slide1 n.
Skip this Video
Loading SlideShow in 5 Seconds..
Dr R V S N Sarma MD MSc Consultant Physician & Chest Specialist PowerPoint Presentation
Download Presentation
Dr R V S N Sarma MD MSc Consultant Physician & Chest Specialist

Loading in 2 Seconds...

play fullscreen
1 / 73

Dr R V S N Sarma MD MSc Consultant Physician & Chest Specialist - PowerPoint PPT Presentation

  • Uploaded on

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. The Outline of Discussion. What are Stem Cells ? Why Stem Cells ? What are the sources of Stem Cells (SC) ?

I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
Download Presentation

PowerPoint Slideshow about 'Dr R V S N Sarma MD MSc Consultant Physician & Chest Specialist' - kin

Download Now An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
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

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 –

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.

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

hierarchy of stem cells
Hierarchy of Stem Cells


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

hectic competition
Hectic Competition

The ultimate in love

week 1 of gestation

4 cell stage

Late Day 2


Day 3

2 cell stage

Day 2


Day 5


Day 6 - 7


Day 1

Week 1 of Gestation

the blastocyst day 5
The Blastocyst – Day 5

The Blastocyst consists of

Inner Cell Mass




week 2 of gestation day 7 5
Week 2 of Gestation - Day 7.5

Uterine Artery

Uterine Gland




Uterine Epithelium

Inner Cell Mass



week 2 early development
Week 2: Early Development


Amniotic Cavity


Inner Cell Mass :



Primary Yolk Sac




sexual reproduction
Sexual Reproduction


germline engineering
Germline Engineering
  • Combines the application of
  • Stem Cell use
  • Genetic Engineering
  • Embryo Cloning and
  • To Produce Designer Babies

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

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

methods of stem cell generation
Methods of Stem Cell Generation



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

therapeutic applications of stem cells
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

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

EMG of Stem Cell

stem cells in cad
Stem Cells in CAD


Coronary Angiogenesis

umbilical cord blood banking
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

ucsc rx for which diseases
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

promising applications of ucsc
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

the placental barrier
The Placental Barrier

why use cord blood stem cells
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

stem cell therapy centres in india
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

storage of umbilical blood for stem cells
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

cord blood collection
Cord Blood Collection

thawing method
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

cord blood preservation
Cord Blood Preservation

Under liquid N2 at – 192o C


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.

  • 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

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

  • Remove from storage
    • Verify all label information with 2nd technologist
    • Detach segment and store

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

connect to wash bags
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 2

Bag 2

diluting cells
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

diluting cells1
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 1

Bag 2

first centrifugation
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.

expressing supernatant
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

spinning the supernatant
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 the original bag.

final pooling
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)

qc testing
QC Testing
  • QC Samples
    • Nucleated Cell
    • Hematocrit
    • Viability
    • ABO/Rh
    • CFU
    • CD34
    • Sterility

checking the cord blood for disease
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

final product
Final Product

potential safety issues
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

To learn more about the

Stem Cell Research visit

National Marrow Donor Program


This presentation is

on the our website


International Stem Cell Forum

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

cord blood banking facilities
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 by
    • LifeCell of ACCPL (Asia Cryo Cell P ltd)
      • Affiliate of Cryo-Cell International (USA)