Dr. Faisal Al-Allaf Assistant Professor of Genetics and Molecular Medicine

1. Dr. Faisal Al-Allaf Assistant Professor of Genetics and Molecular Medicine Umm Al-Qura University Faculty of Medicine, Makkah, Saudi Arabia fallaf@uqu.edu.sa Tel/Fax: 5270000 Ext: 4198 Introduction to stem cell therapy Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

2. Introduction to Stem Cells • Basic properties of stem cells • The hierarchy of stem cells • Various Sources of Human Stem Cells • Five possible mechanisms of adult stem cell plasticity • The different stem cells types and their properties • Representative adult stem cells differentiation properties • Use of Human Stem Cells to Develop Rational Human Therapies • Technical Challenges in the Stem Cells Space • Why do we need more human embryonic stem cell lines for research? • Biology of stem cells Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

3. Basic properties of SCs • Pluripoteny: stem cells are unspecialized cells that capable of generating all the mature cell types found in the adults • Self renewal: potentially unlimited rounds of replication. Unlike post-mitotic terminally differentiated cells (which are in G0 of the cell cycle), SCs are cycling constantly, thereby replenishing their pool in vivo • Migration: homing to the site of injuries • Plasticity: Potential ability to be shaped or formed. It differs from "elasticity", which refers to ability to change temporarily and revert back to original form. Stem cell hierarchy (Wobus and Boheler, 2005) Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

4. The hierarchy of stem cells • Totipotent: Totipotency is the ability of a single cell to divide and produce all the differentiated cells in an organism, including extraembryonic tissues • Pluripotent: give rise to any cell type in the body and the ability for self-renewal • Multipotent: committed cells that give rise to multiple but limited number of cell types. An example is a hematopoietic cell • Fully differentiated: include fully differentiated with limited division (self renewal) or non-mitotic Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

5. What’s so special about stem cells? • Because of the above properties, SCs serve as the body’s repair system by: • Self renewal, they can replicate themselves over and over for a very long time • Replenishing other cells, they have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses • Under culturing condition, stem cells can divide to form more of their own kind, thereby creating a stem cell line. The research aims to: • Induce these cells to generate healthy tissue needed by patients • Understand how stem cells develop • Healthy and diseased cells will also assist the search for cures Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

6. Potential use of hESCs to develop rational therapies • Cell and tissue replacement therapies • Vehicle for gene therapy • Cell-based models of human disease • Drug discovery and toxicology tests Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

7. Various sources of human SCs • Embryonic SCs: (ES cells; ESCs) from IVF clinic • Fetal SCs: from developing tissues (aborted fetuses), stem or progenitor cells from developing tissues (such as cord blood stem cells, placenta and Wharton’s Jelly) • Adult SCs: Stem, progenitor or precursor cells from mature tissues in the adult are less versatile and more difficult to identify, isolate, and purify. Examples; Hematopoietic, Mesenchymal, MAPCs, Neural, Muscle Derived, Pancreatic, Hepatic, Epithelial Stem Cells. Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

8. The different stem cell types and their properties Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

9. The different stem cells types and their properties Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

10. Sources of human embryonic stem cells (hESCs) • IVF clinic: • Cryopreserved of patients who have completed their IVF treatment. Blastocysts are donated by couples after in vitro fertilization (IVF) cycles and the ESCs are derived from a 5-7 days old blastocyst • Fresh embryos unsuitable for clinical IVF use • Somatic cells nuclear transfer (SCNT):asexual reproduction involved nuclear transfer from a mature cell into a donor egg. Functionally different from regular fertilized egg • Cells with poor in vitro development or genetically abnormal will be discarded Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

11. Stem cells from IVF and SCNT Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

12. Potential of hESCs to differentiate into all adult cell types • Differentiation is a process involving unspecialized cells progressing to become specialized cells with restricted developmental potential. Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

13. Potential of hESCs to differentiate into cardiomyogenic cells • Human adult heart is composed mainly of post-mitotic and terminally differentiated cells. The hESCs represent an alternative unlimited source of functional cardiomyocytes that can be used for therapies • Please double click on the picture below to watch the video clip of beating cells Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

14. Problems of current hESC lines:Why do we need more hESC lines? • Genetic diversity • Immunological diversity • Karyotypic stability • Differential potential • New lines not exposed to animal cells or products • Disease-specific lines targeting specific disorders • Patient-specific lines Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

15. Technical Challenges in the Stem Cells Space • Ability to obtain source material (ethical concerns, abundance vs rarity) • Ability to direct differentiation; to select out and purify desired phenotypes • Engraftment and integration vs migration • Tolerance versus rejection • Tumor formation in vivo Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

16. In favor of ESCs research: ESCs research 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 ESCs research: 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 ESCs research. Slippery slope argument: ESCs research will lead to reproductive cloning. The ethical debate of ESCs research Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

17. Key Ethical Issues • The blastocyst used in stem cell research is microscopically small and has no nervous system. Does it count as a “person” who has a right to life? • What do various religions say about when personhood begins? Does science have a view on this? • In a society where citizens hold diverse religious views, how can we democratically make humane public policy? • The future of stem cell research is insecure. The research is strongly supported by scientists and very much needed by patients. • What is the Islamic perspective? Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

18. Federal and international legislation for ESCs • Government funding in USA is prohibited for research using cell lines developed after Aug 9, 2001. Efforts to regulate the research are currently stalemated in USA. • Embryonic stem cell research is highly controversial not only in the United States but worldwide. • In the past two years, many nations have begun to tolerate, if not to support, the research. Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

19. Stem Cell Research Worldwide Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

20. Dedifferentiation and Nuclear Reprogramming • The ability to dedifferentiate (reverse lineage-committed cells back to multipotent or even pluripotent cells) might overcome many of the obstacles associated with using ESCs and adult stem cells • Recent studies suggest that tissue-specific stem/progenitor cells may overcome their intrinsic lineage restriction upon exposure to a specific set of signals in vitro and in vivo, although such reprogramming may not reflect potentials that are normally exercised in vivo • An example is the reprogramming of a somatic cell to a totipotent state by nuclear transfer cloning (Dolly the sheep), where the nucleus of a somatic cell is transferred into an enucleated oocyte Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

21. Potential of SCNT-therapeutic cloning • Patient will receive his own stem cells therefore, no need for donor match and consequently avoiding rejection • SCNT stem cells are alive in a Petri dish and can be differentiated into multiple lineages • Unless implanted in uterus, SCNT cannot generate a fetus • Mounting evidence suggests that SCNT stem cells may NOT develop into a human even when implanted • No reputable scientist wants to clone human beings Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

22. Potential of adult SCs to differentiate into other cell types • Stimulation of post-mitotic differentiated adult cells to re-enter the cell cycle and proliferate may provide new therapeutic approaches for treating various types of diseases • Epigenetic modification can regulate lineage specification by imposing a specific and heritable pattern of gene expression on the progeny of cells, without altering the DNA sequence • Major epigenetic modifications include DNA methylation, histone acetylation and methylation, and histone phosphorylation • DNA methylation, occurring progressively during differentiation of stem cells, typically induces gene silencing • DNA demethylation has been associated with increased cellular plasticity of lineage-committed cells Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

23. Markers which expressed in hESCs and defined stemness • Alkaline Phosphatase • Stage-specific embryonic antigens (SSEA-3, SSEA-4) • POU domain class 5, transcription factor 1 (POU5F1) • Enhancer of rudimentary homolog (ERH) • High mobility group AT-hook 1 (HMGA1) • Octamer-binding protein (Oct4) • SRY box-containing gene 2 (Sox-2) • STELLAR • Nanog • TRA-1-60, TRA-1-8 • Can we induced pluripotent stem cells from committed or differentiated cells by gene transfer? iPS Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

24. Potential of adult mesenchymal stem cells (MSCs) to differentiate other cell types • MSCs are multi-potent stem cells with significant cellular plasticity, and can differentiate into a variety of mesenchymal tissues such as osteoblasts, adipocytes and chondrocytes as well as other tissue types such as neuronal and skeletal muscle cells under specific differentiation conditions (Caplan and Bruder, 2001) Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

25. Representative adult stem cells differentiation properties Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

26. Five possible mechanisms of adult stem cell plasticity Wagers and Weissman, 2004). • A) Trans-differentiation • B) De-differentiation • C) Multiple stem cells in a population • D) Pluripotent stem cells in a population • E) concept of cell-fusion • Orange or green ovals = tissue-specific cells, blue ovals = pluripotent stem cells, red oval = differentiated cells of the orange cells, green hexagons = differentiated cells of the green ones Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

27. Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

28. Major milestones in stem cell research and associated timeline • 1981. First mouse ES cell lines isolated and grown in culture • 1981. First transgenic animals produced • 1988. First cord blood transplant • 1989. A clonal line of human embryonal carcinoma cells derived • 1990. Britain passes the Human Fertilization and Embryology Act • 1994. Human embryonic stem (ES)-like cells generated • 1995. Evidence found for neural stem cells • 1996. Dolly, the first cloned sheep, born in Scotland • 1998. Scientists at University of Wisconsin-Madison and Johns Hopkins • University isolate the first human stem cells • 2000. Scientists in Singapore and Australia derive human ES cells from blastocysts • 2001. Advanced Cell Technology creates the first cloned human embryos • 2001. US President George Bush blocks federal funding for creation of new stem cell lines • 2001. Human ES cells successfully developed into blood cells • 2002. Neural stem cells successfully developed into functional neurons • 2003. Institute of Stem Cell Research, Edinburgh, discovers key gene that keeps ES cells in a state of youthful immortality • 2003. Dolly dies after developing progressive lung disease • 2003. UK Stem Cell Bank Established • 2004. Web-based resource for international stem cell researchers launched • 2004. Californians approve Proposition 71 to spend US$3 Billion over 10 years on stem cell research • 2005. The U.S. House of Representatives approves a bill to loosen restrictions on federal funding for stem cell research • 2006. Judge rules in favor of proceeding with the financing of California’s 10-year stem cell project (based on Proposition 71) Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

29. References and Private Reading • Emery’s Elements of Medical Genetics, 13th edition 2007, by Peter TURNPENNY and Sian ELLARD. Churchill Livingstone ELSEVIER. ISBN: 978-0-7020-2917-2 • Medical Genetics at a Glance, 2nd edition 2008, by Dorian PRITCHARD and Bruce KORF. Blackwell Publishing. ISBN: 978-1-4051-4846-7 • Elsevier's Integrated Genetics, 2007, by Linda Adkison and Michael Brown. MOSBY ELSEVIER. ISBN: 978-0-323-04329-8 • Genetics for Dummies, 2005, by Tara Robinson, Wiley Publishing, Inc. ISBN: 978-0-7645-9554-7 • New Clinical Genetics, 2007, by Andrew Read and Dian Donnai. Scion. ISBN: 978-1-904842-31-6 • Cell Biology and Genetics, Crash Course, 2nd edition 2006, by Manson, Jones, Morris, Michael STEEL and Dan HORTON-SZAR. MOSBY ELSEVIER. ISBN: 0-7234-3248-1 • Human Molecular Genetics, 3rd edition, 2003, by STRACHAN T. and A. READ. Garland science/Taylor and Francis group. ISBN: 978-0-8153-4182-6 • Human Genetics: Concepts and Applications, 7th edition 2007, by Ricki LEWIS. McGraw Hill international. ISBN: 978-0-07-110779-2 • Genomes, 3rd edition 2006, by T.A. BROWN. Garland science, ISBN: 978-0-8153-4138-3 • Colour Atlas of Genetics, 2007, by Eberhard Passarge. Thieme. ISBN: 978-1-58890-3365 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa

30. Acknowledgments • For the providers of all the educational materials (video clips, pictures, diagrams and charts) including publishers, pharmaceutical companies or unknown internet users who made their material available for use, in this and other presentations, I offer heartfelt thanks and deep appreciation. • I feel particularly grateful to faculty, staff, and our brilliant students who provided a unique intellectual and wonderful environment for work. Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa