Stem cell therapy, Basics , History of First stem cell product.

1. Stem Cells
SUDARSAN AGARWAL
sushiagarwal@gmail.com
Pinnamineni medical college, vijayawada

2. Definition of Stem Cells
• A cell that has the ability to continuously divide and
differentiate (develop) into various other kind(s) of cells/tissues
• Biological cells found in all multicellular organisms.
• Blank or Unspecialized and Undifferentiated cells.

3. What can be done with Stem Cells?
• Have immune potential and can help to
treat a wide range of Medical problems.
• Discovery of stem cells lead to a whole new
branch of medicine known as …..
Regenerative medicine

4. Historical perspectives
• 1908: The term "stem cell" was coined by Alexander Maksimov
• 1963: McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow.
• 1968: Bone marrow transplant between two siblings successfully treats SCID.
• 1978: Haematopoietic stem cells are discovered in human cord blood.
• 2000: Several reports of adult stem cell plasticity are published.
• 2007: The Nobel Prize was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver
Smithies "for their discoveries of principles for introducing specific gene modifications in
mice by the use of embryonic stem cells".
• 2008: Development of human cloned blastocysts following somatic cell nuclear transfer with adult
fibroblasts
• 2012: The Nobel Prize was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the
discovery that mature cells can be reprogrammed to become pluripotent"

5. Mouse embryonic stem cells with fluorescent marker

6. Human embryonic stem cell colony on mouse
embryonic fibroblast feeder layer

7. Human embryonic stem cells
Cell colonies that
are not yet
differentiated
Nerve cell

8. Properties of Stem Cells
• Self.renewal.Can regenerate into
only the specific tissue from which
they are isolated (Proliferation
and renewal)
• Potency (ES cells) : Capacity to
differentiate into specialized cell
types of stem cells.

9. Properties of Stem Cells
• Totipotent – Can differentiate into embryonic and
extra embryonic cell types. Can construct a
complete viable organism.
Ex: Cell produced
by fusion of an egg and
sperm
• Pluripotent – Can differentiate into nearly all
cells, cells derived from any of the three germ
layers.
• Multipotent : Can differentiate into a number of
cells, but only those of a closely related family of
cells.
• Oligopotent : Can differentiate into only a few
cells, such as lymphoid or myeloid stem cells.
• Unipotent : Can produce only one cell type of their
own, property of self renewal.

10. Stem cell division
and
Differentiation
1: Symmetric stem cell division
2: Asymmetric stem cell division
3: Progenitor division
4: Terminal differentiation
A: Stem
Cell
B: Progenitor C: Differentiated
Cell
Cell

11. Types of Cultured Stem Cells
• Embryonic stem cells
• Embryonic germ cells
• Trophoblast stem cells
• Extra embryonic endoderm cells
• Embryonic carcinoma cells
• Mesenchymal stem cells
• Epistem cells
• Multipotent adult stem cells
• Spermatogonial stem cells
• Germ line stem cells
• Neural stem cells
• Unrestricted somatic stem cells
• Multipotent adult germ line stem
cells
• Induced pluripotent stem cells

13. Embryonic Stem Cells
• Derived from inner cell mass of a
blastocyst or earlier morula stage.
• Blastocyst is an early stage
embryo.
• 4 – 5 days old – 50 – 150 cells.
• „ES‟ cells – Pluripotent – gives rise
all three primary germ layers.
• Do not contribute to extra
embryonic membranes or the
placenta.

14. Adult Stem Cells
• It is undifferentiated cells found among
differentiated cells in a tissue or organ after birth.
• Do not disappear following birth.
• Remain, play a role in the recovery of damaged
tissue.
• Decreased reserve and loss of vitality as with age.
• Adult stem cells can also develop into other types
of cells and are mostly recovered by bone
marrow.
• The use of Adult stem cell in research is not
controversial as the use of „ES‟ cell, as it does not
require the destruction of an embryo.

15. Adult Stem cells
Normal differentiation
• Hematopoietic stem
cells – give rise to all
types of blood cells.
• Epithelial stem cells – in
GIT – Absorptive
cells, goblet
cells, entero endocrine
cells.
• Skin stem cells.
Adult Stem Cells Plasticity/
Trans Differentiation
• Ability to differentiate into multiple cell
types is called plasticity.
• Haemopoietic stem cells – differentiate
into Brain
cells, neuron, oligodendrocytes, skelet
al muscle cells, cardiac muscle
cells, liver cells etc.,
• Bone Marrow Stem cells – into
cardiac, skeletal or muscle cells.
• Brain stem cells – into Blood cells and
skeletal muscle cells.

16. PROS AND CONS OF EACH TYPE
EMBRYONIC
•
•
•
•
CELL LINES LAST LONG
MULTIPOTENT
EASY TO FIND
ETHICAL ISSUES - WHEN
DOES LIFE BEGIN?
ADULT
•
•
•
•
CELL LINES DO NOT LAST
NOT MULTIPOTENT
HARD TO LOCATE
NO ETHICAL ISSUES

17. Nuclear reprogramming
• Development naturally progresses from totipotent fertilized eggs to pluripotent
epiblast cells, to multipotent cells, and finally to terminally differentiated cells.
• According to Waddington's epigenetic landscape, this is analogous to a ball
moving down a slope.
• The reversal of the terminally differentiated cells to totipotent or pluripotent cells
(called nuclear reprogramming) can thus be seen as an uphill gradient that never
occurs in normal conditions.

18. Nuclear reprogramming
• However, nuclear reprogramming has been achieved using nuclear
transplantation, or nuclear transfer (NT), procedures (often called
"cloning"), where the nucleus of a differentiated cell is transferred into an
enucleated oocyte

19. DOLLY
(5 July 1996 – 14 February 2003)

20. Dolly
Sex
Born
Died
Female
5 July 1996 (Roslin Institute)
14 February 2003 (aged 6)
Nation
United Kingdom
Known for
First mammal to be cloned from an adult somatic cell
Offspring
6 lambs
Named after Dolly Patron

21. Keith Campbell
Dolly
Ian Wilmut

22. • Dolly the sheep, first mammal to
be cloned from an adult somatic
cell
• Even though Dolly was not the
first animal to be cloned, she
gained this attention in the
media because she was the first
to be cloned from an adult cell

23. Somatic
Cell
Nuclear
Transfer

24. • In Dolly, nucleus was
transferred came from
mammary gland cells from a
6-year-old ewe
• In Polly & molly
fibroblast cells were used

25. Species cloned
•
•
•
•
Tadpole: (1952)
Carp: (1963)
Mice: (1986)
Sheep: first mammal being cloned
(1984) from early embryonic cells.
Megan and Morag from
differentiated embryonic cells
(1995)
Dolly from a somatic cell (1996)
• Rhesus Monkey: (2001)
• Cattle: (2001)
• Rat:
• Mule:
• Horse:
• Dog:
• Wolf:
• Water Buffalo:
• Pyrenean Ibex (2009) was the first
"extinct" animal cloned
• Camel: (2009)
• Pashmina goat: (2012) Kashmir
, India

26. Human
cloning

27. Human cloning
• Human cloning is the creation of a genetically identical copy of
an existing or previously existing human.
• The term is generally used to refer to artificial human cloning;
human clones in the form of identical twins are commonplace,
with their cloning occurring during the natural process of
reproduction.

28. • Therapeutic cloning involves cloning adult cells for use in
medicine and is an active area of research.
• Reproductive cloning would involve making cloned humans. A
third type of cloning called replacement cloning is a theoretical
possibility, and would be a combination of therapeutic and
reproductive cloning.
• Replacement cloning would entail the replacement of an
extensively damaged, failed, or failing body through cloning
followed by whole or partial brain transplant

30. Induced
Pluripotent stem
cells

31. Induced Pluripotent Stem Cells
• Not adult stem cells.
• Reprogrammed cells (Ex. Epithelial cells) will have pluripotent capabilities.
• Using genetic reprogramming with protein transcription factors, pluripotent stem
cells equivalent to ES cells have been derived from human adult skin tissue.
• Little is known about factors that induce this reprogramming.
• Induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by
introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture
conditions.

32. Nobel prize in physiology or medicine 2012 for the discovery that
“mature cells can be reprogrammed to become pluripotent"
John B Gurdon
Shinya Yamanaka

33. Reprogramming cells

34. Induced Pluripotent Stem Cells

35. Stem Cells
APPLICATIONS

37. Tissue Repair
Regenerate spinal cord, heart tissue or any other major tissue
in the body.

38. Heart Disease
Adult bone marrow stem cells injected into the hearts are believed
to improve cardiac function in victims of heart failure or heart
attack

39. Leukemia and Cancer
• Studies show leukemia patients treated with stem cells
emerge free of disease.
• Injections of stem cells have also reduces pancreatic
cancers in some patients.
Proliferation of white cells

40. Rheumatoid Arthritis
Adult Stem Cells may be helpful in jumpstarting repair of
eroded cartilage.

41. Parkinson's disease
Major motor features of disorder results from the loss of a
single cell population i.e., dopaminergic neurons with in
stratum nigra. This suggests that cell replacement should
be adequate to treat it.

42. Type I Diabetes
• Pancreatic cells do not produce insulin
• Embryonic Stems Cells might be trained to become pancreatic
islets cells needed to secrete insulin.

43. Diseases that are treated by stem cells are:
1) Acute Leukemia
• Acute Lymphoblast Leukemia (ALL)
• Acute Myelogenous Leukemia (AML)
• Acute Biphenotypic Leukemia
• Acute Undifferentiated Leukemia
2) Chronic Leukemia
• Chronic Myelogenous Leukemia (CML)
• Chronic Lymphocytic Leukemia (CLL)
• Juvenile Chronic Myelogenous Leukemia (JCML)
• Juvenile Myelomonocytic Leukemia (JMML)
Syndromes
• Myelodysplastic Syndromes
• Amyloidosis
• Chronic Myelomonocytic Leukemia (CMML)
• Refractory Anemia (RA)
• Refractory Anemia with Excess Blasts (RAEB)
• Refractory Anemia with Excess Blasts in Transformation
• (RAEB-T)
• Refractory Anemia with Ringed Sideroblasts (RARS)

44. Disorders
1) Stem Cell Disorders
• Aplastic Anemia (Severe)
• Fanconi Anemia
• Paroxysmal Nocturnal Hemoglobinuria
• Congenital Cytopenia
• Dyskeratosis Congenita
2) Myeloproliferative Disorders
• Acute Myelofibrosis
• Agnogenic Myeloid Metaplasia
• Polycythemia Vera
• Essential Thrombocythemia
3) Lymphoproliferative Disorders
• Non-Hodgkin’s Lymphoma
• Hodgkin’s disease
• Prolymphocytic Leukemia
4) Phagocyte Disorders
• Chediak-Higashi Syndrome
• Chronic Granulomatous Disease
• Neutrophil Actin Deficiency
• Reticular Dysgenesis
5) Inherited Metabolic Disorders
• Mucopolysaccharidoses (MPS)
• Hurler’s Syndrome (MPS-IH)
• Scheie Syndrome (MPS-IS)
• Hunter’s Syndrome (MPS-II)
• Sanfilippo Syndrome (MPS-III)
• Morquio Syndrome (MPS-IV)
• Maroteaux-Lamy Syndrome (MPS-VI)
• Sly Syndrome, Beta-Glucuronidase Deficiency
• Adrenoleukodystrophy
• Mucolipidosis II (I-cell Disease)
• Krabbe Disease
• Gaucher’s Disease
• Niemann-Pick Disease
• Wolman Disease
• Metachromatic Leukodystrophy
6) Histiocytic Disorders
• Familial Erythrophagocytic Lymphohistiocytosis
• Histiocytosis-X
• Hemophagocytosis
• Langerhans’ Cell Histiocytosis

45. 7) Inherited Immune System Disorders
• Ataxia-Telangiectasia
• Kostmann Syndrome
• Leukocyte Adhesion Deficiency
• DiGeorge Syndrome
• Bare Lymphocyte Syndrome
• Omenn’s Syndrome
• Severe Combined Immunodeficiency
• SCID with Adenosine Deaminase Deficiency
• Absence of T & B Cells SCID
• Absence of T Cells, Normal B Cell SCID
• Common Variable Immunodeficiency
• Wiskott-Aldrich Syndrome
• X-Linked Lymphoproliferative Disorder
Other Inherited Disorders
• Lesch-Nyhan Syndrome
• Cartilage-Hair Hypoplasia
• Glanzmann Thrombasthenia
• Osteopetrosis
• Adrenoleukodystrophy
• Ceroid Lipofuscinosis
• Congenital Erythropoietic Porphyria
• Sandhoff Disease
9) Plasma Cell Disorders
• Multiple Myeloma
• Plasma Cell Leukemia
• Waldenstrom’s Macroglobulinemia
• Amyloidosis
Abnormalities
1) Inherited Platelet Abnormalities
Congenital Thrombocytopenia
2) Inherited Erythrocyte Abnormalities
• Beta Thalassemia Major
• Sickle Cell Disease
• Blackfan-Diamond Anemia
• Pure Red Cell Aplasia
Other Malignancies
• Ewing Sarcoma
• Neuroblastoma
• Renal Cell Carcinoma
• Retinoblastoma
• Brain tumor
• Ovarian Cancer
• Small Cell Lung Cancer
• Testicular Cancer

46. Strategies for transplantation of stem cells
1. Undifferentiated or partially differentiated stem cells may be
injected directly in the target organ or intravenously.(HSC)
2. Stem cells may be differentiated ex vivo prior to injection into
the target organ. (Beta cells, Cardiomyocyte)
3. Growth factors or other drugs may be injected to stimulate
endogenous stem cell populations(EPO, GM-CSF)

48. HEMATOPOITIC CELL
TRANSPLANTATION

49. Bone marrow transplantation was the original term used to
describe the collection and transplantation of hematopoietic stem
cells, but with the demonstration that the peripheral blood and
umbilical cord blood are also useful sources of stem
cells, hematopoietic cell transplantation has become the
preferred generic term for this process.

50. Properties of Hematopoietic stem cells
• Dies
• Self renewal
• Potency
• Plasticity
-- Apoptosis
-- Stem cell
-- Differentiated blood cells(Differentiation)
-- Neurons/ Germ cells( Trans Differentiation)

51. The procedure is usually carried out for one of two purposes:
(1) to replace an abnormal but nonmalignant lymphohematopoietic system with one from a normal donor, or
(2) to treat malignancy by allowing the administration of higher
doses of myelo suppressive therapy than would otherwise be
possible
The time it takes for hematopoietic progenitors to become mature
cells is ~10–14 days in humans,

52. In humans, transplantation replaces recipient's entire lymphohematopoietic system, including
1. all red cells,
2. granulocytes,
3. B and T lymphocytes,
4. platelets,
5. Kupffer cells of the liver,
6. pulmonary alveolar macrophages,
7. osteoclasts,
8. Langerhans cells of the skin, and
9. brain microglial cells.

53. Pre transplant
• Whole Body Irradiation to remove endogenous immune system and tumor cells
• Injection of bone marrow from a well matched donor to re-establish immune
system
• Regulation of immune response to prevent graft versus host reaction.
• Autologous donation possible if one can purify and remove tumor cells, enriching
for stem cells..
• Allogeneic donors have advantage of graft versus tumor reaction to kill any
remaining tumor cells.
• Allogeneic donors have the disadvantage of graft versus host reaction if they are
not well matched.

54. Purify from
Tumor cells
Unfractionated Bone marrow
Transplant
Transplant
Donor blood
formation, graft-vstumor effect

55. Complications of Allogeneic Transplants
• Regimen related toxicity
• Infectious complications
• Engraftment failure (resistance)
• Graft-versus-host disease
• Transplant related mortality = 10 - 15%

57. Bone Marrow / Ph Blood
• Found in spongy bone where blood cells form
• Bone marrow aspirated from the posterior and anterior iliac crests has
traditionally been the source of hematopoietic stem cells for transplantation.
Typically, anywhere from 1.5 to 5 x 108 nucleated marrow cells per kilogram
• Used to replace damaged or destroyed bone marrow with healthy bone
marrow stem cells.
• Hematopoietic stem cells circulate in the peripheral blood but in very low
concentrations.
• Following the administration of certain hematopoietic growth factors, (EPO,GCSF,GM-CSF) , the concentration of hematopoietic progenitor cells in blood
increases markedly.

58. Umbilical cord Blood Stem Cells
• Also Known as Wharton‟s Jelly
• Adult stem cells of infant origin
• Less invasive than bone marrow
• Greater compatibility
• Less expensive
• Obtained from cord immediately after birth.
• Rich source of haemopoietic stem cells
• These are referred to as neonatal stem cells, less mature than those found in adults
bone marrow..
Three important functions:
1. Plasticity: Potential to change into other cell types like nerve cells
2. Homing: To travel to the site of tissue damage
3. Engraftment: To unite with other tissues

59. Benefits of banking
• Cord blood stem cells are not just for the baby, but may also helps
the whole family
• Siblings have a 25% chance of being a suitable match.
• Stem cell transplants are twice as successful when the stem cells
come from a family member rather than from a non-relative. (63%
vs. 29%)
• Once in a life time opportunity to preserve a biological resource
that could be a lifesaver for the child/ other family members.
• A lifesaving alternative to bone marrow transplants.
• The lower probability of graft Vs. host disease (GVHD) and a
greater likelihood of finding an appropriate tissue type match.

60. Cord Blood Processing
1. HLA and Infectious Disease testing.
2. Removal of RBCs
3. Addition of Dimethyl sulphoxide and Dextran.
4. Enrichment and reduction of volume to 25ml.
5. Controlled rate freezing.
6. Cryogenic storage.(Liquid nitrogen at -1960 C)

61. Process of collection and storage
• Collection is same, in vaginal or in caesarean.
• Can be done before or imm., after the delivery of the placenta.
• Procedure is short (<10 mts)
• Sterilize the umbilical cord with iodine & alcohol swabs
• Sterile needle of the bag is inserted into the maternal side of the
cord & blood is allowed to flow by gravity into bag.
• Tubing emerging from the bag is striped, clamped and sealed/
knotted twice.
• The cord blood bag is packed between the two flaps of the gel
packs to maintain the temperature

62. BONE MARROW/PERIPHERAL BLOOD
Umbilical CORD BLOOD
Requires surgery under general anesthesia. It‟s a painful and
tedious process.
Obtained from the delivered placenta and umbilical
cord. It‟s a painless procedure
Requires a quart or more of bone marrow for transplant
A few ounces can be used for transplantation.
Large dose of stem cells. Rapid engraftment.
Smaller dose of stem cells. Slower engraftment.
After a formal search is begun, takes an average of 4 months to
transplant, even if a donor is available.
When a match is found, can take only few hours for
the confirmatory and special tests.
The fate of the recipient depends on the will of donor.
Stem cells once stored is available
Shelf life ranges from hours(bone marrow) to few
months(peripheral blood).
Long periods
Latent viral infection in the donor common (CMV>50% in U.S)
Rare (iCMV<1% in U.S.)
Severe graft Vs. host disease (GvHD) common.
Rare
Generally requires a perfect match between donor and recipient
for 6/6 HLA-A, -B and –DRBI antigens. Additional HLA factors
(HLA-C, -DQ and –DP) increasingly used to improve prognosis.
HLA mismatched cord blood transplants are
possible, making it easier to find a suitable match.

63. Tissue engineering

64. Tissue engineering
• Tissue engineering has broad goals including
1. Organ development
2. Elimination of waiting time for transplants
3. Creation of living tissue replacements

65. • In the past several decades, the limitations of non living mechanical solutions to
organ and tissue dysfunction are now recognized and include …
1. Dialysis,
2. Mechanical heart valves,
3. Metallic orthopedic implants,
4. Non re-absorbable hernia mesh.
• Current research is focused on
1. Resorbable synthetic polymers (polyglycolic acid, polyurethanes,
polyglycerol)
2. Naturally occurring polymers(collagen, fibrin)
3. Minerals ( calcium triphosphate)

66. • The principal mechanical supporting
structure of any engineered tissue is the
SCAFFOLD.
• The ideal scaffold materials for engineered
tissues are Resorbable materials that
break down over time. During resorption,
the engineered tissue is remodeled by
normal healing processes, leaving only
living cellular tissue with natural supporting
connective tissue.
• Engineered skin substitutes were the first
true clinical success of tissue engineering.

68. Key Ethical Issues
• The blastocyst used in stem cell research is microscopically small and has no
nervous system. Does it count as a “person” who has a right to life?
• What do various religions say about when personhood begins? Does science
have a view on this?
• Humans Playing God ?

69. References
• http://stemcells.nih.gov/info/scireport/2001report.htm
• http://www.ulb.ac.be/sciences/biodic/biodic/images/bio_animale/embryologie/fecondation/baefec_01_01.j
pg
• http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.3748
• http://www.news.wisc.edu/packages/stemcells/illustration.html
• http://www.drugs.com/enc/images/images/en/17010.jpg
• http://embryology.med.unsw.edu.au/Notes/placenta.htm
• http://www.dnalc.org/stemcells.html
• http://gslc.genetics.utah.edu/units/stemcells/
• http://en.wikipedia.org/wiki/Main_Page
• Harrison Internal Medicine Edition 18
• Robbins Pathology Edition 8

70. !!! Thank You !!!