Stem cell & therapeutic cloning Lecture

Caption: This image depicts a colony of human embryonic stem cells grown over a period of 10 months in the absence of mouse feeder cells. The cell nuclei are stained green; the cell surface in red. Photo Credit: Courtesy Ren-He Xu/University of Wisconsin

Caption: Cloned Mouse

Description: A colony of embryonic stem cells. 10X. Image in the Public Domain.Description: (A) Human ESCs; (B) Neurons derived from Human ESCs. Images courtesy of Nissim Benvenisty.

What is stem cell research?Experimental model systems, understanding

more about development

Cell-based therapies

Pharmaceutical research and testing

Stem Cell History1998 - Researchers first extract stem cells from human embryos 1999 - First Successful human transplant of insulin-making cells from

cadavers2001 - President Bush restricts federal funding for embryonic stem-

cell research2002 - Juvenile Diabetes Research Foundation International creates

$20 million fund-raising effort to support stem-cell research2003?? - California ok stem cell research–terminated!2004 - Harvard researchers grow stem cells from embryos using

private funding2004 - Ballot measure for $3 Billion bond for stem cells

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”.

The next few slides are pictures to remind you of the various stages of embryonic development=creation of life!

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

Implantation

What Are Stem Cells?

Stem cells are the raw material from which all of the body’s mature, differentiated cells are made. Stem cells give rise to brain cells, nerve cells, heart cells, pancreatic cells, etc.

What’s So Special About Stem Cells?

They have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses.

They can replicate themselves over and over for a very long time.

Understanding how stem cells develop into healthy and diseased cells will assist the search for cures.

SCAN – Stem Cell Action Network

Two Kinds of Stem Cells Embryonic (also called “pluripotent”) stem cells (ESC) are capable of developing into all the cell types of the body.

From blastocysts left over from In-Vitro Fertilization in the laboratory

From aborted fetuses

Adult stem cells are less versatile and more difficult to identify, isolate, and purify.

Stem cells have been found in the blood, bone marrow, liver, kidney, cornea, dental pulp, umbilical cord, brain, skin, muscle, salivary gland . . . .


ESCs are pluripotent

They retain the ability to develop into nearly any cell type

All stem cells are not alike!• Some stem cells have more potential than others.POTENCY describes this flexibility.• Unipotent stem cells form only one type ofspecialized cell type.• Multipotent stem cells can form multiple types ofcells and tissue types.• Pluripotent stem cells can form most or all celltypes in the adult.• Totipotent stem cells can form all adult cell typesas well as the specialized tissues to supportdevelopment of the embryo (e.g., the placenta)

Generates every cell in the body including the placenta and extra-embryonic tissuesCan form the entire

human being

Cannot form the entire human being

Can generate every cell in the body except placenta and extra-embryonic tissues

Become specific cell types; may or may not have plasticity

Transdifferentiation?! WHAT?

STEM CELL (DEFINITION)

A cell that has the ability to continuously

divide and differentiate (develop)

into various other kind(s) of cells/tissues

Stem cell type Description Examples

TotipotentEach cell can develop into a new individual

Cells from early (1-3 days) embryos

PluripotentCells can form any (over 200) cell types

Some cells of blastocyst (5 to 14 days)

MultipotentCells differentiated, but can form a number of other tissues

Fetal tissue, cord blood, and adult stem cells

Special characteristics of ALL stem cells Self-renewal (proliferation)- the ability of a stem

cell to clone itself indefinitely by cell division.

Relocation and Differentiation are abilities of stem cells to “migrate” to where they’re needed in the body and specialize into a particular type of mature cell

Two Sources of Embryonic Stem Cells

1. Excess fertilized eggs from IVF (in-vitro fertilization) clinics

2. Therapeutic cloning (somatic cell nuclear transfer)


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

differentiateDerived from the inner cell mass of the blastocyst

stage of the embryo

What is a blastocyst?Trophoblast - a hollow sphere of cells that

develops into the extra-embryonic membranes such as the placenta, umbilical cord, and amnion.

Inner cell mass (ICM) - embryonic stem cells are the ICM

How Big Is a Blastocyst?

A. In vitro fertilization - current method of deriving an hESC line

Eggs and sperm donated and fused to create a fertilized egg in a petri dish

Fertilized egg matures into a blastocyst

Embryonic stem cells extracted from blastocyst

Cells re-plated on another petri dish and grown in culture

Q. Where would researchers get the embryos that are the Q. Where would researchers get the embryos that are the source of embryonic stem cells?source of embryonic stem cells?

Fertility clinicsA 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

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

In vitro fertilization - current method of deriving an hESC line

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.


Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment.

These surplus embryos can be used to produce stem cells.

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

Regenerative medical research aims to develop these cells into new, healthy tissue to heal severe illnesses.

Other types of stem cells and current stem cell therapiesAdult 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 cells Adult stem cells are cells

found in post-natal tissue that can yield only the specialized cell types of the tissue from which they originated.

hematopoietic stem cells mesenchymal stem cells umbilical cord stem cells amniotic fluid stem cells

http://www.artsalive.ca/upload/dan/Articles_anatomy_full.jpg

Reprinted with permission of Do No Harm. Click on image for link to website.

Bone Marrow Stem Cells

Early Successes – Adult Stem CellsHuman neural stem cells can migrate extensively in the

brain after injection. Adult stem cells have been isolated from amniotic fluid,

peripheral blood, umbilical cord blood, umbilical cord, brain tissue, muscle, liver, pancreas, cornea, salivary gland, skin, tendon, heart, cartilage, thymus, dental pulp, and adipose tissue.

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

Medical ApplicationsRepair 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?

Parkinson’s DiseaseAlzheimer'sMuscular DystrophyParalysisDiabetesBurnsGenetic DisordersSpinal Cord injuryHeart DiseaseInfertility

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

just a brief list:

People with illnessesParents of children with illnessesPhysicians and scientistsResearch Institutes: NIH, Universities, Corporations and shareholders (those who might profit)Government (s)

USA, California, UK, Australia, Czech Republic,South Korea

TaxpayersChurches and clergyThe cells themselves

Reprinted with permission from the Univ. of Kansas Medical Center. Click on image for link to originating website.

Principle of Cell-based therapy

Cell-based therapy-Type 1 diabetes

Cell-based therapy-Spinal Cord Injury

Differentiate(+ growth factors)

START

*Treatment may not work for the chronically paralyzed

time

Oligodendrocytes

Clinical trials starting for treatment of spinal cord injury* in humans(after much data gathered using ratsas an animal model)

injuredmarkedlyrecovered

Drug Development-Cancer Stem Cells

Reya, T., et al. Nature, 2001

Cell surface markers are a key difference.

Traditional solutionsStatin drugs, blood

thinning, beta-blockers, ACE inhibitors, angioplasty / stents

These solutions are worthwhile but do not address the existing damage to the myocardium

http://www.nlm.nih.gov/medlineplus/ency/imagepages/17004.htm

Cell-based therapy-Cell sources for the myocardium

Smooth muscles cell, skeletal myoblasts, endothelial progenitors

Adult stem cells

Embryonic stem cells

Cardiac progenitors

D. Srivastava & K.N. Ivey Nature 441, 1097-1099(29 June 2006)

Note myofibrils in finger–like projections attaching to microrod. Also note all myofibrils are highly oriented.

Cell-based therapy-Delivery chemotherapeutic agents

Medical ApplicationsMany 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)?

SCNT and Stem Cells Therapeutic Cloning or Nuclear Transplantation (SCNT = Somatic Cell Nuclear Transfer)

Notice that UNLIKE reproductive cloning, all we are doing here is cloning embryonic cells and then coercing them to differentiate into specific cells the patient needs.


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.

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

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”

(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

Asexual reproduction=Reproductive Cloning

The Association of Reproductive Health Professionals

1998 – Mice cloned 1998 – Cows cloned

History of Somatic Cell Nuclear Transfer (Cloning)

1952 – Briggs and King cloned tadpoles

1996 – The first mammal cloned from adult cells was Dolly, the sheep.

2000 – Pigs cloned

2001 – Cat cloned 2002 – Rabbits cloned 2003 – Mule cloned

“CC” Carbon Copy

2004 – Bull serial-cloned 2005 – Dog cloned

History of Somatic Cell Nuclear Transfer (Cloning)

Cloning A Sheep

(Roslin Institute http://www.roslin.ac.uk/library/)

Challenges of Reproductive Cloning

Many animals were cloned after Dolly Cats, pigs, mice, goats, cattle, rabbits

Obstacles:Very inefficient processMost clones have deleterious effects & die earlySurviving clones show premature aging signsSigns of abnormal embryonic development:

Clones & their placentas grow much faster than expected in surrogate mom

Human Clones?

Are there human clones?

Yes – Identical Twins

(Time 2.19.01)

Learn from History-don’t repeat natures mistakes!

3 goals of therapeutic cloning by SCNT in humans

Use embryo as source for ES cellsUse ES cells to generate an organ

In this case the organ generated will carry cells with the same genetic markers as the patient (recipient)

Correct genetic error in ESC in blastula stage

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 SCNTREASON: 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 SCNTTo harvest large number of eggs:

Excessive hormone treatment of females to induce high rate of ovulation

Surgery to retrieve eggsBoth can be harmful to female humanCow/pig females may be used

Cow/pig eggs will carry species-specific mitochondrial genesMixing species is reason for concern!-The island of Dr The island of Dr

Moreau!Moreau!

Common OpinionsReproductive 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? ORthe clone is explanted into culture dish to generate ES cells

Early Successes – Human Cloning2001 – First cloned human embryos (only to six cell stage) created by

Advanced Cell Technology (USA)2004* – Claim of first human cloned blastocyst created and a cell line established (Korea) – later proved to be fraudulent

Creating a human stem cell by cloning is next to Creating a human stem cell by cloning is next to impossible due to the complexity of primates-impossible due to the complexity of primates-

Thank God!Thank God!

*Hwang, W.S., et al. 2004. Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst. Science 303: 1669-1674.

James Watson (L) and Francis Crick (R), and the model they built of the structure of DNA (based on the data garnered by Rosalind Franklin!).

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.

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 cloned previously).

Why the Controversy Over Stem cells?ESCs are derived from extra blastocysts that would

otherwise be discarded following IVF.

Extracting stem cells destroys the developing blastocyst (embryo).

-Questions for Consideration-Is an embryo a person?Is it morally acceptable to use embryos for research?When do we become “human beings?”

For further information: http://bioethics.gov/cgi-bin/bioeth-counter

The Ethical DebateIn favor of ESCR:

Embryonic stem cell research (ESCR) fulfills the ethical obligation to alleviate human suffering.

Since excess IVF embryos will be discarded anyway, isn’t it better that they be used in valuable research?

SCNT (Therapeutic Cloning) produces cells in a petri dish, not a pregnancy.

Against ESCR:

In ESCR, stem cells are taken from a human blastocyst, which is then destroyed. This amounts to “murder.”

There is a risk of commercial exploitation of the human participants in ESCR.

Slippery slope argument: ESCR will lead to reproductive cloning.


That’s all folks!

Education

Stem cell & therapeutic cloning Lecture