Embryonic Stem Cells and Cloning
March 22 and 24, 2004

Note: Web notes only! We will NOT have time to go through all these notes and you will not be tested on all the notes (see objectives below). However, you may want to use these notes as a resource for your own knowledge of 'current events' as they relate to biology, politics, ethics, etc...

I. What is meant by the term 'cloning'? Cloning refers to making an exact copy of an original form.

• The root of the word comes from the greek word for 'twin' or 'breaking in two' - klon.
• Computers or electronic devices that look like an original are said to be 'clones'.
• In science fiction books and movies, 'clones' implied replicated copies of an individual, usually resulting in some sort of 'slave army' working for an evil genius, or an evil scientist.... ; ) See this week's Warm Up 7 to cast a vote for the cloning academy awards! !
• In biology, the term usually refers to asexual reproduction (like bacteria)
• In biotechnology, the term often refers to 'cloned genes' - making multiple, identical copies of a gene (or protein) in bacteria or animal cells.
• In 1996, cloning took on a new meaning: an new individual grown from a single somatic cell, and genetically identical to it... This technique is also called somatic cell nuclear transfer....

II. Hello Dolly: Cloning Mammals...

July 5, 1996: First mammal cloned from adult cells: A surrogate mother sheep gives birth to Dolly, a lamb cloned from an udder cell of an adult sheep born 6 years earlier. Ian Wilmut and colleagues at the PPL Theraputics / Roslin Institute in Scotland quietly announce the birth of Dolly in February, 1997 in the journal Nature (Viable offspring derived from fetal and adult mammalian cells. Nature 1997 Feb 27; 385(6619) Wilmut I, et al. Roslin Institute (Edinburgh), Roslin, Midlothian, UK.)

Dolly is a later-born genetic twin of the somatic cell she was cloned from (a female Finn Dorset Ewe).

Since then, much work has been done to streamline and refine the cloning process (Don't memorize this 'laundry list'; it is just to show you the variety of work that has been done!):

• (6/97)Hello PollyCloned sheep bearing a human gene (blood clotting factor VIII), by PPL Theraputics / Roslin Institute.
• (8/97) 'Gene' the Bull - cloned by ABS Global.
• (10/97) 22 Cloned Mice: "Cumulina" and some of her cloned siblings (some of whom were cloned from clones). Author Teruhiko Wakayama concludes that "contrary to previous opinion, mammals can be reproducibly cloned from adult somatic cells" [Image]
• 1999, Roslin Bio-Med becomes Geron Bio-Med. Gotta love their patent position: telomerase, hES cells, AND somatic cell nuclear transfer!
• (12/99) Mice cloned from ES cells , Teruhiko Wakayama demonstrates that embryonic stem (ES) cells can be used to clone animals. It thus may be possible to clone from a single cell a large number of individuals over an extended period...."
• (8/00) First cloned Piggies: Roslin Institute / Geron and National Institute of Animal Industry in Japan (cute picture!) simultaneously publish in Science and Nature.
• (2/02) First cloned kitty - Cc - see below (very cute pic!)
• (8/02) First cloned pigs designed for human transplants The Roslin Institute - Piglets lack the genes to display the sugar alpha 1,3 galactose on organs that usually cause hyperacute organ rejection in humans, a vital step in producing pigs with organs and cells which can be used in humans.
• (2/03): "Dolly the sheep, the first cloned mammal, is dead at age 6, of a lung infection. Snif!
• Cloning primates...still not there yet! Problems cloning monkeys 12/12/01 - cloned rhesus macaque embryos show numerous abnormalities, Failed Monkey Clonings Cast Doubt on Human Cloning Claim 4/9/02 - "It is extremely unlikely that human reproductive cloning is going to happen any time soon," said Richard Paulson at the University of Southern California.

III. Somatic Cell Nuclear Transfer: (aka cloning). A how-to:

Ingredients:

• 1 somatic (diploid) donor cell, containing the complete genome of that individual +
• 1 unfertilized recipient egg with its haploid nucleus removed (or 'e-nucleated') +
• 1 electric shock to get them together...

Images from Time Magazine, March 10, 1997

More detail on those Early Steps: Scientific American, November 2000
Quiescence: "The state in which all but the most basic functions of a cell or group of cells has stopped. This is usually a response to an environment in which the food supply is low or absent. The cell becomes dormant until its surroundings are more favourable. In this state the genes that define the specialist function of a cell "switch off" making the cell suitable for nuclear transfer."

IV. Why do scientists want to clone animals?!?!?

1. The Potential of cloned livestock: (September 1997) Scientific American. Cloning livestock allow for the genetic replication of animals that are expecially high producers of a desired product, allowing for the creation of animal herds that can be farmed for milk, blood and organs: [Image]

* Human therapeutic proteins (hormomes, clotting factors, etc)
* Organs and tissues for transplants
* 'Humanized' cows milk
* Animal models of disease (diabetes, muscualr dystrophy, etc)

The big livetock cloners:
The Roslin Institute; Advanced Cell Technology, Cyagra; ABS Global

2. A second reason: Saving endangered species: Cloning Noah's Ark (November 2000) Scientific American. The inspiring (but sad) story of the cloning of the baby Guar, Noah, a Cow-Guar hybrid. See Good For 7! (Even sadder than NOT being able to clone endangered species? - the daily extinction of species due to the continual, daily habitat destruction for numerous species worldwide....thanks to humans...)

3. A third reason: Cloning your deceased Pet?!?!? Lassie, come home.

• The Missyplicity Project is a project funded by John Sperling's Genetic Savings & Clone to produce the first cloned dog. They have invested $2.3 million to produce a clone of a beloved pet dog, Missy, a mixed breed border collie.
• 2/14/02 Operation Copycat First Cloned Cat is born. The kitten is a calico-and-white shorthair that looks similar to, but not exactly like, her genetic donor (genetics contribute to an animal's markings, but so do conditions in the womb and environment).
• 1/21/03Copied cat hardly resembles original CC and genetic twin Rainbow, are different in many ways...
• Wondering what to give that special someone who has everything?? Why not a gift certificate good at any Genetic Savings and Clone?

V. Cloning Human Beings...? (Will there ever be another 'ewe'?)

"There is no clinical reason why you would clone humans. Why would you make another human being? We think it would be ethically unacceptable and certainly would not want to be involved in that project.'' Dr. Ian Wilmut, The Roslin Institute

"We will get there, because very simply it's a matter of determination. And I think we are determined to get there," Dr. Panos Zavos, University of Kentucky, who leads one of the 3 groups attempting to clone the first human.

Two reasons some scientists and physicians want to pursue human cloning:

1. Reproductive cloning: making babies for infertile couples created from a single somatic cell without sexual reproduction.

2. Therapeutic cloning: making human embryonic stem (hES) cells that are immunologically compatible for people with a disease, a disorder, or an injury (diabetes, leukemia, spinal cord injury, etc. etc.). Some scientists advocate calling this technique "nuclear transplantation" to avoid the negative connotations of cloning.

Three reasons to not pursue human cloning:

(1) July 16 2001: H.R. 2505, Human Cloning Prohibition Act of 2001, House of Respresentatives approves a ban on all human cloning. If passed into law, would make cloning a crime punishable by up to 10 years in prison.

(2) April 10, 2002 President Bush Calls on Senate to Back Human Cloning Ban "I believe all human cloning is wrong, and both forms of cloning ought to be banned..."

January 28, 2003: State of the Union Address: "...And because no human life should be started or ended as the object of an experiment, I ask you to ... pass a law against all human cloning."

(3) June 14, 2002: Anti-Cloning Bills Stall in Senate; Vote Unlikely Soon "The Democratic-controlled Senate appears divided over whether to impose a ban on all cloning or to allow cloning aimed at producing stem cells with the potential for curing many diseases, including Alzheimer's and Parkinson's."

November 6, 2002: Election Update 2002: G.O.P. Retakes Control of the Senate In a Show of Presidential Influence. Will this have any impact on the future of human cloning research?

Mini-project: Cloning movies and cloning statements.

VI. Future Games: Reproductive Cloning:

Time Magazine, February 2001 Human Cloning is closer than you think
Time Magazine, August 2001 Is Cloning an Inevitability? (Time: moral compass)

First human clone bid planned: 3 privately-funded US / European groups vow to contunue their programs on Human Cloning.

Dr. Severino Antinori: of Italy, best known for helping a 63-year-old woman to have a baby, making her the oldest known women in the world to give birth.    Plans to clone babies for couples using the man's genetic material and the woman's egg (and egg cytoplasm, which contains mitochondria).

Dr. Panos Zavos: of Lexington KY, director of the Andrology Institute of America. Would like to impregnate up to 200 women from infertile couples with cloned embryos. (Note from Dr. Marrs: In the immortal words of Buzz Lightyear, from Toy Story (1995), "I don't believe that man has ever been to medical school".)

Dr. Brigitte Boisselier: scientific director of Clonaid, "The First Human Cloning Company". Founded by Rael, a spiritual leader of the Raelian Movement (and former french auto racer) who believes that human life is the result of extraterrestrial genetic experiments. (Note from Dr. Marrs: In the immortal words of Fozzie Bear, from The Muppet Movie (1979), "We picked up a weirdo...!")

• December 27, 2002: Baby Eve, the first cloned baby, the first step towards human immortality? OR, maybe just a media circus.... Without scientific data, we'll never know...
• Feb 2003 Nature Biotechnology: Scary thought: "A secretive group like Clonaid might be the perfect organization for carrying out a reproductive cloning program. The main requirements would be: 1) a small team of researchers willing to carry out the work, irrespective of the appalling risks to the mother and child, 2) an initial outlay of equipment necessary for IVF, 3) lots and lots of human eggs...."
• March 2003 Scientific American: The Rael Thing. Funny....

VII. Human Embryonic Stem Cells and Therapeutic Cloning:

1. What are Human Embryonic Stem Cells (hES cells) and why are we talking about them in Contemporary Biology !??

A review of Human embryonic development, days 1-5... hES cells are cells derived from the Inner Cell Mass of surplus blastocysts from In Vitro Fertilization (IVF). If implanted in a woman's uterus at 4-5 days after fertilization, IVF blastocysts may develop into a baby (see Good For 6), but if the couple does not use them, the extra (or 'surplus') frozen blastocysts can be donated, after informed consent, to obtain Human Embryonic Stem Cells (hES cells) from the Inner Cell Mass cells.

Why is the inner cell mass important? Remember from our last discussion....these are the cells that have the potential to become the baby, and the placenta. When YOU were a blastocyst, the Inner Cell Mass developed (by mitosis, of course, as well as lots of developmental instructions from your cells) to become YOU!

However, the hES cells that can be derived from the inner cell mass have tremendous potential for treatment of many types of diseases, including Parkinsons, Alzheimers, spinal cord injuries, diabetes, transplants, etc.... There are definitely ethical implications in the derivation and use of hES cells, because the blastocyst does not survive the process needed to obtain the inner cell mass cells.

On August 9, 2001, In his first primetime address to the nation President Bush decided to allow federal funding of ES cell research to go forward, but only onthe 64 hES cell lines already in existence, because destruction of an embryo has already taken place. He refused to allow research on any cell line created in the future to prevent the federal government from encouraging the destruction of human embryos.

2. The 4 amazing characteristics of Human Embryonic Stem Cells:

• Pluripotent. hES cells can form virtually any cell in the body, including specific cell types like Cardiomyocytes (heart) Hematopoietic (blood forming) cells, Endothelial (blood vessel) cell, Islet cells (pancreas), Neurons, Skin cells and Chondrocytes (Cartilage forming cells). Having a reliable source of these cells could treat diseases and injuries in millions of people each year...
• Self-replicating. Under the right conditions, hES cells propagate indefinitely in the undifferentiated state.
• Telomerase expression. Telomerase is an enzyme that allows for cell division. Usually, as cells age, they lose telomerase activity - and die. hES cells continually express telomerase and are potentially immortal.
• Normal chromosome structure (karyotype). hES cells have normal set of chromosomes, even after prolonged growth. They do not have any additions, deletions or rearrangements in their chromosomal structure.

3. Therapeutic cloning What is it, and how does it relate to hES cells and to cloning? Therapeutic cloning involves the creation of an blastocyst via somatic cell nuclear transfer (cloning) from an animal (ie: a person) with a disease, to derive hES cells immunologically identical to donor, in hopes of treating the an injury or a disease. The cloned blastocyst would exist only until day 5, when it would be used to isolate hES cells.

• The nucleus of a somatic cell from the donor would be removed and placed into an enucleated human egg.
• "Fertilization" would be simulated with electric shock, and the zygote would be cultured for 4-5 days. .
• The inner cell mass would be removed from the 5-day old blastocyst, which is genetically identical to the donor.
• hES cells would be cultured into the tissue type needed by the donor (heart cells, neurons, etc).
• This immunologically identical tissue would be placed back into the donor, curing or treating the donor's disease
• (Remember that this has not YET been done for humans; scientists are currently working on it in other animals)

Eventually, growing replacement organs (heart, liver, pancreas, skin, etc) MAY be possible - however I think we can conservatively say we are many years away from producing needed tissues, let alone whole organs.

A step forward...or back?

• 11/25/01Sunday, Thanksgiving weekend, 2001, 11 pm news: " The First Cloned Human Embryo Advanced Cell Technologies (you know...the livestock cloners...) reported that they created create several clomed human embryos. Unfortunately, only one of the embryos progressed to a "6-cell" stage, at which point it stopped dividing and died and none were able to get to the 5-day blastocyst stage. (E-biomed Rapid Communication: Somatic Cell Nuclear Transfer in Humans: Pronuclear and Early Embryonic Development the Journal of Regenerative Medicine  2 (25 - 31) Jose B. Cibelli et al.)
• 11/26/01 Scientific criticism: Experts rip cloning 'story' (Very irresponsible "press release" science)
• 11/27/01 Presidential criticism: Bush denounces ACT (OUCH!)

IV. Safety Issues:

Cloned Cows Dropping Like Flies April 2, 200; Study Raises Human Cloning Doubts July 6, 2001; Imprinting marks clones for death. Nature, July 2001; Clone Snafu suspected Wired, May 2001

The rate of success of cloning is currently very low - cloned animals rarely make it to adulthood - or sometimes even birth.

• It took 277 attempts (failed pregnancies) to get 1 cloned sheep - Dolly.
• It took 188 tries (failed pregnancies) to get just one 'Cc'.
• Some labs are better at it than others, but typically a hundred or so attempts are made before one 'successful' clone is born alive and well.
• Many times, the placenta of the animal is abnormal, contributing to a miscarriage.
• Many animal embryos spontaneously miscarry or the animal dies shortly after birth.
• Cloned animals often have problems with their immune systems, lungs, and livers, causing death at an early age. They also have problems with obesity.
• FInally, a close look at the DNA of cloned animals shows that there are subtle changes in a genetic process called 'imprinting' that are seem between 'identical' clones...and scientists do not know how to explain these changes yet...

V. Ethical Issues

What are the benefits of and who benefits from reproductive cloning?
What are the potential drawbacks or harmful things that could result from reproductive cloning?

Imagine a child growing up as a 'clone' of an adult from a member of her family. What are some of the 'issues' she might have to deal with (besides the usual issues like "nobody understands me" and "why can't I get my ears pierced?")

What are the benefits of and who benefits from therapeutic cloning?
What are the potential drawbacks or harmful things that could result from therapeutic cloning?

Groups Supporting Embryonic Stem Cell Research: No surprises here
Groups Opposed to Embryonic Stem Cell Research: No surprises here either!

Read if you are interested: The Vatican: Reflections on Cloning; 21 arguments against human cloning and their responses

Objectives: Ones in purple are the MOST IMPORTANT to study for the test!

1. Please be able to state 'who' these cloned animals are and why we are discussing them: Dolly, Polly, Cumulina, Noah, and Cc. Who is Ian Wilmut? (no, he is not a cloned mammal!)
2. Define 'cloning' as it relates to mammals. What is a more precise term for this procedure?
3. Describe the 'ingredients' needed to make a cloned mammal.

• What is the purpose of the diploid nucleus?
• Why is the recipient egg enucleated before the procedure?
• What is left in the recipient egg after e-nucleation and why is this important for the developing animal?
• What is the purpose of the electric shock?
• What is quiescence and why does it appear to be crucial to cloning mammals?

4. List three reasons scientists are interested in cloning livestock animals or research animals like mice, and two reasons scientists are interested in cloning animals like guars, and like cats and dogs.
5. What is the Human Cloning Prohibition Act of 2001?
6. What is human Reproductive Cloning? Who is pursuing this procedure?
7. Where do hES cells come from (specifically), and what are the 4 amazing characteristics of hES cells?
8. What is human Therapeutic Cloning and how does it relate to hES cells?
9. List 4 medical and safety issues concerning cloned animals.