Stem Cells

What happens when we get injured?

Immune cells come in and clear away the debris and any infection.
Tissues can repair themselves, filling the void with tough but
otherwise non-functional material using the body’s equivalent of
duct tape; the scar.
Regeneration is the complete restoration of the original tissue.
Damaged tissue is replaced by new functional cells.
Injured tissues heal by a blend of repair and regeneration.
Tissues regenerate new functional cells by:
either 1- by dividing the existing functional cells
2- or by activating an adult stem cell population.

A stem cell must make more copies of itself (self-renewal ) and

give rise to more capable offspring (differentiation).

A stem cell is a cell that has the ability to divide for

indefinite periods.
SO…..WHAT ARE STEM CELLS?
• CELLS THAT CAN MAKE MORE OF THEMSELVES
• CELLS THAT CAN BECOME ALMOST ANY CELL - MULTIPOTENT

What are the characteristics of a stem cell?

1) POTENCY
• The ability to differentiate into any type of cell in response to cell
signaling.
• Pluripotency: This is why stem cells have the potential to "cure any
disease“.

2) SELF RENEWAL
o The stem cell can divide over and over and remain in this
undifferentiated state.

3) CONTROVERSY
o No matter what, they seem to cause controversy
Stem Cells types

Stem cell
type Description Examples

Each cell can develop Cells from early (1-

Totipotent
into a new individual 3 days) embryos

Some cells of
Cells can form any cell
Pluripotent blastocyst (5 to 14
type
days)

Cells differentiated, but Fetal tissue, cord

Multipotent can form a number of blood, and adult
other tissues stem cells

Totipotent - having unlimited capability. Totipotent cells have the

capacity to specialize into extraembryonic membranes and all cells
of the embryo.
Pluripotent -capable of giving rise to most tissues of an organism.
Multipotent -capable of giving rise to many tissues of an organism.
 WHERE DO STEM CELLS COME FROM?
Adult stem cells
An adult stem cell is an undifferentiated cell that occurs in a differentiated
tissue,
The primary role of adult stem cells in a living organism is to maintain and
repair the tissue in which they are found.
• Adult tissues reported to contain stem cells include: brain, bone
marrow, peripheral blood, blood vessels, skeletal muscle, skin and
liver.
• There are a very small number of stem cells in each tissue
Embryonic stem cells (ES)
Embryonic stem (ES) cell lines are cultures of cells derived from the inner
cell mass (ICM) of a blastocyst or earlier morula stage embryos (a
blastocyst is an early stage embryo, approximately four to five days old in
humans and consisting of 50–150 cells)
General properties of embryonic stem cells
- Derived from the inner cell mass of the blastocyst.
- Can undergo for an unlimited number of symmetrical divisions without
differentiating.
- Exhibit and maintain a stable, full (diploid), normal complement of chromosomes.
- Can integrate with all fetal tissues during development.
- Can be induced to continue proliferating or to differentiate.
- ES cells spend most of their time in the S phase
of the cell cycle, during which they synthesize
DNA. Unlike differentiated somatic cells,
ES cells do not require any external stimulus to
initiate DNA replication.
- Clonogenic, that is a single ES cell can give rise
to a colony of genetically identical cells,
or clones, which have the same properties as
the original cell.
Blastocyst
Two Sources of Embryonic Stem Cells
1-In Vitro Fertilization (IVF)
1. Eggs are removed from ovary of female
2. Sperm is obtained from male
3. Egg and sperm are combined in test tubes/Petri dish
4. Fertilized eggs (zygotes) are allowed to divide for a few days.
5. Blastocysts are then transferred to uterus of woman
2- Somatic Cell Nuclear Transfer
- The nucleus of a donated
egg is removed and replaced
with the nucleus of a mature,
"somatic cell" (a skin cell,
for example).
- No sperm is involved in
this process, and no embryo
is created to be implanted in
a woman’s womb.
- The resulting stem cells
can potentially develop into
specialized cells that are
useful for treating severe
illnesses.
Differences between embryonic and adult stem cells
• They have different self-renewal capabilities
– Embryonic stem cells: near indefinite self-renewal
– Adult stem cells: limited self-renewal

• They have different differentiation potentials

– Embryonic stem cells: differentiate into all cell types in an organism
– Adult stem cells: differentiate into restricted cells types.

• They differ in how they respond to external stimuli

– Embryonic stem cells are readily to change upon stimulation
– Adult cells emphasize on stability and need to be activated by cues,
e.g. injuries.
 Importance of Stem Cells
• Perhaps the most important potential application of human stem cells
is the generation of cells and tissues that could be used for cell-
based therapies.

• Today, donated organs and tissues are often used to replace

destroyed tissue, but the need for transplantable tissues and organs
far outweighs the available supply.

• Stem cells, directed to differentiate into specific cell types, offer

the possibility of a renewable source of replacement cells and
tissues to treat diseases including Parkinson's and Alzheimer's
diseases, spinal cord injury, stroke, burns, heart disease, diabetes,
osteoarthritis, and rheumatoid arthritis.

. It is important to understand how diseases can occur. By watching

stem cells mature into cells that eventually become bones, heart
muscle, nerve cells and other organs and tissue, researchers and
doctors may better understand how a variety of diseases and
conditions develop.
Gene therapy
Gene therapy is an experimental technique that uses genes to treat or
prevent disease (It is a technique for correcting defective genes that
are responsible for disease development)

This technique might allow doctors to treat a disorder by inserting a

gene into a patient’s cells instead of using drugs or surgery.

Researchers are testing several approaches to gene therapy, including:

1- A normal gene inserted to compensate for a nonfunctional gene.
2- Inactivating, or “knocking out,” a mutated gene that is functioning
improperly.
3- Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of

diseases (including inherited disorders, some types of cancer, and
certain viral infections), the technique remains risky and is still under
study to make sure that it will be safe and effective. Gene therapy is
currently only being tested for the treatment of diseases that have no
other cures
 The First Case
• The first gene therapy was performed on September 14th, 1990
– Ashanti De Silva was treated for SCID
• Severe combined immunodeficiency
– Doctors removed her white blood cells, inserted the missing
gene into the WBC, and then put them back into her blood
stream.
– This strengthened her immune system
– Only worked for a few months

How does it Work

• A vector delivers the therapeutic gene into a patient’s target

cell
• The target cells become infected with the viral vector
• The vector’s genetic material is inserted into the target cell
• Functional proteins are created from the therapeutic gene
causing the cell to return to a normal state
 Viruses
• Replicate by inserting their DNA into a host cell
• Gene therapy can use this to insert genes that encode for a
desired protein to create the desired trait
Four different types
1- Retroviruses
Created double stranded DNA copies from RNA genome
-The retrovirus goes through reverse transcription using reverse
transcriptase and RNA
-The double stranded viral genome integrates into the human genome
using integrase
• integrase inserts the gene anywhere because it has no specific
site
• May cause insertional mutagenesis
-One gene disrupts another gene’s code (disrupted cell division
causes cancer from uncontrolled cell division)
-Vectors used are derived from the human immunodeficiency
virus (HIV) and are being evaluated for safety
 2-Adenoviruses
• Are double stranded DNA genome that cause respiratory,
intestinal, and eye infections in humans
• The inserted DNA is not incorporated into genome
• Not replicated though
– Has to be reinserted when more cells divide
• Ex. Common cold

3-Adeno-associated Viruses
• Adeno-associated Virus- small, single stranded DNA that
insert genetic material at a specific point on chromosome 19
• From parvovirus family- causes no known disease and
doesn't trigger patient immune response.
• Low information capacity
• gene is always "on" so the protein is always being expressed,
possibly even in instances when it isn't needed.
• hemophilia treatments, for example, a gene-carrying vector
could be injected into a muscle, prompting the muscle cells to
produce Factor IX and thus prevent bleeding.
 4-Herpes Simplex Viruses
• Double stranded DNA viruses that infect neurons
• Ex. Herpes simplex virus type 1

Non-viral Options
• Direct introduction of therapeutic DNA
– But only with certain tissue
– Requires a lot of DNA
• Creation of artificial lipid sphere with aqueous core, liposome
– Carries therapeutic DNA through membrane
• Chemically linking DNA to molecule that will bind to special
cell receptors
– DNA is engulfed by cell membrane
– Less effective
• Trying to introduce a 47th chromosome
– Exist alongside the 46 others
– Could carry a lot of information
But how to get the big molecule through membranes?
Problems with Gene Therapy
• Short Lived
– Hard to rapidly integrate therapeutic DNA into genome
– Would have to have multiple rounds of therapy
• Immune Response
– new things introduced leads to immune response
– increased response when a repeat offender enters
• Viral Vectors
– patient could have toxic, immune, inflammatory response
– also may cause disease once inside
• Multigene Disorders
– Heart disease, high blood pressure, Alzheimer’s, arthritis and
diabetes are hard to treat because you need to introduce more
than one gene
• May induce a tumor if integrated into a tumor suppressor gene
because insertional mutagenesis
Recent Developments
• Genes get into brain using liposomes coated in polymer called
polyethylene glycol
• Create tiny liposomes that can carry therapeutic DNA through pores
of nuclear membrane
• Sickle cell successfully treated in mice
Gene Regulation
includes the processes that can be used to regulate the way that the
information in genes is converted into gene products.
Although a functional gene product can be an RNA, the majority of known
mechanisms regulate protein coding genes.
Regulated stages of gene expression
The following is a list of stages where gene expression is regulated, the
most extensively utilised point is Transcription Initiation:
1-Chromatin domains
2-Transcription
3-Post-transcriptional modification
4-RNA transport
5-Translation
6-mRNA degradation

Concepts
Gene: A DNA segment that contains all the genetic information required
to encode RNA and protein molecules.
Genome: A complete set of genes of a given species.
Gene expression: A process of gene transcription and translation.
 Types of gene expression

a. Constitutive expression
Some genes are essential and necessary for life, and therefore are
continuously expressed.
These genes are called housekeeping genes.
b. Induction and repression
The expression levels of some genes fluctuate in response to the
external signals.

Some genes demonstrate higher expression level once being activated.

It is called induced expression.
On the other hand, some genes are repressed and their expression
levels are lower. It is called repressed expression.
 Regulatory Elements
• Gene expression is a multiple-level process.
• Transcription initiation is a key point of controlling gene expression.

Basic elements that regulate the transcription include:

a. Special DNA sequences
b. Regulatory proteins
c. DNA-protein interaction and protein-protein interaction
d. RNA polymerase
Special DNA sequence
For prokaryotic systems:
Operon is composed of structural genes, promoter, operator, and other
regulatory sequences.
The lactose operon model has the following feature:
The lactose-operon system consists of two kinds of components—
A- Structural genes (lacZ, lacY, and lacA), which encode proteins needed
for the transport and metabolism of lactose;
B- Regulatory elements include the repressor gene I, the promoter P, and
the operator O.
regulatory site structural gene

I P O Z Y A
Structure
of lac operator transacetylase
operon
promoter permease
CAP-binding site
beta-galactosidase
regulatory gene
 Promoter
The DNA sequence that RNA-pol can bind to and initiate the transcription.
Promoter: a promoter is a region of DNA that facilitates the transcription
of a particular gene through providing a site for RNA polymerase to bind
and initiate transcription.

Operator
The DNA sequence adjacent to the structural genes that the repressor
protein can bind to and prevent the transcription of structural genes.

Operator – a segment of DNA that a repressor or activator binds to. It

is classically defined in the lac operon as a segment between the
promoter and the structural genes of the operon.

promoter
RNA pol repressor
operator structural gene
For eukaryotic systems:
Cis-acting elements is the special DNA sequence that can affect
the expression of its own gene.

is a region of DNA or RNA that regulates the expression of genes located

on that same molecule of DNA (often a chromosome)

Trans-regulatory elements are diffusible factors, usually proteins, that

may modify the expression of genes distant from the gene that was
originally transcribed to create them. For example, a transcription
factor which regulates a gene on chromosome 6 might itself have been
transcribed from a gene on chromosome 11

Cis-regulatory elements are present on the same molecule of DNA as

the gene they regulate, whereas trans-regulatory elements can
regulate genes distant from the gene from which they were
transcribed.
 Trans-acting factors

DNA a

mRNA
A b

protein A A
Regulatory proteins
For prokaryotic systems:
• Specific factor: It facilitates the binding of RNA-pol to particular DNA
sequence.
• Repressor: It binds to the operator and prevent the transcription,
known as negative regulation.

• Activator: It associates with DNA near the initiation point, resulting in

the increase of RNA-pol binding affinity and the enhancement of the
transcription efficiency.

For eukaryotic systems:

• The regulatory proteins are called transcription factors (TF).
• After expression, TF will interact with the cis-acting elements to
activate another genes. Therefore, they are referred to as trans-
acting factors.
DNA-protein interactions
• The regulation is implemented through numerous interactions between
cis-acting elements and trans-acting factors.
• They are non-covalent bond.

Protein-protein interactions
• Proteins may have to interact with each other prior to the DNA binding.
• Proteins can form a homo or hetero-dimer form to function properly.
• Present in prokaryotes as well as eukaryotes.
 Regulation of
Eukaryotic Transcription
In eukaryotic organisms like ourselves there are several methods of
regulating protein production
Most regulatory sequences are found upstream from the promoter
Genes are controlled by regulatory elements in the promoter region that
act like on/off switches or dimer switches
Specific transcription factors bind to these regulatory elements and
regulate transcription
Regulatory elements may be tissue specific and will activate their gene
only in one kind of tissue
Sometimes the expression of a gene requires the function of two or more
different regulatory elements
Eukaryotic DNA differs from prokaryotic DNA in that the coding
sequences along the gene are interspersed with noncoding sequences
The coding sequences are called
EXONS
The non coding sequences are called
INTRONS
After the initial transcript is produced the introns are spliced out to form
the completed message ready for translation
Introns can be very large and numerous, so some genes are much bigger
than the final processed mRNA
Structural features
• Large genome: 3 x 109 bps, 35 000 genes
• Repeated sequences: different lengths and different
frequencies. Often inverted repeats
• Splite genes: separated by introns and exons alternatively

Regulation features
1. RNA-pol: 3 forms (I, II, and III) for different RNAs
2. Changes of chromosomal structure
• Hypersensitive site . Base modification
• Isomer-conversion . Histone changes

3. Positive regulation
4. Transcription and translation are separated
5. Post-transcriptional modification
6. Regulation through intercellular and intracellular signals
lac Operon Gene Function
Gene
I Gene for repressor
protein
P Promoter

O Operator

lac Z Gene for ß-galactosidase catalyzes the hydrolysis

of lactose to glucose and
galactose
lac Y Gene for ß-galactoside is a carrier protein in the
permease bacterial plasma
membrane that moves
the sugar into the cells
lac A Gene for ß-galactoside role in the process is not
transacetylase yet clear.
 Negative and Positive Regulation

• The lac operon is negatively regulated: the regulatory protein

(repressor) causes transcription to stop.

• Positive regulation, where the regulatory protein causes transcription

to start, is more common.

• The lac operon also contains an example of positive regulation, called

“catabolite repression”. E. coli would prefer to use glucose as its
food source. In the presence of glucose, the lac operon (and other
similar genes) are turned off, even if lactose is present in the medium.
 Catabolite Repression
• Catabolite repression uses a regulatory protein called CAP
(catabolite activator protein). It also uses the small molecule cyclic
AMP (cAMP).

• cAMP is made from ATP. When the glucose level in the cell is high,
the cAMP level is low, because glucose inhibits synthesis of cAMP.
When the glucose level is low, the cAMP level is high.

• cAMP combines with the CAP protein to form a complex that binds
to part of the lac operon promoter. This complex bends the DNA in
a way that makes it much easier for RNA polymerase to bind to the
promoter. This allows transcription to occur, but only if the lac
repressor isn’t present.

• Thus, low glucose levels cause high cAMP levels. When cAMP is
high, it combines with CAP. The CAP-cAMP complex then binds to
the promoter to allow transcription to occur.

• This is positive regulation because the binding of CAP to the DNA

causes transcription to occur.