
Adult Stem Cells (ASC)  Limited developmental potential  Potential for undesired  Better behaved, easier to manage development  Lose their ability to (teratomas) proliferate or  Infinite lifespan, differentiate after a unlimited supply time in culture  High ethical burden  Less moral ambiguity  Uncertain legal status  Less legal controversy Embryonic Stem Cell (ESC)  High malleability
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Methods of Stem Cell Generation

IVF SCNT

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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 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

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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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EMG of Stem Cell

Programming of Stem Cells – ex vivo

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Stem Cells in CAD

Cardiomyogenesis
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Coronary Angiogenesis
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Let us Understand

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The Enormous Power of the Umbilical Cord37 37

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 www.drsarma.in match
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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 www.drsarma.in

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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 Blood for 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 2

Bag 3
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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 37°C 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 22 Bag

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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

Bag 1

Bag 2

The total volume in the Bag 2 should be approximately 200 mL www.drsarma.in 

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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 4°C.

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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

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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 4°C. Express the supernatant leaving approximately 10-20 mL of cell suspension in

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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 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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International regulations regarding embryonic stem cell research
A: Prohibition B: Utilisation C: Utilisation and extraction A ustralia Canada Greece Japan Spain Sweden The Netherlands France D: Utilisation, extraction and creation Singapore UK Israel China

Austria Brazil Costa Rica Denmark Equador Ireland Peru Poland

Germany USA

[Taken from Towns C.R. Embryonic Ste Ce Re arch: Unde m ll se rstanding and Analysis at the Inte rface of Scie , Me s nce dicine and Ethics. PhD Thesis, University of Otago, 2222 ]
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American Association for Advancement of Science

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To learn more about the 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
http://stemcellforum.org Currently representatives from the following countries: • Japan •
Australia • Canada • Czech Republic • Finland • France • Germany • Israel • 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 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 www.drsarma.in by

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