Embryonic Stem Cells and Cloning
March 22 and 24, 2004

Note: Web notes only! Wow - lots of notes here. 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 Polly Cloned 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 simultaneously publish in Science and Nature.
• (2/02) First cloned kitty - Cc - see below (very cute pic, see below!)
• (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. Her death probably had nothing to do with the fact that she was a clone, said Dr. Ian Wilmut. "The fact the she isn't there any more will be brought home to us when we go near that barn and she's not there," Dr. Wilmut said. Snif!
• (5/03): Way to go, Idaho!: A healthy mule named Idaho Gem is the first member of the horse family to be cloned. Yee-hah!
• (8/03): World's first cloned horse born to its genetic twin: LONDON, England (Reuters) -- "Italian scientists said Wednesday they had created the world's first cloned horse, Prometea, from an adult cell taken from the horse who gave birth to her. Prometea is the first animal known to be carried and born by the mother from which she was cloned...".
• (9/03): Rat joins list of cloned animals Rats have been much more difficult to clone than sheep, cattle or even mice because their oocytes (necessary for cloning) speed through meiosis. Zhou et al succeeding in cloning Ralph (the rat) by using a drug to block the first metaphase-anaphase transition, a technique they call delayed oocyte activation. Published in Science 26 September 2003
• 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. How ARE animals cloned? Somatic Cell Nuclear Transfer (SCNT)

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?!?!? No 'evil scientist' scenarios needed...

1. Main reason: 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 (pigs with blood cell sugars 'knocked out')
* '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 this week's Good For! (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...

Overview: 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 direct 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 branches of government and their views on human cloning: The two different goals of human cloning have divided politicians (and physicians, clergy, the general public...) into two camps: those who want to ban both forms of human cloning and those who want an exception for research (therapeutic cloning).

1. President Bush 28 January 2003: In his State of the Union Address, President Bush states to congress that "...because no human life should be started or ended as the object of an experiment, I ask you to set a high standard for humanity and pass a law against all human cloning."

(Previous statement from Bush: Remarks from 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, for the following reasons. First, anything other than a total ban on human cloning would be unethical. Research cloning would contradict the most fundamental principle of medical ethics, that no human life should be exploited or extinguished for the benefit of another.")

2. The House of Representatives has passed an act to ban cloning in 2003:
28 February 2003: House Passes Ban on All Human Cloning: "The Weldon-Stupak Human Cloning Prohibition Act (HR 53) bans all human cloning — for reproduction or research — and imposes a $1 million fine and a prison sentence of up to 10 years for violators. PS. The Human Cloning Prohibition Act of 2001 passed by a vote of 265 to 162 in the US House of Representatives in 2001 but stalled in the Senate)

3. The Senate prepared two rival bills for 2003:

• 1) Senator Diane Feinstein (D-CA) said the 241-155 House vote sends the wrong message to America's medical research community. 'I remain confident that similar legislation will not pass the United States Senate,' Feinstein introduced the Human Cloning Prohibition Act of 2003 (S.1758), on Dec 2, 2002, that bans reproductive human cloning but permits the use of SCNT (somatic cell nuclear transfe)r for therapeutic purposes (research on Alzheimer's, Diabetes, Parkinson's, spinal cord injury, etc).
• 2) Senator Sam Brownback (R-KS) introduced the Human Cloning Prohibition Act of 2003 (S. 245) on Jan 29, 2003, with U.S. Sen. Mary Landrieu (D-LA). The bill would ban both forms of human somatic cell nuclear transfer.
• Neither of these bills came forward in 2003 because the Senators knew they did not have the required votes in support of either bill.
• Optional: S. 1758 or S. 245 - Which do YOU support? Let our Indiana Senators know how you feel about cloning! After all, our esteemed Senators are in Washington DC to represent you ! Yes you ! You elected them, you pay their salary... Don't delay - the Senate will act on cloning legislation soon!!
  Evan Bayh (D-IN) homepage and webmail address
  Dick Lugar (R-IN) homepage and e-mail: senator_lugar@lugar.senate.gov

Cloning in an Election year?

President Bush: Opposes federal funding of both forms as above, and has stated  he would sign bill that bans and criminalizes all cloning, including therapeutic cloning research if passed by Congress.

• June 30, 2003: GOP Moderates Question Bush Stem Cell Policy Will Bush hold his stand - a number of republican lawmakers are urging the Pres to consider revising his stand on stem cells.

Sen. John Kerry: (D-MA) Supports full federal financing of stem cell research : "If, as he says, the president believes that stem cell research may have lifesaving potential for millions, he should give scientists the tools to explore it rather than have the government impose burdensome restrictions which close the door to medical advances."
Quote: New York Times,  August 10, 2001

Mini-project: Cloning movies and cloning statements.

VI. Reproductive Cloning: Beyond the fringes of ethical behavior and responsible science...

"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.

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 maternal mitochondria). Still waiting!

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".) Still waiting!

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: The Raelians announce by press release (on a very quiet news day) Baby Eve, 'the first cloned baby': Is this the first step towards human immortality? OR, maybe just a media circus.... Without scientific data, we'll never know...
  • Great quote: "Nobody should get a story until they produce evidence. No matter how telegenic they are, no matter how many Star Fleet Command uniforms they have, if you don’t show up with a baby or a parent or a DNA test, or some witnesses who are credible, you shouldn't’t have a story." -- Arthur Caplan Director, Center for Bioethics, University of Pennsylvania
• 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...."
• Enough said on this topic.

VII. Human Embryonic Stem Cells and Therapeutic Cloning: high potential for saving millions of lives and curing disease, but not without ethical dilemma...

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 were first derived from the Inner Cell Mass of surplus blastocysts from In Vitro Fertilization (IVF) [history]. If implanted in a woman's uterus at 4-5 days after fertilization, IVF blastocysts may develop into a baby, 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 federal funding of research on any cell line created in the future to prevent the federal government from encouraging the destruction of human embryos. Lots of resources and info at: NIH's Stem Cell Information home page

• Most scientists now realize that there never actually were '64 stem cells lines', and even those few lines - maybe 10 - that exist will have very limited or NO usefulness for human therapeutics. PLUS, it is difficult for the NIH to produce enough of these cells for researchers who want to use them, with scientists needing to wait for 9 months - 1 year to obtain the cells they request....

Update: March 3, 2004: Harvard Researcher Doug Melton derives new stem cell lines that promise new medical treatments. From Wired, March 3, 2004: "Faced with limitations on stem-cell research from the Bush administration, a Harvard scientist has raised his own money and developed 17 new batches of stem cells, which he's offering to any researcher who needs them for just the cost of shipping. Melton is motivated by more than just scientific curiosity -- his 9-year-old son has type 1 diabetes, which requires daily insulin injections. He saw stem cells as a potential cure for his son's disease. Now, he heads up one of the leading labs in the country, working to create insulin-producing pancreatic beta cells from stem cells."

• Melton and his colleagues used private money donated by the Juvenile Diabetes Research Foundation and the Howard Hughes Medical Institute. They extracted the stem cells from embryos donated by the fertility clinic Boston IVF. " Scientists who would like to use these cells must also have private research funding, not federal (NIH) funds.

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 in a petri dish.
• 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
• Research Shows Therapeutic Cloning Can Cure Parkinson's-like Disease In Mice Nature Biotechnology, October 2003

• News Update: Thursday, Feb. 12, 2004 Researchers Clone Human Embryo for Stem Cells "Scientists in South Korea report that they have created human embryos and extracted human embryonic stem cells, the universal cells that hold great promise for medical research. Their goal, the scientists say, is not to clone humans but to advance understanding of the causes and treatment of disease. Patients with diseases like Parkinson's and diabetes have been waiting for the start of so-called therapeutic cloning to make embryonic stem cells that are an exact genetic match of the patient. Then those cells, patients hope, could be turned into replacement tissue to treat or cure their disease without provoking rejection from the body's immune system". (Quote: NY Times)

VIII. What about Adult Stem Cells or Umblilcal Stem Cells? From Adult Stem Cells: November 2001 Tech Review

Adult Stem Cells may be the answer to the ethical dilemma! "While the hES story has been unfolding, adult stem cells, have quietly been been developing in many academic labs and biotech companies. Adult stem cells are found in tissues throughout the body, from just below the surface of the skin to places like the liver and bone marrow. BUT - adult stem cells are not, critics say, the answer to every ill. "For certain diseases, adult cells appear very promising, for hepatic and cardiac diseases in particular," says Ronald McKay, a researcher at the National Institutes of Health. "However, if you're asking for a solution to Parkinson's disease or diabetes, I would say the cells that offer the best way are fetal and embryonic." Still, adult stem cells are already being tested in human clinical trials.

"Adult stem cells are much more biologically versatile, and capable of adopting many more cellular fates, than anyone previously thought. Adult stem cells plucked out of the skin, an easily accessible site for harvest, can develop into fat, muscle and neural cells. The bone marrow mesenchymal stem cell possesses the ability to form not only bone and cartilage, but also muscle, tendon, fat and stroma, the weblike matrix of tissue inside bones. In mice, transplanted bone-marrow-derived stem cells can migrate to the brain and develop into cells with characteristics of neurons and other types of brain cells. These studies reinforce the notion that the adult body maintains a reserve of stem cells, certainly in the bone marrow and probably in many other tissues as well—although the supplies seem to dwindle with age." [Image from 10/26/01 article from HMS Beagle article about Curis]

Pregnant? Thinking about banking your baby's umbilical cord blood? Read more here!

IX. 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

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...

X. Ethical Issues in hES Cells

Opponents of hES cell research: human life begins as soon as an egg is fertilized, a human embryo is a human being. Any research that involves the destruction of a human embryo, regardless of the life-saving potential of the treatments derived from these cells, is morally wrong.

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

Proponents of hES cell research: a fertilized egg may have the potential for human life but not until it has at least been successfully implanted in a woman's uterus. It is morally permissible to use surplus embryos, which would be discarded otherwise, or blastocysts created for the purpose of obtaining hES cells, for potentially life-saving biomedical research.

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

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?

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

Objectives: Ones in bold are the ones to study for the test! Don't worry about the ones in purple for the exam...

1. Please be able to state 'who' these cloned animals are and why we are discussing them (what makes them unique as cloned animals): Dolly, Polly, Cumulina, Noah, Cc, Prometia. Who is Ian Wilmut? (no, he is not a cloned mammal!) You do not need to know about any other cloned animals for the exam.
2. 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?
• Explain EACH term as it relates to cloning: Somatic Cell Nuclear Transfer

3. List three reasons scientists are interested in cloning livestock animals or research animals like mice. You do NOT need to know the details about cloning pets like cats and dogs.
4. Be able to differentiate between the two types of cloning (reproductive vs.therapeutic), but do NOT worry about specific legislation relating to cloning.
5. Regarding Reproductive Cloning, and who is pursuing this procedure: there will NOT be exam questions on the people involved, only on some of the SAFETY issues described later in the notes.
6. Where do hES cells come from (specifically), and what are the 4 amazing characteristics of hES cells? Why did President Bush agree to allow federal funding of research on stem cell lines that existed BEFORE August 2001 but not after?
7. What is human Therapeutic Cloning? How does it relate to hES cells and how does it relate to human cloning?
8. What is the potential usefulness of adult stem cells or umbilical stem cells?
9. List 4 medical and safety issues concerning cloned animals.
10. There will NOT be exam questions on ethical issues regarding stem cells