www.drsarma.in 1. 2 2 dr r v s n sarma md msc consultant physician & chest specialist stem cell...
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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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