Stem Cells

Stem Cells
- a recent approach
DR. D. SUDARSANAM
PROFESSOR
Dept. of Advance Zoology and Biotechnology
Loyola College, Chennai-34
STEM CELL UPDATE: TELLING HYPE FROM HOPE
Stem cells Outline
 Introduction
 Time line
 Types
 Embryonic stem cells (source)
 Adult stem cells - HSC, NSC, MSC (source)
 Culture
 Applications - General
 Therapeutics
 Medicine
 Dentistry
 Challenges
 Alternatives
 Current approaches
 iPS
Stem cells Introduction
 Self replicate, throughout the life of
the organism.
 Homeostatsis (renew & replenish old and
dead cells)
 Unspecialized, pluripotent cells
 Under physiological or experimental

conditions 
into any type (>250) of tissue / organ specific cell.
 In some organ (gut and bone marrow), stem cells regularly

divide to repair and replace worn out or damaged tissues. In
other organs (pancreas & heart), stem cells only divide under
special conditions.
Stem cells Introduction
In vitro differentiation of ES cells.

Undifferentiated mouse ES cells (A) develop in
vitro via three-dimensional aggregates (embryoid
body, (B) into differentiated cell types of all three
primary germ layers. Shown are differentiated cell
types labeled by tissue-specific antibodies (in
parentheses). C: cardiomyocytes (titin Z-band
epitope). D: skeletal muscle (titin Z-band epitope).
E: smooth muscle (smooth muscle -actin). F:
neuronal (III tubulin). G: glial (glial fibrillary
acidic protein, GFAP). H: epithelial cells
(cytokeratin 8). I: pancreatic endocrine cells
[insulin (red), C-peptide (green), insulin and C
peptide colabeling (yellow)]. K and L: hepatocytes
Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005)

Stem cells Time line
NATURE REVIEWS | GENETICS VOLUME 7 | APRIL 2006 |
James Thomson
University of Wisconsin-Madison, US isolated cells from the inner cell
mass of the early embryo, called the blastocyst, and developed the first
human Embryonic Stem (hES) cell lines. Isolation of human ES cells. James Thomson, et al.
in 1998 – Science 282:1145–1147 (1998)
Human ES stem cells & embryonic germ cells are

John Gearhart
different in some characteristics and are not the same
Johns Hopkins University, US reported the first derivation of human

embryonic germ cells from an isolated population of cells in fetal gonadal
tissue, known as the primordial germ cells, which are destined to become
the eggs and sperm.
Science 282:1145–1147 (1998)
Stem cells Types
Types of of stem cells from animals and humans:
 Embryonic stem cells (ES cells)
 somatic or adult stem cells (AS cells) : Non-embryonic
 Induced pluripotent stem cells (iPS cells) : since 2006
Adapted from Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005) & BD.
Types Stem cells
Embryonic stem (ES) cells Types
The embryonic stem (ES) cell is defined by its origin - from the
earliest stages of the development of the embryo – blastocyst.
Derived from the inner cell

mass of the blastocyst at a
stage before it would implant
in the uterine wall.
Pluripotent : it can give rise

to cells derived from all 3
germ layers.
Source : Mouse Source : Human
EG : Embryonic germ cell

Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005) ES : Embryonic stem cells
Examples for the in vitro differentiation capacity of
hES
mouse and human ES cells
mES

Adult stem cells Stem cells
 Undifferentiated, (unspecialized) cell that occurs in a
differentiated (specialized) tissue, renews itself, and becomes
specialized to yield all of the specialized cell types of the
tissue from which it originated.
 Self-renewal
 Multipotent (NOT PLURIPOTENT).
 Exhibit plasticity
 Adult stem cells usually divide  generate progenitor or

precursor cells, which then differentiate or develop 
“mature” cell.
 Plasticity is the ability of
an adult stem cell from one
tissue to generate the
specialized cell type(s) of
another tissue).
 Sources of adult stem cells
include
 bone marrow, blood,
the cornea and the
retina of the eye,
brain, skeletal muscle,
dental pulp, liver, skin,
the lining of the
gastrointestinal tract,
and pancreas.
Preliminary Evidence of Plasticity Among Nonhuman Adult Stem Cells.
Hematopoietic and Stromal Stem Cell Differentiation

Adult stem cells types Stem cells
 Hematopoietic
 Source: bone marrow  Differentiate  T cell, B cell,
Erythrocyte, etc.,
 Maintenance of undifferentiated HSCs  stem cell factor (SCF),
IL-3, IL-6.
 Differentiation  EPO, G-CSF
 Mesenchymal
 Source umbilical vein, bone marrow, adipose, tissue, human
embryonic stem cells
 Differentiate  osteogenic, chondrogenic, adipogenic cells
 Differentiation  FGF, EGF, ITS+ Premix, TGF-β,
Hydrocortisone
 Neuronal
 Source: brain cortices  neuron, astroglial
 Differentiation  FGF, BDNF
Culture Stem cells
 Traditional method – Mouse Embryonic Fibroblast (MEF) feeder layer
 Use MEF feeder layer as a substrate that provides essential growth and
attachment factors
 Alternative feeder layer method – human foreskin fibroblast feeder layer
 Undefined media and surface components, but no animal-derived factors
 Feeder-free hESC culture
 BD Matrigel™ or extracellular matrix (ECM) proteins are used as a
substrate or Matrigel hESC qualified matrix.
 May use conditioned media from MEFs or a defined media
Feeder layer cells Matrigel ES cells
ES cells
Molecular aspects Stem cells
Specific Molecular Markers

Molecular aspects Stem cells
Specific Molecular Markers
Ecat1, Dppa5(Esg1), Fbox15, Nanog, Eras, Dnmt31, Ecat8,

Gdf3, Sox15, Dppa4, Dppa2, Fthl17, Sall4, Oct3/4, Sox2,
Rex1, Utf1, Tcl1, Dppa3, Klf4

Applications Stem cells
With stem cells therapy we hope to achieve the following

 Improve the prognosis of diseases  quality of life
 Predictable therapeutic management
 Affordable
Applications Stem cells
Adapted from http://stemcells.nih.gov/index.asp

Therapeutics Medicine : Stem cells
 Transplant cells  tissues  organs
 Therapeutic delivery systems
 explored as a vehicle for delivering genes to specific
tissues in the body.
 Research
 exploration of the effects of chromosomal abnormalities in
early development
 Regenerative medicine
 Pancreas, liver, muscle, brain, etc
 Autoimmune disease
 Diabetes
 Neurological
 CVS
Therapeutic delivery systems
Autoimmune disease
 Rheumatoid arthritis, Systemic lupus erythematosis (lupus),
Type 1 diabetes, Multiple, sclerosis, Sjogren’s syndrome
and Inflammatory bowel disease.
 Current therapy : anti-inflammatory agents, steroids.
 hematopoietic stem cell therapy for lupus is to destroy the
mature, long-lived, and autoreactive immune cells and to
generate a new, properly functioning immune system.
 Autologous hematopoietic stem cell transplantation.
 Genetically Modified Stem Cells in Experimental 
reduced inflammatory response and inflammation induced
tissue damage.
 Diabetes
 Restore glucose-
responsive insulin-
secreting cells either
by transplantation of
stem cell-derived
cells
 Reprogramming of
existing cells.
Neuro-degenerative diseases
Spinal injury
Parkinson’s Disease : stem cell therapy
Heart Muscle Repair with Adult Stem Cells
When injected into the damaged wall of the ventricle, these cells led to the
formation of new cardiomyocytes, vascular endothelium, and smooth muscle
cells, thus generating de novo myocardium, including coronary arteries,
arterioles, and capillaries
NATURE REVIEWS | DRUG DISCOVERY VOLUME 6 | AUGUST 2007 |
• ANEMIAS & OTHER BLOOD CONDITIONS:
• Sickle cell anemia
• Sideroblastic anemia
• Aplastic anemia
• Red cell aplasia (failure of red blood cell development)
• Amegakaryocytic thrombocytopenia
• Thalassemia (genetic [inherited] disorders all of which
involve underproduction of hemoglobin)
• Primary amyloidosis (A disorder of plasma cells)
• Diamond blackfan anemia
• Fanconi’s anemia
• Chronic Epstein-Barr infection (similar to Mono)
AUTO-IMMUNE DISEASES:
• Systemic lupus (auto-immune condition that can affect skin, heart, lungs,
kidneys, joints, and nervous system)
• Sjogren’s syndrome (autoimmune disease w/ symptoms similar to arthritis)
• Myasthenia (An autoimmune neuromuscular disorder)
• Autoimmune cytopenia
• Scleromyxedema (skin condition) , Scleroderma (skin disorder)
• Crohn’s disease (chronic inflammatory disease of the intestines)
• Behcet’s disease
• Rheumatoid arthritis, Juvenile arthritis, Multiple sclerosis
• Polychondritis (chronic disorder of the cartilage)
• Systemic vasculitis (inflammation of the blood vessels)
• Alopecia universalis
• Buerger’s disease (limb vessel constriction, inflammation)
• Juvenile (Type-I) Diabetes
CARDIOVASCULAR:
• Acute Heart damage
• Chronic coronary artery disease
IMMUNODEFICIENCIES:
• Severe combined immunodeficiency syndrome
• X-linked lymphoproliferative syndrome
• X-linked hyper immunoglobulin M syndrome
LIVER DISEASE:
• Chronic liver failure
• Liver cirrhosis
NEURAL DEGENERATIVE DISEASES & INJURIES:

• Parkinson’s disease
• Spinal cord injury
• Stroke damage
OCULAR:
• Corneal regeneration
WOUNDS & INJURIES:

• Limb gangrene
• Surface wound healing
• Jawbone replacement
• Skull bone repair
OTHER METABOLIC DISORDERS:

• Hurler’s syndrome (hereditary genetic disorder)
• Osteogenesis imperfecta (bone/cartilage disorder)
• Krabbe Leukodystrophy (hereditary genetic disorder)
• Osteopetrosis (genetic bone disorder)
• Cerebral X-linked adrenoleukodystroph
CANCERS: • Cancer of the lymph nodes:

• Brain tumors—medulloblastoma and Angioimmunoblastic lymphadenopathy
glioma • Multiple myeloma (cancer affecting white
• Retinoblastoma (cancer) blood cells of the immune system)
• Ovarian cancer • Myelodysplasia (bone marrow disorder)
• Skin cancer: Merkel cell carcinoma • Breast cancer
• Testicular cancer • Neuroblastoma (childhood cancer of the
• Lymphoma nervous system)
• Non-Hodgkin’s lymphoma • Renal cell carcinoma (cancer of the kidney)
• Hodgkin’s lymphoma • Soft tissue sarcoma (malignant tumor that
• Acute lymphoblastic leukemia begins in the muscle, fat, fibrous tissue, blood
• Acute myelogenous leukemia vessels)
• Chronic myelogenous leukemia • Ewing’s sarcoma
• Chronic myelomonocytic leukemia • Various solid tumors
• Juvenile myelomonocytic leukemia • Waldenstrom’s macroglobulinemia (type of
lymphoma)
• Hemophagocytic lymphohistiocytosis
• POEMS syndrome (osteosclerotic myeloma)
• Myelofibrosis
Therapeutics Dentistry : Stem cells
Craniofacial Tissue Engineering by Stem Cells
 Biologically, mesenchymal cells are primarily responsible for the
formation of virtually all dental, oral, and craniofacial structures.
 Mesenchymal stem cells, the reservoir of mesenchymal cells in the adult,

have been demonstrated, in tissue engineering, to generate key dental, oral,
and craniofacial structures.
 The stem cell populations that generate native

craniofacial structures, such as the mandibular joint,
are heterogeneous and likely include both
mesenchymal and hematopoietic stem cells. J Dent Res.
2006 November ; 85(11): 966–979.
 The morphogenesis of the articular condyle requires

stem cells, chondrocytes, and osteoblasts in addition
to angiogenesis (Mao, 2005).
Source of stem cells from dento-alveolar tissues
 Recently, dental pulp stem cells (DPSCs) have been isolated

from extracted human third molars (Gronthos et al., 2000; Shi et al.,
2001).
 Stem Cells from Human Exfoliated Deciduous Teeth (SHED)
 A recent report identified stem cells in human PDL

(PDLSCs) and found that PDLSCs implanted into nude mice
generated cementum/ PDL-like structures that resemble the
native PDL as a thin layer of cementum that interfaced with
dense collagen fibers, similar to Sharpey’s fibers (Seo et al.,
2004).
 Stem cells from the apical part of the papilla (SCAP).
 Stem cells from the dental follicle (DFSC).

International Journal of Paediatric Dentistry 2009; 19: 61–70
 DPSCs were capable of forming ectopic dentin and associated pulp
tissue in vivo.
 Stromal-like cells were reestablished in culture from primary DPSC

transplants and retransplanted into immunocompromised mice to
generate a dentin-pulp-like tissue, demonstrating their self-renewal
capability.
 DPSCs were also found to be capable of differentiating into

adipocytes and neural-like cells.
 DPSC generated ectopic dentin in vivo,
J Dent Res 81(8) 2002

Two current ways of tooth
tissue engineering.
a Dissociated tooth germs grow

on a tooth-shaped scaffold and
produce small complex tooth-like
structures.
b Epithelial and mesenchymal

(stem) cells, either from tooth
germs or other sources, are
grown in organ culture and,
through epithelial-mesenchymal
interactions, can form organized
teeth
Cell Tissue Res (2008) 331:359–372 367

Construction of a bioengineered tooth. Stem cells for tooth formation in vitro
and ex vivo.
The association of tooth-derived stem cells with defined

scaffolds in the presence of growth factors allows the A tooth germ can be created in vitro after co-culture of
creation of tooth specific constructs such as crown and isolated epithelial and mesenchymal stem cells. This germ
root of missing parts of an injured tooth. These biological
could be implanted into the alveolar bone and finally
constructs could be used in dental clinics as substitutes develop into a fully functional tooth.
for metal implants, crowns and restorative dental
 TISSUE ENGINEERING OF THE TEMPOROMANDIBULAR JOINT
 PERIODONTAL TISSUE ENGINEERING
 TISSUE ENGINEERING OF CRANIOFACIAL BONE

 Adipose-derived Stem Cells Heal Calvarial Defects
 Craniofacial Bone Regeneration after Ablative Cancer Surgery and
Radiation Therapy in oral cancer patients.
Therapeutics Ophthalmology : Stem cells
EYES, A GREAT SOURCE OF STEM CELLS !

CORNEAL TRANSPLANTS FAIL IF

NO LIMBAL CELLS
“In 10 years, we will
have eliminated the
need for cell therapies.
If we find the signals to
regenerate the cornea,
we can deliver them in
drops.”
Sajjad Aahmad
Northeast England Stem Cell
Institute
Retinal pigment epithelium

• From vitrectomy +
cadavers (no mice)
• Normally RPE yields 6
types of retinal cells
• ES medium yields all 3
germ layers.
• Divide in culture up to
11 times.
• In clinical trials to treat
Parkinson’s disease.
In a world-first breakthrough by Australian scientists, the

eyesight in a damaged eye has been restored by using stem
cells from the eye itself.
The medical researchers from UNSW's School of Medical Sciences used

stem cells cultured on a simple contact lens to restore sight to sufferers
of blinding corneal disease and they say within weeks of the simple,
inexpensive procedure, sight was significantly improved and only a
minimal hospital stay was required.
The research team harvested stem cells from patients' own eyes which
were then cultured on a common therapeutic contact lens - this was then
placed onto the damaged cornea for 10 days and the cells were able to
re-colonise the damaged eye surface and rehabilitated the damaged
cornea.
The trial was conducted on three patients;
 two with extensive corneal damage resulting from multiple

surgeries to remove ocular melanomas, and
one with the genetic eye condition aniridia (a congenital condition

affecting both eyes) - corneas can also be damaged by chemical or
thermal burns, bacterial infection and chemotherapy.
Challenges Stem cells
 Culture / Source
 Simulating physiological conditions in-vitro.
 Standardized protocol - world wide acceptance
 Time consuming : characterization new culture conditions
 Spontaneous differentiation.
 Efficiency in transfection of hES cells.
 Virus, bacterial, fungal, mycoplasma contamination
 Xenotransplantation issues – due to the use of mouse feeder cells
 Mass production – specific
 Comparing data from different laboratories
 Safety – use of ES cells / differentiated ES cells for therapy in
humans
 Further the understanding of stem cell biology.
 hES cells – chromosomal abnormalities – in culture
 Predictable and controlled differentiation in side humans
 Ethical considerations
Alternatives Stem cells
Somatic cell nuclear transfer (SCNT)
The nucleus from an adult cell is transferred to an enucleated
egg (the nucleus was removed)
 Advantages over ES cells
 No embryo derived cells
 Well characterized system in mice
 No Federal funding restrictions
 Potential for generating stem cells from any individual
 Disadvantages
 Limited success in primates
 Human egg donation
 Labor intensive
 In some organ (gut and bone marrow), stem cells regularly divide
to repair and replace worn out or damaged tissues. In other organs
(pancreas & heart), stem cells only divide under special conditions.
Alternatives Stem cells
Induced pluripotent stem cells (iPS)
Human cells are infected with genes that make them behave like
hES cells
 Advantages over ES cells
 No embryo derived cells
 Adult cells only
 No Federal funding restrictions
 Potential for generating stem cells from any individual
 Challenges
 New : the potentials are being researched.
 The infection process makes the genes integrate randomly into the
DNA. (Potential for cancer in clinical applications.)
Current approaches iPS cells
Principal strategies to obtain human iPS cells.
A. Rolletschek and A.M. Wobus 2009

Improved knowledge on STEM CELLS
Efficient & predictable therapeutics

& improved prognosis
Improved quality of life

Stem Cells - A Recent Approach: Dr. D. Sudarsanam Professor

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 Under physiological or experimental

into any type (>250) of tissue / organ specific cell.

 In some organ (gut and bone marrow), stem cells regularly

In vitro differentiation of ES cells.

Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005)

NATURE REVIEWS | GENETICS VOLUME 7 | APRIL 2006 |

Human ES stem cells & embryonic germ cells are

Johns Hopkins University, US reported the first derivation of human

Derived from the inner cell

Pluripotent : it can give rise

EG : Embryonic germ cell

Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005)

 Multipotent (NOT PLURIPOTENT).

 Adult stem cells usually divide  generate progenitor or

Hematopoietic and Stromal Stem Cell Differentiation

Feeder layer cells Matrigel ES cells

Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005)

Ecat1, Dppa5(Esg1), Fbox15, Nanog, Eras, Dnmt31, Ecat8,

Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85:635-678 (2005)

With stem cells therapy we hope to achieve the following

Adapted from http://stemcells.nih.gov/index.asp

NEURAL DEGENERATIVE DISEASES & INJURIES:

WOUNDS & INJURIES:

OTHER METABOLIC DISORDERS:

CANCERS: • Cancer of the lymph nodes:

 Mesenchymal stem cells, the reservoir of mesenchymal cells in the adult,

 The stem cell populations that generate native

 The morphogenesis of the articular condyle requires

 Recently, dental pulp stem cells (DPSCs) have been isolated

 Stem Cells from Human Exfoliated Deciduous Teeth (SHED)

 A recent report identified stem cells in human PDL

 Stem cells from the apical part of the papilla (SCAP).

 Stem cells from the dental follicle (DFSC).

 Stromal-like cells were reestablished in culture from primary DPSC

 DPSCs were also found to be capable of differentiating into

 DPSC generated ectopic dentin in vivo,

J Dent Res 81(8) 2002

a Dissociated tooth germs grow

b Epithelial and mesenchymal

Cell Tissue Res (2008) 331:359–372 367

The association of tooth-derived stem cells with defined

 PERIODONTAL TISSUE ENGINEERING

 TISSUE ENGINEERING OF CRANIOFACIAL BONE

EYES, A GREAT SOURCE OF STEM CELLS !

CORNEAL TRANSPLANTS FAIL IF

Retinal pigment epithelium

In a world-first breakthrough by Australian scientists, the

The medical researchers from UNSW's School of Medical Sciences used

 two with extensive corneal damage resulting from multiple

one with the genetic eye condition aniridia (a congenital condition

A. Rolletschek and A.M. Wobus 2009

Efficient & predictable therapeutics

Improved quality of life

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