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Current topics in stem cell research. Dr. Houda Darwiche UF CPET 23 July 2013. Disclaimer: This presentation contains graphic visuals. Unlimited Potential?. ES Cells and Reproductive Cloning. Reproductive cloning is the process of making an identical genetic copy of an existing organism - PowerPoint PPT Presentation
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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