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Jason Ip Graduate Student

The end of the beginning for pluripotent stem cells Peter J. Donovan * & John Gearhart † * Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania † The Institute of Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland. Jason Ip

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Jason Ip Graduate Student

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  1. The end of the beginning for pluripotent stem cellsPeter J. Donovan* & John Gearhart†*Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania†The Institute of Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland Jason Ip Graduate Student

  2. “If we do the work that we can do in this country, the work that we will do when John Kerry is president, people like Christopher Reeve will get up out of that wheelchair and walk again” -Senator John Edwards October 12, 2004

  3. Background • Pluripotent stem cells • def. cells in a stem cell line capable of differentiating into several different final differentiated types • First recognized in teratocarinomas • Primordial germ layers • The ectoderm, endoderm, and mesoderm, are the three major cell lineages • Formed during gastrulation (cell migration resulting in cleavage)

  4. Background • The three types of embryonic tissue • Embryonic stem (ES) • Embryonic germ (EG) • Embryonal carcinoma (EC) • Attributes of EC, ES, and EG cells • Transcription factor Oct4 • Alkaline Phosphatase • Telomerase …Upregulation sustains pluripotency

  5. Agenda • The Science of Pluripotency • Developmental potential • The basic biology of human development • Embryonic stem cells vs. adult stem cells • Bringing stem cells to the clinic • Expansion and differentiation • Safety considerations in cell-based therapies • The future of stem cells

  6. The Science of Pluripotency

  7. Developmental potential • Assessment in three independent assays • In vitro differentiation in a Petri dish • Differentiation into teratomas or teratocarcinomas within histocompatible mice • In vivo differentiation within blastocoel cavity of a pre-implantation embryo

  8. Developmental potential • Directing differentiation • Manipulation of cellular environment • Growth of cells at high density • Growth of cells on different types of feeder cells • Addition of growth factors • Growth on crude or defined ECM substrates …differentiation varies and lacks robustness • Suspended three dimensional aggregation • Development of embryoid bodies

  9. Developmental potential • Embryoid bodies • Capable of differentiating into any of the three primordial germ layers • Germ layer cells are multipotent, as opposed to pluripotent

  10. The basic biology of human development • Pluripotent stem cells can… • Aid in deciphering developmental gene-expression • Survival • Proliferation • Differentiation • Migration • Lend insight to tumorigenesis and genetic diseases

  11. Embryonic stem cells vs. adult stem cells • Ethical consideration • What marks the beginning of life? • Main differences • The number of potential derivatives; embryonic > adult • Feasibility • Lack of publication on adult stem cell research (add more?)

  12. Bringing stem cells to the clinic

  13. Successful transplantations of mouse ES cells Cardiomyoctes form stable and functional grafts Glial precursors interact with host neurons to replenish lost myelin in the brain and spinal cord Embryoid bodies differentiate into neurons in the spinal cord, promoting motor recovery Insulin-producing cell line implanted into mice resulted in normalized glycemia

  14. • • • ? Successful transplantations of mouse ES cells Rats with motor injury and stroke treated with neuronal cells derived from human EC cells resulted in partial recovery of motor function

  15. • • • Successful transplantations of mouse ES cells Transplanted cells are replacing lost cells such as neurons or glia Transplanted cells providing factors facilitating the regeneration of host cells Cell-based therapies may be useful in abating the effects of injury and disease

  16. Stem cell expansion and differentiation • Requirements leading to clinical therapy • Growth in large quantities • Controlled homogeneous differentiation • Histocompatibility • Limitations • Stem cell survival in long-term culture • Stem cell genetic mutations

  17. Stem cell expansion and differentiation • Possible solutions • Progenitor cells • Advantages • Derived from embryo bodies • Easier to grow and expand • Possess normal karyotype • Disadvantages • Limited self-renewal capability • Can be unipotent or multipotent • Example: • Neural progenitors can be formed from human ES cells in high density culture, become neurons

  18. Stem cell expansion and differentiation • Genomics • Microarray technology can reveal expression of growth factors, growth-factor receptors, and cell-adhesion molecules • Expression profiles allow for optimal conditioning of stem cell growth environment

  19. Safety considerations in cell-based therapies • Three key safety issues: • Histocompatibility • Tumorigenesis • Infection from serum-containing culture

  20. Safety considerations in cell-based therapies • Histocompatibility • Immune suppression • Slows immune response • Nonspecific • Tolerance induction • Antigen-induced • Specific • Embryo-derived compatible cells • Therapeutic Cloning (somatic cell nuclear transfer) • Genetic recombination of existing stem cell lines to match patient

  21. Safety considerations in cell-based therapies Therapeutic cloning Genetic Recombination

  22. Safety considerations in cell-based therapies • Tumorigenesis • Conflicting arguments • Imprinted genetic loci are erased in EG cell lines • EG cell lines behave normally in chimeras • Key questions • How can cells be ensured to migrate to designated sites? • At what stage of differentiation should transplantation occur? (hmmm?)

  23. Safety considerations in cell-based therapies • Infection from serum-containing culture • Infection caused by blood-borne bacteria • Serum contains necessary growth factors • Human ES cells require fetal calf serum or conditioned medium via mouse feeder cells for growth

  24. The future of stem cells • Mouse ES cells have contributed much to our understanding of embryogenesis • Prospects • Stem cell therapeutics • Deeper understanding of human growth and development • Treatment on non-human primates likely to be a next step before use in the clinic

  25. References http://www.biology-online.org/dictionary.asp http://www.csa.com/discoveryguides/stemcell/images/pluri.jpg http://en.wikipedia.org/wiki/Image:Cloning_diagram_english.svg http://alignmap.com/wp-content/Graphics/JohnEdwards(098).jpg http://upload.wikimedia.org/wikipedia/commons/d/d4/Cell_differentiation.gif http://www.bio.miami.edu/dana/pix/gastrulation.jpg http://abcnews.go.com/Health/wireStory?id=4313450 http://www.brown.edu/Courses/BI0032/adltstem/stem-cell.gif http://www.wormbook.org/chapters/www_germlinegenomics/germlinegenomicsfig1.jpg http://static.howstuffworks.com/gif/stem-cell-therapeutic.gif http://regentsprep.org/Regents/biology/units/reproduction/crossingover.gif http://omegascientific.com/catalog/images/fetal-bovine-fam.jpg

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