fall 2008 1110

Embryonic Stem Cells and Therapeutic Cloning

Review Embryonic Development from the Reproductive Cloning PP

The next few slides are pictures to remind you of the various stages of

embryonic development

A fertilized egg

Notice the nuclei from the egg and sperm fusing

4 day old embryos

5 day old embryo at the blastocyst stage

So what happens from here? • In the adult, most cells are specialized for

specialized tasks:– Red blood cells carry oxygen– Neurons transmit electrical signals– Pancreatic cells secrete insulin

• Cells of the embryo will then need to “commit” to a particular fate (decide which specialized cell type they will become).

• This process is called differentiationdifferentiation

(Remember our cells only do this once! Once a brain cell has decided to become a brain cell, it can not “change its mind” and differentiate into a muscle cell. That is good for us and our brains!)

You may never read some stories. You may read some stories only once. You might read other stories over and over.

All the cells in your body contain the same exact DNA. How your cells pick and choose the genes that will be expressed or “turned on” via transcription and translation is what makes cells different.

These books represent different chromosomes in all your somatic cells. The stories within are the genes. The words are the genetic code.

During development, most cells of the body “differentiate”.

So, what do stem cells have to do with all this?So, what do stem cells have to do with all this?

• Embryonic stem cells (ESC) are cells that have yet to differentiate

• Derived from the inner cell mass of the blastocyst stage of the embryo

Embryonic Stem Cells can be cultured in different laboratory environments to develop into a specific cell type.

Culturedembryonicstem cells

(developing an ESC line)

Different cultureconditions

Liver cells

Nerve Cells

Muscle Cells

Different types ofdifferentiated cells

Stem Cell Animation

http://www.stemcellresearchfoundation.org/WhatsNew/EmbryonicStemCells.htm

Watch the:

Medical ApplicationsMedical Applications

Medical Applications

• Repair a damaged tissue or group of cells that can't heal itself. – This might be accomplished by transplanting

ESCs into the damaged area and directing them to grow new, healthy tissue.

– It may also be possible to coax stem cells already in the body to work overtime and produce new tissue.

– Tissue/Organ growth and transplants?

Medical Applications

• Many of these therapies are still in the infant stages and we don’t know what other challenges will be faced as this technology progresses. – For example: How long will a stem cell

therapy last? Will these cells “behave” themselves (differentiate into the correct cell type, act as the intended cell type, and also not form tumors)?

• Parkinson’s Disease

• Alzheimer's

• Muscular Dystrophy

• Paralysis

• Diabetes

• Burns

• Genetic Disorders

What sort of diseases or disorders could stem cell therapies be utilized for?

just a brief list:

Where would researchers get the embryos that are the source of

embryonic stem cells?

• Fertility clinics– A couple undergoing fertility treatments using in vitro

fertilization generally will produce many more embryos than needed to bring about a pregnancy

– With a couple’s consent, embryos that would have been discarded as medical waste are utilized for research

– See the next page about a relatively recent study (June 2007) that says many couples would donate their unused embryos for this research

• Therapeutic cloning (SCNT) – we will talk about this later.

What is in Vitro Fertilization (IVF)?

• IVF is the process of fertilization by manually combining an egg and sperm in a laboratory setting

• There are a variety of ways this can be done

In vitro fertilizationSome procedures involved with IVF manually inject the sperm into the egg, and others simply allow fertilization to occur by

adding the sperm to the egg in the lab setting.

Why use IVF as a source of stem cells?

• According to a survey conducted in 2003, there are approximately 400,000 unwanted pre-embryos in the United States. (Source: Hoffman, D.I., et al. 2003. Cryopreserved embryos in the United States and their availability for research. Fertility and Sterility 79: 1063-1069.)

• These may no longer be needed for fertility purposes and remain frozen or could be destroyed

Other types of stem cells and current stem cell therapies

• Adult Stem cells: These stem cells (can give rise to a small number of different cell types) but are already "committed" to differentiating along a particular cellular development pathway (for example, the “stem cells” we all have in our bone marrow that can differentiate only into various types of blood cells). Current therapies utilize them from the bone marrow.

• Umbilical Cord Blood (Core Blood) Stem cells: similar to “adult” stem cells above but collected from the umbilical cord of a newborn.

Adult Stem CellsYou can also find these same type of stem cells in the blood system:

Peripheral Blood Stem Cells (PBSCs)

Used to treat leukemia, other cancers and various blood disorders

Less invasive than collecting bone marrow, but are sparse!

The bone marrow is the spongy core found in the bones and is a source of adult stem cells.

These stem cells are the precursor cells responsible for the formation of the blood cells (red blood cells, platelets, and white blood cells).

Multipotent stem cell rich blood found in the umbilical cord has proven useful in treating the same types of health problems as those treated using bone marrow stem cells

Umbilical Cord Blood Stem Cells

In 2005, there were more than 1,400 cord blood transplantations in adults, according to NETCORD, an international network that coordinates umbilical cord blood banks

Why would a parent consider this blood collection and why might it be considered to have advantages over bone marrow stem cells?

The process to collect and store a child’s cord blood doesn’t come cheap. The company “Cord Blood Registry”, for example, charges and “initial fee” of $1975 and then it is $125 per year for storage.

Differences between embryonic stem cells and adult stem cells:

-ESC can differentiate into any cell type (totipotent/pluripotent) while adult SC have already “committed” to a particular fate (multipotent).

Some Challenges in Research:-Adult stem cells are often present in only minute quantities and can therefore be difficult to isolate and purify. -There is also evidence that they may not have the same capacity to multiply as embryonic stem cells do. -They do not have the development potential that a ESC-Finally, adult stem cells may contain more DNA abnormalities—caused by sunlight, toxins, and errors in making more DNA copies during the course of a lifetime. -These potential weaknesses might limit the usefulness of adult stem cells.

Still so many questions about adult stem cells…

• How many kinds of adult stem cells exist, and in which tissues do they exist?

• What are the sources of adult stem cells in the body? Are they "leftover" embryonic stem cells, or do they arise in some other way?

• Why do they remain in an undifferentiated state when all the cells around them have differentiated?

• Is it possible to manipulate adult stem cells to enhance their proliferation so that sufficient tissue for transplants can be produced?

• Does a single type of stem cell exist—possibly in the bone marrow or circulating in the blood—that can generate the cells of any organ or tissue (a totipotent adult stem cell)?

• What are the factors that stimulate stem cells to relocate to sites of injury or damage

• We’ll talk about some new developments in adult stem cells later on

Therapeutic Cloning or Nuclear Therapeutic Cloning or Nuclear Transplantation Transplantation

(SCNT = Somatic Cell Nuclear (SCNT = Somatic Cell Nuclear Transfer)Transfer)

&&Stem CellsStem Cells

SCNT and Stem CellsNotice that UNLIKE reproductive cloning, all weare doing here is cloning embryonic cells andthen coercing them to differentiate into specificcells the patient needs.

SCNT Process (1)

Remove the nucleus from an unfertilized egg cell (A). (Because the egg cell is only 100 micrometers, or one-tenth of a millimeter wide, this is done under a microscope.)

Use a suction pipette (B) to hold the egg cell steady and a glass needle (C) to remove the cell’s nucleus.

http://www.pbs.org/wgbh/nova/sciencenow/3209/04-clon-nf.html

SCNT Process (2)

Gently push the glass needle through the tough shell that surrounds the egg cell.

Here, the glass needle is in the process of removing the nucleus from within the egg. If you look closely at the tip of the needle, you can just make out the genetic material being drawn out.

SCNT process (3)• The egg cell’s nucleus (A) has

been released outside of the egg. This nuclear material will no longer be needed.

• What remains is an “enucleated” egg (B). The nucleus, with the important genetic information, no longer remains but the enucleated egg still have certain molecules and other important factors that will ultimately help to establish embryonic stem cells.

SCNT process (4)

Ease the tip of the glass needle deep into the enucleated egg cell. Then, deposit the donor nucleus.

Inject the nucleus (at arrow) from a donor celldonor cell into the enucleated egg cell. (Such a donor cell might be a skin cell from a disease sufferer whom doctors hope to treat using the patient’s own stem cells grown in culture)

SCNT process (5)

After completing the nuclear transfer, the unfertilized egg cell is “activated” using a chemical or electrical treatment that stimulates cellular division.

The first division results in two cells (left image), the next makes four cells, and so on. This structure is now termed an embryo.

Now, at this point, if this was a sheep embryo and we implanted it into a surrogate ewe……what would we get?

SCNT process (6)The proliferating cells form a

structure called a blastocyst within days. It is roughly the same size as the egg cell.

The right-hand image shows the blastocyst “hatching”

An embryonic stem cell line has now been synthesized, the cells have the same DNA as the donor. These cells can be “customized”; they can be made into any cell/tissue/organ

of the body and transplanted (in theory) to the donor without immune rejectionwithout immune rejection.

(The cells may have their genes “corrected” before being transplanted to an individual.)

Therapeutic cloning (SCNT): Could the technique “fix” a gene

(alternative to gene therapy)?

• Theoretically a person with a gene defect can have a transplant of new cells/tissue/organ that are customized for them (no immune rejection because the transplanted cells are their own cells) EXCEPT a “good” copy of the gene they are defective in has been inserted

First things first…Can we make ESCs into the cells of

specific organs?• Scientists at Imperial College London reported in

2005 that they successfully coaxed embryonic stem cells to change into specialized lung cells. Researchers describe their study in the journal Tissue Engineering. – "Although it will be some years before we are able to

build actual human lungs for transplantation, this is a major step towards deriving cells that could be used to repair damaged lungs," the study's lead author, Anne Bishop, said in a press statement.

• Interesting and exciting, but again, still in infant stages

How big of a role does the media play in our impressions on stem

cell research?Hint: The answer is that the media plays a HUGE role!

The Media had a field day reporting on:Hwang Woo-Suk

Hwang Woo-Suk was a professor and prominent researcher in the College of Veterinary Medicine at Seoul National University. In February 2004, Hwang and his team announced that they had successfully created embryonic stem cells with the somatic cell nuclear transfer method, and published their paper in Science (one of the most prestigious scientific journals there is) in March 2004. A second paper, published in May 2005, reported the creation of 11 stem cell lines that genetically matched nine patients with spinal cord injury, diabetes, and an immune system disorder.

• This, the first breakthrough of its kind, renewed hopes that such stem cells could someday lead to insights into many hereditary conditions as well as the creation of replacement tissues genetically matched to patients and treat degenerative diseases.

• Hwang Woo-Suk even made TIME Magazines list of “People Who Mattered 2004”.

• Those hopes, however, began to unravel in June 2005, when someone sent a message to the "tip off " mailbox on the Web site of a long-running investigative TV news program in Seoul.

• The writer said his conscience had been bothering him over problems he knew of with Hwang's research…..

• Reporters interviewed co-authors of the 2005 paper and found that the majority had never actually seen the cloned embryonic stem cells.

• The university in Seoul investigated and concluded that the data reported in both the 2004 and 2005 Science papers was fabricated.

• Science magazine retracted the papers.• Hwang Woo-Suk was accused of "research

misbehavior” and was dismissed from his position in 2006.

What did he really do, and what was fabricated? • Hwang “did not have any proof to show that cloned

embryonic stem cells were ever created,” the panel said in a report, disputing the central claim in Hwang’s 2004 paper in the journal Science.

• In the paper, Hwang said he had cloned a human embryo and extracted stem cells from it. But the university cast doubt on whether an embryo was cloned, saying there is a high possibility it could have merely been a mutated egg, which could appear to have similar qualities of an embryo.

• “The 2004 paper was written on fabricated data to show that the stem cells match the DNA of the provider although they didn’t,” the report said.

• The panel upheld Hwang’s claims last year to have created the world’s first cloned dog, an Afghan hound named Snuppy. The journal Nature (another very prestigious scientific journal), which published Hwang’s cloned-dog article, said results from its independent tests also showed Snuppy was indeed a clone. (This accomplishment not as noteworthy because many other animals have been closed previously).

What was the effect of this on our society?

• Some say the reputation of science and stem cell research suffered, other aren’t so bleak. – AAAS:

Stem Cell Experts Assess the Impact of Hwang Fraud on Research, Public Trust

ESCs and PoliticsESCs and Politics

• Bush placed a federal ban on using federal funding to make any new human ES cell lines. (Only those lines made before 2001 can be used.)

• California and New Jersey have facilities that use state and private funds.

• Other states that can use state funds (with limits): Connecticut, Illinois, Indiana, and Maryland

July 2006: Stem Cell Research Enhancement Act: Passed by the Senate

• The bill expands the number of stem cell lines that are eligible for federally funded research.  Currently, federal policy on human embryonic stem cell research permits only the use of stem cell lines derived before August 9, 2001 for federally funded research.

• The Stem Cell Research Enhancement Act lifts that restriction. Stem cell lines shall be eligible for federally funded research regardless of the date on which they were derived.

July 2006: Stem Cell Research Enhancement Act: Passed by the Senate

• The bill expands the number of stem cell lines that are eligible for federally funded research.  Currently, federal policy on human embryonic stem cell research permits only the use of stem cell lines derived before August 9, 2001 for federally funded research.

• The Stem Cell Research Enhancement Act lifts that restriction. Stem cell lines shall be eligible for federally funded research regardless of the date on which they were derived.

VETO!!!By President Bush July 19, 2006

• This veto was in 2006 and another one followed in June, 2007

• There is already a lot of chatter concerning how quickly President Obama will move on making changes to the current legislation

You might be thinking:• What’s the problem with using existing stem cell lines?

– Those against stem cell research say:• Still destroying an embryo for profit

– Scientists say:• Too little genetic diversity• Some cell lines contaminated• The existing cell lines have been grown with mouse “feeder cells” and

therefore Under Food and Drug Administration rules, such transplants with existing cells would be classified as "xenotransplants," or transplants of animal tissue. These would be subject to strict requirements on both researchers and patients

• Why not just use Adult Stem Cells?– The potential is not present in these type of cells than what is

available in a totipotent or pluripotent cell like and ESC. We are still learning quite a bit about adult stem cells and their potentials. (See back to Adult Stem Cells)

• What’s the big deal about not approving federal funds for stem cell research?– See next several slides.

Why Does Federal Funding Matter?

• Regardless of the veto, stem cell research will continue, but federal funding would dramatically change the scope of this research and widen the circle of scientists involved.

• This type of research requires big time $$$ and mostly large government organizations (such as the NIH) can provide that support.– According to the President’s Council on Bioethics, “While there

are currently no federally legislated constraints on the use of private funds for this research, there is a consensus opinion in the scientific community that without NIH support for newly created ESC lines progress in this important realm of research will be severely constrained. “

• Federal regulations would be able to be enacted with

rules for oversight.

Federal Funding• If President Obama Says...• Yes

The floodgates would open. Right now most scientists steer clear of stem-cell research because they have to: if any part of their lab receives federal money (and most do), they can't touch this research. If that changes, hundreds of labs across the country, including medical powerhouses like those at Harvard and M.I.T., would probably begin work on stem cells. Scientists would be able to share findings freely and review one another's conclusions. The government could choose to regulate how embryos are cultivated, handled and ultimately destroyed. Treatments would probably come sooner. (Of course, there are no guarantees!)

• NoResearch would proceed but only in the handful of labs willing to fund it on their own. These labs are subject to minimal oversight. They rarely consult with one another, research doesn't get peer-reviewed, and studies may be unknowingly (and unnecessarily) duplicated. Many of the nation's top scientists who would otherwise lead the research effort would remain on the sidelines. And commercial pressures could make private labs focus more on research that might turn a profit than on studies that advance general knowledge. Says James Thomson, the stem-cell pioneer: "Industry and other countries will go forward. The field will progress without federal funding, but very, very slowly."

If that political debate isn’t confusing enough…let’s throw stem cell research via SCNT in!

(from the AAAS, American Association for the Advancement of Science) • Arguments Against Nuclear Transplantation Research

– Proponents of a comprehensive ban on nuclear transplantation for research and reproductive purposes raise two main arguments. Religious conservatives argue that human embryos should be afforded a moral status similar to human beings and should not be destroyed, even in the course of conducting research. They also argue that permitting nuclear transplantation would open the door to reproductive cloning, because a ban only on implantation would be difficult to enforce. In this second argument, conservatives are joined by a coalition of environmental, women's health, and bioethics groups who are not unalterably opposed to nuclear transplantation, but believe that it should not be permitted until strict regulations are in place.

• Arguments For Nuclear Transplantation Research– Proponents of a ban solely on reproductive cloning that would permit nuclear

transplantation research, include a coalition of science organizations, patient groups, and the biotechnology industry. These groups argue that the moral status of a human embryo is less than that of a full human being, and must be weighed against the potential cures that could be produced by research using nuclear transplantation. They contend that a ban on implantation on the product of nuclear transplantation would be no more difficult to enforce than a ban on nuclear transplantation itself. They argue further that criminalizing scientific research, which has been done only very rarely in the past, would set a bad precedent.

• Listen or read to find out more at:http://www.pbs.org/newshour/bb/health/jan-june07/stemcell_06-07.html