Embyonal Stem Cells - Properties and Classification

Embryonal Stem Cells
Dr. Reenaz Shaik
MD PATHOLOGY

The Battle between Mind and Hearts
Ethics and embryos
Success stories

Contents
 Stem cells - Introduction
 Features of Stem cells
 Properties
 Classification
 Embryonic Stem cells
How to derive them
Advantages and disadvantages of embryonic stem cells
Difference between adult and embryonic stem cells
Embryonic Stem cell markers

Stem Cells Introduction
 Cells having the capability of self-renewal and differentiation are called Stem
cells.
 Self-renewal: Ability of cells to proliferate without the loss of differentiation
potential and without undergoing senescence.
 Differentiation: Ability to give rise to multiple tissue types such as a skin, muscle,
or nerve cell.
 Plasticity: Ability to differentiate into cell types beyond those of the tissues in
which they normally reside.
 A stem cell is essentially the building block of the human body.

Features of Stem cells
 Unique
 Ability to develop into several distinct cell types in the body.
 Repair system for the body.
 Replenish the lost cells.
 Divides to remain as a stem cell or have a specialized function.

Properties: Unspecialized and Uncommitted
 Capable of dividing and renewing themselves for long periods.
 Unspecialised: They can give rise to specialized cell types.
 Uncommitted: Until it receives a signal to develop into a specialized cell.

Classification
Stem Cells
Based on Source
of origin
Embryonic
Adult
Based on Potency
Totipotent
Pluripotent
Multipotent
Unipotent

Potency of Stem cell
 Based on the number of types of differentiated cells that one stem cell can make.
Totipotent cells: Produces all cell types : embryonic and extraembryonic (placenta) cells.
Pluripotent cells: Can only make cells from any of the germ layers.
Multipotent cells: Can only make cells within a given germ layer.
Unipotent cells: Can make only cells of a single cell type.

Source of Origin
 Embryonic Stem cells: Pluripotent stem cells derived from the inner cell mass
of a blastocyst, an early stage preimplantation embryo.
 Adult Stem cells: Undifferentiated cells which are found among differentiated
cells in a tissue or organ. Can form major specialized cell types of the tissue or
organ.

Embryonic Stem cells
 Derived from the inner cell mass of a blastocyst (4–5 days post fertilization and consist of 50–150
cells).
 Human ES cells measure approximately 14 μm.
 Pluripotent: Can differentiate to form ectoderm, endoderm, and mesoderm.
 Capable of propagating themselves indefinitely in an undifferentiated state.
 When provided with the appropriate signals to differentiate they transform via the formation of
precursor cells to almost all mature cell phenotypes.
 This allows embryonic stem cells to be employed as useful tools for both research and
regenerative medicine, because they can produce limitless numbers of themselves for continued
research or clinical use.

Inner Cell Mass
 The first differentiation event : Fifth day of development, when an outer layer of cells
committed to becoming part of the placenta (the trophectoderm) separates from the
inner cell mass (ICM).
 After implantation, they are quickly depleted as they differentiate to other cell types
with more limited developmental potential.
 However, if the ICM is removed from its normal embryonic environment and cultured
under appropriate conditions, the ICM-derived cells can continue to proliferate and
replicate themselves indefinitely and still maintain the developmental potential to form
any cell type of the body.

Ethics and consent
 Ethical issue:
Isolating the embryoblast or inner
cell mass (ICM) results in destruction
of the blastocyst, which raises
ethical issues, including whether or
not embryos at the pre-implantation
stage should be considered to have
the same moral or legal status as
more developed human beings.

History of Embryonic Stem Cells
 1981: Derivation of mouse ES cells was first reported in 1981.
 1998: Derivation of human ES cell lines was first reported.
 Early 1980s: Culture media and species specific cells production was suboptimal.
 In 1990s: ES cell lines from two non-human primates
 Rhesus monkey 5
 Marmoset 6
were derived as they are closer models for the derivation of human ES cells.
 March 2004: A South Korean group reported the first derivation of a human ES cell line
(SCNT-hES-1) using the technique of somatic cell nuclear transfer (SCNT).

Advantages of Embryonic Stem cells
 Flexible - appear to have the potential to make any cell.
 Immortal - one embryonic stem cell line can potentially provide an endless
supply of cells with defined characteristics.
 Availability - embryos from in vitro fertilization clinics.
 Preimplantation genetic diseases can be known.

Disadvantages of Embryonic Stem cells
 Difficult to differentiate uniformly and homogeneously into a target tissue.
 Immunogenic - embryonic stem cells from a random embryo donor are likely to
be rejected after transplantation
 Tumorigenic - capable of forming tumors or promoting tumor formation.
 Destruction of developing human life.

Stem Cell Research- ESC
• Human embryonic stem cell
(HESC) research offers great
promise of cures for
otherwise incurable
conditions:
Spinal cord injuries
ALS
Alzheimer’s
Parkinson’s, etc

Isolating embryonic stem cells

Harvesting ESC by In-vitro fertilization
Poor Quality
Discarded
Early arrested
Dead embryos
Cultured on top of the MEF feeder layer.
When they are removed from the feeder layer and cultivated in
suspension as 3D cell aggregates (EBs), the ES cells differentiate
into specialized cells, including neuronal, hematopoietic, skeletal
muscle, smooth muscle, and cardiac tissue.

Nuclear transfer- ESC
 The differentiated cells are not rejected
by the donor's immune system
Human somatic nucleus
taken
Its genetic material is
stripped off
Transferred into human
oocytes
Cultured in vitro to
blastocyst stage

Somatic Cell Nuclear Transfer (SCNT)

Culture of ESC: Growth factor addition
 Origin of Mouse ES cells and human ES cells : Grown on layer of mouse fibroblasts in the
presence of bovine serum.
Cytokine
Leukaemia Inhibitory
factor(LIF)
Activation of
STAT3 pathway
Proliferation of
cells even with
no feeder cells.
BMP
• Inhibits differentiation
• Inhibits receptor kinase response
• Inhibits p38 mitogen activated
protein kinases.

Culture of ESC
 It is reported that bFGF, TGFβ, and LIF could support some human ES cell lines in
the absence of feeders.
bFGF • Allows clonal growth by helping with serum replacement.
6Bromoindirubin3’oxime
(BIO)
• Promotes self renewal of ES Cells in presence of bFGF

Neuronal Rosettes
Neural rosettes, derived from human
embryonic stem cells, assemble into spheres
in culture.
Credit: Gist Croft/Ali Brivanlou/Rockefeller
University

Genetic Manipulation of ESC
 To direct the differentiation of human ES cells towards specific cell types or to tag
an ES cell derivative with a certain marker gene.
 Techniques for manipulation of gene:
Electroporation
Transfection by lipid-based reagents
Lentiviral vectors.
RNA Interference: Small pieces of DsRNA
are introduces in cells
Causes degradation of
mRNA
No translation of mRNA
to specific protein

Differences between Adult and embryonic stem cells

Embryonic Stem Cell Markers
 Embryonic stem cell (ESC) markers are molecules specifically expressed in ES cells.
 The marker-based flow cytometry (FCM) technique and magnetic cell sorting (MACS)
are the most effective cell isolating methods, and a detailed maker list will help to
initially identify, as well as isolate ESCs using these methods
• Cells are coated with antibodies against a
particular surface antigen.
• Then they are separated by using magnetic
nanoparticles which get attached to the cells
with antigen.
MACS
• Cell population is coated with several antibodies.
• Each is coupled with a different fluorochrome.
• By flow cytometry, each population can be identified
and quantified.
FCM

Enzyme
Markers
Alkaline
Phosphatase
Telomerase

Adult Stem Cells
 Adult stem cells are undifferentiated cells that occur in a differentiated tissue, such as
bone marrow or the brain, in the adult body.
 They can renew themselves in the body, making identical copies of themselves or
become specialized to yield the cell types of the tissue of origin.
 Sources of adult stem cells include
Bone marrow
Blood
Eye
Brain
Skeletal muscle
Dental pulp
Liver
Skin
Lining of the gastrointestinal tract, and
pancreas

Isolation of Adult Stem Cells
1. From the body itself:
 Adult stem cells can be isolated from the body in different ways, depending on
the tissue.
 Blood stem cells can be taken from a donor’s bone marrow, from blood in the
umbilical cord when a baby is born, or from a person’s circulating blood.
 Mesenchymal stem cells, which can make bone, cartilage, fat, fibrous connective
tissue, and cells that support the formation of blood can also be isolated from
bone marrow.
 Neural stem cells have been isolated from the brain and spinal cord and cardiac
stem cells from the heart.

2. From amniotic fluid:
 Amniotic fluid contains foetal cells including mesenchymal stem cells, which are
able to make a variety of tissues.
 Extra fluid left after procedures like amniocentesis is used.
 Stem cells and using them to grow new tissues for babies who have birth defects
detected while they are still in the womb, such as congenital diaphragmatic
hernia
 They can be implanted either in utero or after the baby is born.

3. From other adult stem cells:
Certain kinds of adult stem cells can transform, or differentiate, into apparently
unrelated cell types.
Trans-differentiation: Brain stem cells that differentiate into blood cells or blood-
forming cells that differentiate into cardiac muscle cells.

Adult stem cell harvesting procedure

CORD BANKING
 Cord blood banking means preserving the new born stem cells found in the blood
of the umbilical cord and the placenta.
 Umbilical cord blood (UCB) has emerged as a critical source of cells for
haematology and regenerative medicine applications.
 Advantages:
Easy access and availability
Higher frequency of transplantable stem cells
Higher proliferative capacity
Hosts a variety of adult stem cell populations and progenitor lineages.

Embyonal Stem Cells - Properties and Classification

Application of UC derived blood cells in Vascular medicine
Useful Populations:
1. CD133 positive haemopoietic
progenitor
2. Vascular endothelial
progenitor
3. Mesenchymal Stromal Cells
4. Unrestricted somatic cells
5. Very small embryonic stem
cells.

Therapeutic Applications
 Stroke:
During Stroke: Hypoxia Neuronal death Proliferation of neural precursors in the
Subventricular zone
Olfactory bulb
Hippocampus.
Neurovascular niche: Has neuroblasts, astrocytes and neural stem cells located within a
rich microvascular network.
The administration of UCB-derived therapeutic cells via systemic or local administration has
produced functional recovery in animal stroke models
Neurovascular Niches

 Neurodegenerative diseases:
In animal models of amyotrophic lateral sclerosis (ALS), Alzheimer’s and Parkinson’s
disease, observable behavioural improvement.
UCB CD133+ with
Retinoic acid
Differentiate into
astrocytes,
oligodendrocytes and
glial cells
Express markers for
NSE, Tubulin BIII,
MAP-2, Astrocyte
specific glial protein

 Spinal Cord Injury: In vivo, the administration of fresh CD133+ and expanded
cells enhanced angiogenesis, astrogliosis.
 Cardiovascular Diseases: Transplantation of EPC in restoring blood flow and
improving cardiac function in animal models of ischemia.
Other Advantages:
 Allows HLA matching to minimize immune rejection.
 Not tumorigenic.
 Easy to obtain and amplify.

References
 Umbilical Cord Blood Banking and Transplantation, Stem cell biology and regenerative medicine – Editor
Karen Ballan.
 Stem cell biology by Daniel R. Marshak, Richard L. Gardner and David Gottlieb.
 Stem cells and future of regenerative medicine by Committee on the Biological and Biomedical
Applications of Stem Cell Research Board on Life Sciences National Research Council Board on
Neuroscience and Behavioral Health Institute of Medicine NATIONAL ACADEMY PRESS Washington, D.C.
 Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell
Res Ther 10, 68 (2019).
 Zhao, Wenxiu et al. “Embryonic stem cell markers.” Molecules (Basel, Switzerland) vol. 17,6 6196-236. 25
May. 2012, doi:10.3390/molecules17066196
 Kumar, V., Abbas, A. K., & Aster, J. C. (2017). Robbins Basic Pathology (10th ed.). Elsevier - Health Sciences
Division.

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