Stem Cell biology II

Lecture 10
 Outline
• Cloning
• Types of cloning
• SCNT
• Genetic engineering and stem cells
 Cloning
Cloning describes the processes used to
create an exact genetic replica of another
cell, tissue or organism.
Classified as

• Reproductive cloning
• Therapeutic cloning

Note: Here we are not concerned with gene cloning

 Cloning of whole organisms
• Purpose:
– Reproductive cloning in animals
– Therapeutic cloning
– Breeding animals or plants with favorable traits
– Producing TRANSGENIC animals that:
• Make a therapeutic product (vaccine, human protein etc)
• Act as animal models for human disease
• Deliver organs that will not be rejected (cells lacking cell
surface markers that cause immune rejection)
– Vaccines in biotech industry: steps in cloning a gene
 SCNT: Somatic Cell Nuclear Transfer
• Is a method of cloning an organism that
involves removing of the nucleus from the
somatic cell (full num of chromosome) and
putting it into an egg.
 Steps
1- Eggs are coaxed to
mature in a culture
dish. Each has a
remnant egg cell called
the polar body and
cumulus cells from the
ovary clinging to it.
 Cont.…...
2- While an egg is
held still with a
pipette, a needle is
used to drill through
the zona pellucida,
removing a plug.
3- After ejecting the
zona plug, the needle is
inserted back in the egg
through the hole to
withdraw and discard
the egg's genetic
material.
4- A somatic cell’s
genetic material is taken
up into the needle
5- The genetic material is
injected deep into the
egg that has been
stripped of its genetic
material.
6- The injected egg is
exposed to a mixture of
chemicals and growth
factors designed to activate
it to divide.
7- After roughly 24 hours,
the activated egg begins
dividing. The cells contain
genetic material only from
the injected somatic cell.
8- By the fourth or fifth day, a
hollow ball of roughly 100
cells has formed. It holds a
clump of cells called the inner
cell mass that contains stem
cells.

9- The blastocyst is broken

open, and the inner cell mass
is grown in a culture dish to
yield stem cells.
10- The stem cells, in
turn, can be coaxed to
grow into a variety of
cells that might one
day be injected into
patients.
 World famous Dolly
• In 1996 Scottish scientist in UK created dolly

• The first mammal to be cloned

• It took 277 attempts until successful clone.

• Lived until she was six years Old

• Dolly reproduced as well giving s

 SCNT: Somatic Cell Nuclear Transfer

• SCNT is a method used for:

– Reproductive cloning such as cloning an embryo
– Regenerative cloning to produce “customized” stem
cells & overcome immune rejection
• Blastula stage cannot continue to develop in vitro
– It must be implanted into surrogate mom
– Surrogate mom is just a container that provides
protection & chemical signals necessary for
development
http://www.kumc.edu/stemcell/early.html Reprinted with permission from the University of Kansas Medical Center.
http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.
 Challenges of Reproductive Cloning

• Many animals were cloned after Dolly

– Cats, pigs, mice, goats, cattle, rabbits
• Obstacles:
– Very inefficient process
– 90% of the clone fail
– Most clones have deleterious effects (DNA) & die early
– Surviving clones show premature aging signs
– Signs of abnormal embryonic development:
• Clones & their placentas grow much faster than expected in
surrogate mom
 Therapeutic Cloning

• goals of therapeutic cloning by SCNT in humans:

– Use embryo as source for ES cells
– Use ES cells to generate an organ
• In this case the organ generated will carry cells with the
same genetic markers as the patient (recipient)
 Pitfalls of therapeutic cloning (1)

Some immune rejection may occur- WHY?

– About 1% of the DNA in the clone will NOT be

identical to donor cell (patient)
– It will be identical to egg cell used in SCNT
– REASON: mitochonrial DNA in eggs
• Human mitochondria carry about 13 genes, some of
which code for surface proteins
 Pitfalls of therapeutic cloning (2)
• Large number of eggs needed for SCNT
• To harvest large number of eggs:
– Excessive hormone treatment of females to induce
high rate of ovulation
– Surgery to retrieve eggs
• Both can be harmful to female human
• Cow/pig females may be used
– Cow/pig eggs will carry species-specific mitochondrial
genes
• Mixing species is reason for concern!
 Common Opinions

• Reproductive cloning is a criminal offense (it is

ILLEGAL worldwide!)
• Therapeutic cloning is acceptable, however
there is still significant controversy over
whether:
the clone is implanted into the uterus of surrogate
mom? OR
the clone is explanted into culture dish to
generate ES cells
 Status of SC research in other countries

• Great Britain
– Very liberal policies on research
– Therapeutic cloning allowed, use of excess embryos & creation of embryos
allowed
– Stem cell research allowed
• France
– Less liberal politics
– Use of excess embryos allowed
– Reproductive AND therapeutic cloning banned
• Germany
– Very strict policies
– Use of excess embryos and creation of embryos banned
– Scientists can IMPORT embryos
 Gene therapy and Stem cells
Gene therapy can be performed either by direct
transfer of genes into the patient or by using
living cells as vehicles to transport the genes of
interest. Both modes have certain advantages
and disadvantages.
 Cont……
• Direct gene transfer is particularly attractive
because of its relative simplicity (viruses)

• biggest strength—simplicity—is
simultaneously their biggest weakness.

• Problem of wrong integration of gene.

• On the other hand, therapeutic genes can be
delivered using living cells.
• relatively complex in comparison because of
isolation and again placement of the cells in
the patient
• Advantages, In the laboratory dish (in vitro),
cells can be manipulated much more precisely
than in the body (in vivo)
 ES for gene therapy
• Embryonic stem cells could be genetically
manipulated to introduce the therapeutic
gene.
Skin cells from an immuno deficient mouse were
used to generate cellular therapy that partially
restored immune function in the mouse.
 How genetic manipulation can take
place
Genes may be introduced into cells by transfection
or chemical or physical methods to introduce new
genes into cells.
Usually, small molecules, such as liposomes are
employed to facilitate the entry of DNA encoding
the gene of interest into the cells.
Brief electric shocks are additionally used to
facilitate DNA entry into living cells
(electroporation). All of these techniques have been
applied to various stem cells, including human
embryonic stem cells.
However, the destiny of the introduced DNA is
relatively poorly controlled using these procedures.
 Cont….
• viral vectors for DNA transfer are also used.
Viruses, by nature, introduce DNA or RNA into
cells very efficiently.
• Engineered viruses can be used to introduce
almost any genetic information into cells.
• However, there are usually limitations in the
size of the introduced gene
 Stem Cell Theory of Cancer
• 1855: Rudolf Virchow developed the Embryonal-
Rest Hypothesis
– Microscopic examination of tumor samples revealed
many morphological (structural & functional)
resemblances to ESC in a developing fetus
• Isolation of teratoma: nonmalignant tumors
– Teratoma represents a ball of almost all cell types
– This indicates that teratoma may originate from
unregulated stem cells that can give rise to almost all
tissues
 Teratoma
• Ovarian Teratoma
– You can see teeth!

http://home.earthlink.net/~radiologist/tf/040802.htm Image courtesy of Leonard J. Tyminski, M.D., Radiologist at earthlink.net

 Current Efforts with SC and Cancer
• Determine difference
between cancer & normal
stem cells
• Identify potential points in
pathways critical for the
survival of cancer SCs
• Develop therapies that
specifically target cancer SC
Tumor stem cell
• Duke University Explanation

Tumor cell

Drawn by Christine Rodriguez

 References
http://learn.genetics.utah.edu/content/cloning/
whatiscloning/
THANK YOU FOR ALL YOUR PATIENCE