Therapeutic Cloning and Stem Cell Therapy

Lea McLaughlin Shelby Seebacher Zach Homitz Dan Lang

Jamie Thomson• Born: Dec. 20, 1958• First person to isolate and culture ESCs. • Developmental biologist and vetetarian

at the university of Wisconsin at Madison.

• In 2008, Time Magazine, named him one of 100 of the most influential people in the world.

• 2007: Thomson's laboratory devised a method for modifying human skin cells in such a way that they appear to be embryonic stem cells without using a human embryo. They are called Induced Pluripotent Stem Cells.(IPS cells)

What is SCNT : Somatic Cell Nuclear Transfer?

• This is also known as therapeutic cloning.• Therapeutic cloning is a technique that would be used to

produce cloned embryos, but only to create embryonic stem cells that can be used to repair damaged or defective tissue in the donor of the cloned cells.

• The process: – remove the nucleus of an unfertilized egg cell– replace it with the genetic material from the nucleus of a

"somatic cell" (usually skin or muscle cell)– stimulate this cell to begin dividing by chemicals or electric

shock – Once the cell begins dividing, stem cells can be extracted 5-6

days later and used for research

SCNT : Somatic Cell Nuclear Transfer

• The embryonic stem cells contain the donor of the somatic cell’s DNA ;therefore, the ESC can be used to generate cells and tissues and they will not be rejected by the patient. (Who donated their somatic cell)

• It is still not clear how to produce whole complex organs using this method.

Let’s watch

What are Embryonic Stem Cells?

What is a stem cell?

• Cells with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

What is an embryonic stem cell?

• Undifferentiated cell derived from a five day preimplantation (before the embryo implants into the uterus wall) embryo that are capable of dividing without differentiating for a prolonged period in culture and are known to develop into cells and tissues of the three primary germ layers.

• Undifferentiated- a cell that has not yet developed into a specialized cell type.

Where are they found?

• Embryonic Stem Cells are found in the inner cell mass of a hollow blastocyst.

• A blastocyst is an embryo that is 3 to 5 days old. The embryos that are used for embryonic stem cells are fertilized in vitro.

4 or 5 day old embryo.

Inner Cell Mass

How do embryonic stem cells grow outside the body?

• For embryonic stem cells to grow outside the body, they must be cultured in the presence of embryonic fibroblasts.

• Fibroblasts are the least specialized cells in the connective-tissues family.

• Fibroblasts are needed because they provide nutrients to keep the embryonic stem cells in an undifferentiated state, and allow the cells to continue to divide.

• When the cells are taken out of the presence of fibroblasts, the cell spontaneously differentiate into a cell type. – Differentiate- the process where a

unspecialized cell acquires the features of a specialized cell. It is controlled by the interaction of a cell’s genes and physical and chemical condition outside the cell, usually through signaling pathways involving proteins imbedded in the cells surface.

Fibroblasts

More on ESCs• Embryonic stem cells are

cells that have almost unlimited developmental potential. They are considered pluripotent.

• Pluripotent-having the ability to give rise to all cell types of the body, but not the extra-embryonic cell types.

• Totipotent- having the ability to give rise to all cell types of the body, plus all the cell types of extra- embryonic cells, like the amnion, chorion and other components of the placenta.

Why do we need them?

• Study cell differentiation• Understanding prevention and treatments of birth

defects • Identify drug targets and test potential therapeutics• ESCs can make testing drugs safer.

– Before, drugs were tested on animals before they were used on humans, but some drugs that tested on animal improved their condition. When tested on humans, the drugs were toxic to human heart.

– Now, human ESCs that differentiate into heart cells can be tested before the drugs go to clinical trials.

–Making an embryonic stem cell line is not very efficient. When the scientists are successful, they will carefully remove the ESCs and plate them on several different plates. They will repeat this process so that they end up with millions of ESCs.

Florescent microscope picture of ESCs.

Advantages and Disadvantages of ESCsAd

vant

ages

Dis

adva

ntag

es Embryonic stem cells are pluriopotent, so they can form any tissue except fetus or placenta.

They can be cultured in vitro to form million of ESCs.

Will not be rejected if cell somatic cell donor is the patient.

Difficult to differentiate uniformly and homogeneously into a target tissue.Capable of forming tumors or promoting tumor formation called tumorigenic. • Why? The

intermolecular mechanics of stem cells are similar to the mechanics of cancerous cells.

Destruction of developing human life

More on ESCs

• Embryonic stem cells have the potential to become all tissues of the body to create tissue to repair or create organs.

• Embryonic stem cells are not used in clinical trials, and are still in a highly experimental stage.

What is an Adult Stem Cells?

What is it?

• Adult Stem Cells are undifferentiated cells found in many organs and differentiated tissues with a limited capacity to self renew and differentiate. These cells vary in in their differentiation capacity, but is usually limited to cell types in the organ of origin.

What do they do in the body?• Their main role in a living organism is to

maintain and repair the tissue in which they where found.

• Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, fat, teeth, heart, gut, liver, ovarian epithelium, and testis. They are thought to be located in in a particular area of each tissue called a "stem cell niche".

• Stem cells may remain non-dividing for periods of time until they are activated by a need for more cells to maintain tissues, disease or tissue injury.

• As a baby adult stem cells are at their highest amount and as we get older, ASCs drop in number.

Adult Stem Cell Analogy • Everybody is born with a certain amount of stem cells,

specifically, "adult stem cells." We can relate this to a "bank account”. Each person can make "withdrawals" throughout his or her life as needed.

• Not all bank accounts are created equal, some people are born “with a silver spoon in their mouth” and others are born poor, but most are middle class.

• Some people are able to "spend" their stem cells more than others because they have more to spend.

• Environmental factors also play a key role in determining how rapidly some one’s "bank account" of (adult) stem cells is used up. However, stem cells continually diminish with age.

Terms

• Adult stem cells can be multipotent stem cells are capable of differentiating into multiple types of cells, but not all possible types.

• Multipotent-having the ability to develop into more than one cell type in the body.

• Many types of adult stem cells exhibit "monopotency", meaning that they are capable of differentiating into only one particular type of cell. This "specialized" feature has typically been considered one of the major drawbacks of adult stem cells.

• Monopotency- Cells capable of differentiating into only one particular type of cell. This "specialized" feature has been considered one of the drawbacks of adult stem cells.

Transdifferentiation

• Experiments have been reported that certain adult stem cell types can differentiate into cell types seen in organs or tissues other than those expected from the cells' predicted lineage

• Example-– brain stem cells that differentiate into blood cells. – blood-forming cells that differentiate into cardiac

muscle cells.

Differentiation of different stem cells

• Hematopoietic stem cells give rise to all the types of blood cells

• Mesenchymal stem cells give rise to a variety of cell types: bone cells, cartilage cells, fat cells, and other kinds of connective tissue cells such as those in tendons.

• Neural stem cells in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal .

Hematopoietic stem cell

Differentiation of different stem cells

• Epithelial stem cells in the lining of the digestive tract occur in deep crevasses and give rise to several cell types.

• Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.

How to collect Adult Stem Cells?

• It depends on the location of where the adult stem cells are extracted from.

• Ex. Peripheral blood stem cells (PBSCs) are collected by an apheresis.

• Apheresis- a process in which the donor is connected to a special cell separation machine by a needle inserted in the vein.

• Blood is taken from one vein and is circulated through the machine which removes the stem cells and returns the rest of the blood and plasma back to the donor through another needle inserted into the opposite arm.

How to collect Adult Stem Cells

• Ex.2• Collecting bone marrow

involves gathering stem cells with a needle placed into the soft center of the bone marrow. Most sites where bone marrow is collected is located in the hip bones and the sternum.

Advantages• Adult stem cells are ethically and politically noncontroversial.• Special adult-type stem cells from bone marrow and from umbilical cord

have been isolated recently which appear to have high plasticity as the embryonic type– Plasticity-the ability of an adult stem cell from one tissue to generate the

specialized cell type or types of another tissue• Already somewhat specialized so inducement may be simpler. • Not immunogenic—recipients who receive the products of their own stem

cells will not experience immune rejection. • Some adult stem cells are easy to gather(skin,muscle, marrow, fat), while

others may be more difficult to obtain (brain stem cells). The umbilical and placental stem cells are readily available.

• Non-tumorigenic—do not usually form tumors • No harm done to the donor

Disadvantages

• Can sometimes be difficult to find in large numbers.

• May not live as long as ES cells in culture • Low Plasticity - (with the exception bone

marrow and umbilical cord)—may be more difficult to reprogram to form other tissue types.

• Once cultured, the cells are slow to grow.

ESCs vs. ASCs

-Can become all cell types of the body because they are pluripotent

-Grown relatively easily in culture-Rejection depends on donor of somatic cell

-Controversial

-Most are thought to be limited to differentiating into different cell types of their tissue of origin, multipotent or monopotent-Rare in mature tissues, so isolating these

cells from an adult tissue is challenging-Less likely to initiate rejection after

transplantation-No ethical problems

-Both differentiate

into more specialized cell

type

Stem Cell Therapy Used Today

Bone Marrow• Bone Marrow transplant is the most well

known. • Bone marrow contains hematopoietic stem

cells which produce blood and immune cells.– Used to treat leukemia or blood or immune

system diseases• Existing bone marrow may have to be killed by

chemotherapy or radiation.

Peripheral Blood stem cells

• Small number of hematopoietic stem cells circulate in the bloodstream.

• It is a less invasive procedure.

• Collecting enough hematopoietic stem cells is difficult because of the small number.

Umbilical Cord Blood

• Cord blood contains hematopoietic stem cells. These hematopoietic stem cells are usually called neonatal stem cells.– They are less mature than those stem cells found in the

bone marrow. • Rejected less often because they have not developed

proteins that can be recognized by patients immune system.

• Lack well developed immune cells, so they are less likely to attack recipient’s body-graft versus host disease.

Types Transplants– In autologous transplants, patients receive their own stem cells. – In syngeneic transplants, patients receive stem cells from their

identical twin. – In allogenic transplants, patients receive stem cells from their

brother, or parent or person who is not related to the patient .

Skin

• Used on patients who suffered from extensive burn damage.

• Skin was made using stem cells from bone marrow.• Once on the patient the dermal layer begins to

regenerate and stem cells were differentiated into skin cells.

• Now skin stem cells found on a hair follicle.– stem cells in the hair follicle receive a signal to divide

therefore a round of hair growth begins.

Adult stem cells used to create windpipe

• Scientist implanted a new windpipe into a woman whose airway was badly damaged by tuberculosis.

• Doctors took adult stem cells from the bone marrow of Claudia Castillo.

• Once in laboratory dishes scientists injected various chemicals to induce the cells to turn into highly specialized cells.

• Scaffold-a trachea , a portion of the windpipe, taken from a 51-year-old man who died. The trachea was rinsed with antibiotics, and the cells were removed with enzymes.

• The stem cells coated the outside and inside of the trachea.

• They applied nutrients and chemicals to promote the growth of new layers of tissues.

Current Experimental Procedures

Experiments Using Adult Stem Cells

• Horses-stem cells are used to heal tendons and ligaments.

–The stem cell used is mesenchymal. (come from bone marrow, umbilical cord, and fat tissue)

-James Andrew Lee, M.D., was the first researcher to demonstrate in vivo(in the body) that exposing adult stem cells derived from fat to different growth factors to generate bone, cartilage and other cells.

Dental stem cells

• Dental adult stem cells were put in the hippocampal areas of mice. Hippocampal is a part of the brain that play an important roles in long term memory and spatial navigation. These cells started the growth of new neural cells.(many of these formed neurons)

• More potential for adult stem cells

Spinal Cord injury

– In a mouse, grafting human neural stem cell(adult stem cells) in the spine, the cell differentiate appropriately and help mice to recover.

Human neural cells

Insulin like Growth Factor

• Uses an insulin like growth factor(IGF)– Promotes muscle repair

• Experiment using a mouse

IPS(Induced Pluripotent Stem Cells)

What is it?

• IPS cells are mature cells that have been genetically reprogrammed to an embryonic stem cell like state.

• IPS cells are very similar to ESCs. – Both cells a had round shape, a large nucleus and

cytoplasm. Colonies of IPS cells were also similar to that of ESCs.

– IPS cells divide at a rate equal to ESCs.– IPS cells expressed cell surface antigenic markers expressed

on ESCs.– Express same genes– Both have high telomerase activity.

Why are they useful?

• IPS cells are valuable tools for drug development and modeling of diseases, and scientist hope to use them in transplantation medicine.

How do you make them?

• Viruses are used to introduce the reprogramming factors into adult cells.

• This process must be carefully controlled and tested before it can be used on humans. In animal studies, the virus used to introduce stem cell factors sometimes cause cancers.

• Researchers are looking for a non-viral way to introduce stem cell factors.

Viral vectors

• In 2006, Shinya Yamanaka used retroviruses to insert four genes into the chromosome of mouse skin cells. This successfully turned the adult stem cells to IPS cells, which could become any of the body tissues.

• The following year he redid his experiment with human cells. He was able to make IPS cells, but it was too risky to take cells grown from IPS cells and transplant them into a human.

• The genes(used to reprogram) themselves are known to trigger cancer. Also when retroviruses land in the chromosome, they can disrupt genes that normally keep cancers from forming.

Genes Used to Convert Cells

• Oct4 and Sox2 play an important role in the maintenance of embryonic stem cell pluripotency.

• Myc and Klf4 are the two other genes scientists have discovered that are necessary to convert somatic cells to IPS cells.

Other Methods

Adenoviruses (instead of retroviruses)These viruses do not normally integrate into the genome of the cell and therefore only have a little risk of cancer.Problem: This method is not as efficient as using retroviruses. Also, in a study by Hochedlinger’s team, 3 out of 13 cell lines had a complete extra set of chromosomes. Cells could be fusing with one another.

Viruses are not used anymore because of the cancer risk.

piggyBac

• This method uses transposons, small pieces of DNA that hops in and out of the genome.

• The four genes used to reprogram are inserted into the transposons (piggyBac vector)

• An enzyme called transposase causes the vector to “jump” into the genome.

• Made IPS cells!!• This system allows you to remove the vector, by adding

more transposase in order to mobilize the vector with in the genome.

• Some piggyBac vectors jumped out and into a different part of the genome, but under certain circumstances, the vector would jump out and never jump back in.

Transposons are cheaper to produce, so, more available to the population. Also, viruses cannot carry large genes.

No genes • This process uses proteins instead of genes because proteins do the

reprogramming.( genes make the proteins)• Polyarginine is attached to the end so that the proteins are positively

charged and can easily cross the cell membrane. • The reprogramming proteins are quickly broken down (repeated cycle

of protein addition is needed)• There is no genetic material left behind that could be reactivated. (no

cancer threat)• Not as efficient as retroviruses and genes but better than

adenoviruses. • Valproic acid is sometimes added to improve the efficiency and other

molecules are being screened.• Preference would be for proteins!!!!

Plasmid

• Does not integrate into chromosomes to reprogram cells.• The vector which is derived from the Epstein-Barr virus

but without viral machenery. It is called an episome. It only replicated once per cell cycle and is lost over time because copies do not always segregate properly during cell division. – Reprogrammed cells that did not lose the plasmid form

teratomas(tumor).– After cells divide often enough so that the episome was lost.

PCR and other tests confirmed that the vector genes were gone

IPS Cells

Disadvantages

• IPS cells are expensive to make.

• Genes used to reprogram are linked with cancer.

• If a patient has a heart attack and needs immediate care IPS cells will not work. They take several weeks to dedifferentiate and several other weeks to grow enough to do a transplant.

Advantages

• Patient derived-autologous transplant(no rejection)

• Don’t require use of embryos.

• If the goal is to eventually replace the heart and there is enough time then IPS cells can work.

• Efficiency of reprogramming cells to IPS cells is low.

• IPS cells is a newer field and needs more research before any treatment humans can be done.

Conclusion

• Ethics – Human embryonic stem cells – Pluripotent stem cells (cells that can develop into many

different cell types of the body) are isolated from human embryos that are a few days old.

– Pluripotent stem cell lines have also been developed from fetal tissue (older than 8 weeks of development).

– Church views-strongly disagree with the use of embryonic stem cells. They view a fertilized egg as baby and any harm done to the fertilized egg as harming a child.

• Adult stem cells-– No controversy because no work is being done

with the embryo.• IPS cells-– No controversy because no work is being done

with the embryo.

Creating Organs• The hope is create whole organs for injury and diseases.• Questions to think about when creating an organ.• Ex. Kidney• First, you need to obtain a type of stem cell that can ultimately become a kidney. Would this be an

embryonic stem cell? A fetal stem cell? Or will an adult stem cell do the job? How do you know? Where will you get the cells?

• Next, you have to coax the stem cells to grow and divide in a lab dish. Which nutrients will help them grow? Do they need other types of cells around to encourage them to divide? If so, can you obtain them?

• After you have enough stem cells, you must simulate the physical environment required for them to differentiate into a functional kidney. How do you make the cells begin differentiating? Can you simulate the physical environment they need? Can you create a physical scaffold which the cells can shape themselves into a kidney? How will you help the developing kidney grow blood vessels to supply oxygen and nutrients to cells on the interior?

• Most of this research is performed in stem cells obtained from other organisms, such as mice, rats and frogs.• The final step, creating a functional organ out of differentiating stem cells, is more challenging. Obstacles to

success include the problem of delivering oxygen and nutrients to cells on the inside of the organ, as well as creating physical scaffolds upon which to grow and differentiate cells.

Making a Heart

Using regeneration to heal a bladder

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