CURRENT TOPICS IN STEM CELL RESEARCH

Disclaimer: This presentation contains graphic visuals

Dr. Houda DarwicheUF CPET

23 July 2013

Unlimited Potential?

Reproductive cloning is the process of making an identical genetic copy of an existing organism Naturally occurring clones – Identical twins Artificial Embryo splitting Somatic cell nuclear transfer

ES Cells and Reproductive Cloning

Somatic Cell Nuclear Transfer (SCNT)

1901 – Hans Spemann split a fertilized 2 cell embryo = 2 tadpoles Artificial embryo splitting

History of Cloning and Nuclear Transfer

Hans Spemann

1977 – John Gurdon cloned Xenopus laevis from differentiated cells from an albino tadpole Injected tadpole intestinal cell nuclei

into enucleated oocytes

1978 – Baby Louise Brown born – first child created through in vitro fertilization by Robert Edwards

1996 - Ian Wilmut cloned Dolly the sheep from adult cells – major breakthrough

History of Cloning and Nuclear Transfer

August 1997 - President Clinton proposed legislation to ban the cloning of humans for at least 5 years

September 1997 - Thousands of biologists and physicians signed a voluntary five-year moratorium on human cloning in the U.S.

January 1998 - Nineteen European nations signed a ban on human cloning

History of Nuclear Transfer and Cloning

Endangered species

Rescue Genetics

Old or deceased animals

Little Nicky, a cat cloned for a woman in Texas by

Genetic Savings & Clone – cost $50,000

Dr. Hwang Woo-Suk with first successfully cloned

dog named Snuppy

RNL Bio will be cloning pets - $150,000 eachGuar, wild ox from

Southeast Asia

Genetically Engineered Livestock

Increase disease resistance

Increase heat tolerance

Increase nutritional value

of meat

Modification of muscle massIncrease reproductive

capacity

Xenotransplants

Protein production for

Pharmaceutical needs

Milk Production

Increase levels of milk

Produce milk with better characteristics (i.e. more calcium, more vitamin D)

Improve digestibility of milk (i.e. for

people with lactose intolerance)

Belgian Blue bull – contain a natural mutation where they lack a protein called myostatin that normally interferes with

muscle development - produce large amounts of meat with very little fat – “doubly muscled”

Genetically Engineered Meat

Jan 2008, Food and Drug Administration approved the sale of food from cloned animals U.S. Department of Agriculture asked producers to keep

the meat off the market because of consumer fears FDA stated that they will not require producers to put

packaging labels on cloned meat or milk products

Cloning Livestock

Genetically engineered cows to produce human immunoglobulins (antibodies) Used for treating a variety of diseases from anthrax

to the virus that causes earaches Important for creating antibodies that can’t be made

in the lab and can only come from human donors Obtain these humanized antibodies from the cows

blood and milk Large quantities that you wouldn’t be able to obtain

from humans

Pharmaceutical uses

Pampa, one of 4 cloned Jersey calves in Argentina that can produce human

antibodies

Because cloning is expensive and inefficient only 1-5% of all cattle will be products of cloning Most clones will be prized bulls/exotic breeds/endangered

breeds Disease won’t wipe out entire herd – only small

percentage of herd actually the result of cloning

Reality of Cloning

Nature vs NurtureClones will not necessarily take on the same

personality or physical characteristics of the original Cc, “CopyCat”, doesn’t have calico markings that

original cat “Rainbow” has

Adult Stem Cells

Also referred to as “somatic cells”Small population of undifferentiated cells

that are found in most adult tissues or organs Self-renew and differentiate to yield all or many

of the specialized cells found in that tissue or organ

Important to maintain or repair the tissue in which they are found

Found in many important organs in the body (skin, liver, brain, pancreas and blood) More adult cell populations being discovered all the time

“adult” stem cell population found in deciduous teeth!

Hematopoietic Stem Cells

Characteristics of HSCs

Large capacity for self-renewal

Multipotent – able to give rise to many different cells types

Used to treat a variety of diseases

Early HSC Experiments

“Discovered” in the mid 1940’s when first bone marrow (BM) transplants were performed Complete

hematopoietic recovery in irradiated patients

What diseases can HSCs be used to treat? (without alteration)

Leukemia and Lymphoma – cancers of the blood characterized by uncontrolled growth of the white blood cells Patient’s own cancerous hematopoietic cells have to be

destroyed (chemotherapy or radiation) and replaced with donor cells (bone marrow transplant)

Inherited blood disorders Anemia – failure to produce normal red blood cells resulting

in low blood iron levels (i.e. aplastic anemia, sickle-cell anemia)

Metabolic disorders - genetic defects in key enzymes needed to perform basic biological functions and breakdown chemical byproducts (i.e. Hunter’s syndrome, Hurler’s syndrome)

Mesenchymal Stem Cells

Also referred to as “stromal stem cells”

MSCs, like HSCs, are also found in the bone marrow MSCs can also be isolated

from umbilical cord blood and adipose tissue

Responsible for bone and cartilage formation in the developing embryo and bone and tissue repair in the adult

MSCs give rise to a variety of cell types bone cells (osteocytes) cartilage cells

(chondrocytes) fat cells (adipocytes) and other kinds of connective

tissue cells such as those in tendons

Use of mesenchymal stem cells to expedite wound repair Repair burn wounds Chronic non-healing

wounds Elderly patients with

diabetes Skin cancer wounds

Clinical Applications for MSCs

MSCs and Wound Repair

Application of bone marrow–derived cultured cells to human chronic wounds

A) Non-healing wound (for more than a year) on ankle of patient

B) Third application of MSC-fibrin spray to the now healing wound

C) Wound at 3 months, the wound is almost healed

D) Wound closure at 6 monthsB D

Other Clinical Implications of MSCs Bone regeneration

OsteoCel – “off-the-shelf” cellular product created by Osiris therapeutics bone graft using MSCs seeded

onto a biomatrix speed bone healing from injury, disease or degeneration

Cartilage and joint repair Chondrogen – another Osiris

product Aimed at regenerating the

meniscus of the knee Slow the progression of

osteoarthritis in the knee Chondrogen

untreatedChondrogen

treated

Stem Cells and Cancer

Cancer – cell or group of cells that display uncontrolled growth and display protection against programmed cell death (apoptosis) Benign – self-limited, do not spread Malignant – spread to other sites in the body via

lymph or blood Sites of tumors different from original tumor site often

referred to as “metastases” Difficult to treat, poor prognosis Patients often unable to tolerate amount of

chemotherapy needed to destroy all tumor sites in the body Especially true for children Also, people with reoccurring cancer

Stem Cell Therapeutics

St. Jude Children’s hospital Researchers created a human neural stem cell line

that expresses a specific enzyme needed to activate an anti-cancer drug When injected into the body - neural stem cells migrate

to the various sites where the tumor has spread to Phenomenon called “tumor-tropism”

Stem cells migrate toward a site of tumor development Shown in neural stem cells and in mesenchymal stem

cells

Stem Cell Therapeutics

Neural stem cells target specific tumor sites throughout the body

Engineered neural stem cells express an enzyme that cleaves a pro-form of an anti-cancer drug

Conventional chemotherapy is systemic Causes damage to healthy tissue as well as the

cancerous tissueThis method allows drug to only work at

specific sites where these neural stem cells are found If stem cells migrate to tumor site targeted delivery

Injected mice with human neuroblastoma cells

• Injected mice with neural stem cells containing the specific enzyme required for the anti-cancer drug to work

• Cells formed multiple tumors to simulate

metastases throughout the body

• Neural stem cells migrate towards metastatic tumor sites

Stem Cell Cancer Therapy

Green = Tumor cells

Red = neural stem cells that express enzyme

Aboody et al., 2006

Stem Cell Cancer Therapy

Animals that received neural stem cells + anti-cancer drug had a higher rate of survival compared to animals that received neural stem cells alone or drug alone

Anti-cancer drug

Neural stem cell line with

enzyme alone

Neural stem cell line with

enzyme + anti-cancer drug

Case Study #1

Spinal cord injury At 24, Susan suffered an

injury to her spine in August, 2001 that left her paralyzed

1 of 7 patients (ranging from 18-32 yrs of age) treated in Portugal in a study by Dr. Carlos Lima

Used her own adult stem cells derived from olfactory mucosa Olfactory mucosa is located in the

upper nasal cavity Neurons that detect smell are found

here Stems here are being used to

produce neurons to replace damaged neurons in spinal cord

“Only part of my dreams has been attained. But I have come farther than my American doctors ever thought. My most recent MRI took place 5 days ago. The doctors were in disbelief at the improvement they saw where my spinal cord had been injured. I have recovered some functional improvement through Dr. Lima's procedure, such as the ability to hold my bladder and at times even void on my own. Sensation has been restored, though it is not completely normal. When concentrating I am now able to contract my thighs slightly; once again, this was also impossible before my surgery in Portugal.But most important on my way to recovery is that I can now walk with the aid of braces. I am now preparing to shed the shell of this wheelchair, which has confined me for over two years, to more and more use my braces and walker for mobility. This is something my doctors here in America told me would never be possible with my level of injury and to accept my fate. With Dr. Lima's adult stem cell based therapy, I have accomplished much more than my U.S. doctors said was possible. But this is only the first step to a complete cure.”

Testimony

Senate Commerce Subcommittee on Science, Technology, and Space

July 14, 2004

Case Study #2

Parkinson’s disease Suffered extreme shaking of the right side

of his body and was unable to use his right arm

Neurosurgeon Dr. Michele Levesque isolated neural stem cells from Mr. Turner’s brain Cells were expanded, induced to differentiate

into nerve cells and injected into the left side of Mr. Turner’s brain

Soon afterwards, the Parkinson’s symptoms began to improve in his right side. trembling decreased neurological evaluation indicated a marked

improvement in his symptoms Lasted about 5 years Expressed willingness to undergo a repeat

surgery to further slow the progression of his symptoms

Dennis Turner

“By early 1991 I suffered extreme shaking of the right side of my body, stiffness in my gait and movements. After some years of medication, I developed fluctuation and poor response to Sinemet. This made daily activities needing the coordinated use of both hands hard or impossible, such as putting in contact lenses. My disability prevented me from using my right arm. And since my only other realistic alternative was to continue growing worse until I eventually died, I decided to have the surgical procedures in 1999, one to remove the tissue and another to inject the cells. I was awake for both procedures, under local anesthesia. Soon after having the cells injected my Parkinson’s symptoms began to improve. My trembling grew less and less, until to all appearances it was gone, only slightly reappearing if I became upset. Dr. Levesque had me tested by a Neurologist, who said he wouldn’t have known I had Parkinson’s if he had met me on the street. I was once again able to use my right hand and arm normally, enjoying activities that I given up hope of ever doing. Because of my improvements through Dr. Levesque’s treatment I’ve been able to indulge in my passion for big game photography these past five years. While on safari in 2001 I scrambled up a tree to avoid being run over by a Rhino. I swam in the South Atlantic with Great White Sharks. Two weeks ago I returned from Africa after photographing Cheetahs and Leopards in the wild. The pictures I took represent memories and experiences I feel I have Dr. Levesque to thank for. I came here to offer him my sincere gratitude, and to offer others with Parkinson’s a concrete reason for hope.”

Testimony

Senate Commerce Subcommittee on Science, Technology, and Space

July 14, 2004

Adult stem cell treatments

Phase I/II study in Brazil involving 15 newly diagnosed patients with Type I diabetes mellitus (ages 14-31 yrs) Patients were given immunosuppressive drugs and their own

stem cells via IV injection. During a 7- to 36-month follow-up, 14 patients became insulin

free 1 for 35 months, 4 for at least 21 months, 7 for at least 6 months

and 2 with late response were insulin-free for 1 and 5 months All displayed increased -cell function

Need to regrow a finger? There’s an app for that!

General Issues in stem cell research

Very-slow going Although most stem cell populations were discovered

over 40-50 years ago – still much we don’t understand about them Signaling pathways? What factors control differentiation/self-renewal?

In vitro vs in vivo conditions What a cell does in culture may not reflect what the cell

can do once in the body How do we know what the cell will do in a new

environment outside its “niche” In vitro = controlled environment In vivo = signals/factors that we can’t account for in vitro

Animal models vs. humans

Wave of the Future?

Induced Pluripotent Stem Cells Adult cells that have been genetically reprogrammed

into immaturity via the induction of 4 genes Oct3/4, Sox2, Klf4, c-Myc Genes initially induced virally, but new methods have

recently been developed Mouse iPS cells first reported in 2006; human iPS cells in

2007

Both mouse and human iPS cells… express stem cell markers are capable of generating cells characteristic of all three

germ layers

Wave of the Future?

Clinical Applications of iPS Cells

Lots of hype, comparitively few results We’re making progress, but can’t cure diseases

overnight

Stigma in the media When most people hear “stem cell research” they

immediately think embryonic stem cells Lack of education and familiarity with the use of

adult stem cells Recent controversies

General Issues in stem cell research

Stem Cell Controversies

Dr. Hwang Koo-Suk of Korea Biomedical scientist and former

Professor of Veterinary Medicine at Seoul National University-

Claimed to have created 11 human ES cell lines by SCNT

Discovered that some data had been fabricated

Accepted egg donations from two of his own junior researchers Violation of scientific ethics

Two Science papers (2004, 2005) retracted

A Long Road Ahead…

http://www.youtube.com/watch?v=TRtlkcQ6brE&feature=related

Stem Cell Resources

www.pubmed.gov Research articles

http://stemcells.nih.gov/ - NIH Stem cell resource center Stem cell background info Current stem cell experiments Current U.S. policies regarding research, funding

etc

www.isscr.org/public/adultstemcells.htm International Society for Stem Cell research

Resources

Links to articles describing use of adult stem cells to treat diseases http://www.stemcellresearch.org/facts/asc-refs.pdf

Cloning Informationhttp://www.ornl.gov/sci/techresources/

Human_Genome/elsi/cloning.shtml