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Controlling Stem Cell Fate with Small Molecules - Applications in Regenerative Medicine

Stauprimide. Neuropathiazol. Controlling Stem Cell Fate with Small Molecules - Applications in Regenerative Medicine. Chantelle Capicciotti Thursday, November 18, 2010. Cardiogenol C. Reversine. Stem Cells. Differ from other cells of the body. Three general properties:

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Controlling Stem Cell Fate with Small Molecules - Applications in Regenerative Medicine

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  1. Stauprimide Neuropathiazol Controlling Stem Cell Fate with Small Molecules - Applications in Regenerative Medicine ChantelleCapicciotti Thursday, November 18, 2010 Cardiogenol C Reversine

  2. Stem Cells • Differ from other cells of the body. • Three general properties: • Capable of self-renewal through cell division. • Unspecialized. • Have the ability to differentiate into specialized cells. Cell Cycle Karp, G. Cell and Molecular Biology, 4th ed.; Wiley: New York, 2004.

  3. Embryonic Stem Cells • ESCs are derived from blastocyst of pre-implanted embryo. • Pluripotent – Able to differentiate into any of the three germ layers and thus all cell types. Blastocyst Blastocoel Trophectoderm Fertilization Zygote Ectoderm Mesoderm Inner Cell Mass Embroynic Stem Cells Endoderm Thomson, J.A.; Itskovitz-Eldor, J.; Shapiro, S.S.; et al. Science. 1998, 282, 1145-1147. http://stemcells.nih.gov/info/2006report/2006Chapter1.htm

  4. Adult Stem Cells • Found in various tissues and organs throughout the body. • Multipotent – Able to differentiate into several distinct cell types of the organ or tissue they originate. • Maintain and repair the tissue in which they are found. Chen, S.; Hilcove, S.; Ding, S. Mol. BioSyst. 2006, 2, 18-24.

  5. Adult Stem Cells http://stemcells.nih.gov/info/2006report/2006Chapter2.htm

  6. Regenerative Medicine • Use of stem cells to repair, regenerate or replace diseased or injured cells, tissues and organs. • Treatment of cardiovascular and neurodegenerative diseases, diabetes and spinal cord injury. Red Blood Cells • Bone marrow transplant – Hematopoietic stem cell transplant done after chemotherapy and radiation for leukemia treatment Marrow White Blood Cells Platelets http://leukemiaawareness.com/

  7. Maintaining Pluripotency Proliferation ESCs remain in pluripotent state ESC Differentiation High Expression Low Expression Hyslop, L.A.; Armstrong, L.; Stojkovic, M.; Lako, M. Expert Rev. Mol. Med. 2005, 7, 1-21.

  8. Differentiation Proliferation ESCs remain in pluripotent state ESC • ESCs must exit pluripotent state to become more specialized. • To differentiate into any of the three germ layers, certain pathways must be activated/deactivated. Differentiation Murry, C.E.; Keller, G. Cell. 2008, 132, 661-680

  9. Differentiation Blastocyst Ectoderm BMP4 Mesoderm ESCs Activin Endoderm Murry, C.E.; Keller, G. Cell. 2008, 132, 661-680

  10. Culture Differentiation of ESC’s Blastocyst • Embryoid bodies spontaneously differentiate into multiple cell lineages. • Increase specificity to certain cell types by using “cocktails” of growth factors and signalling molecules. • Conditions often not completely defined or are non-specific. Inner Cell Mass ESCs Feeder Layer Embryoid Bodies Ectoderm Mesoderm Endoderm Hyslop, L.A.; Armstrong, L.; Stojkovic, M.; Lako, M. Expert Rev. Mol. Med. 2005, 7, 1-21.

  11. Small Molecule Approach • ESC differentiation in vitro is poorly controlled. • Use of “cocktails” does not facilitate studies on the molecular mechanisms involved in development. • Small molecules offer a solution: • Can be more specific and have a high degree of control. • Molecular mechanisms can be studied. Stauprimide Primes ESCs for differentiation Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  12. High-Throughput Screening Identification Image-based high-throughput screening Kinase-Oriented Library Approx. 20 000 Compounds • Stauprimide • Increased efficiency of differentiation towards endoderm lineage. Sox17 All Cells DMSO Stauprimide Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  13. Further Differentiation of Endoderm Cells • Stauprimide-primed endoderm cells could be further differentiated to hepatocytes and pancreatic precursor cells. Hepatocyte Differentiation Factors Hepatocytes Liver Endoderm Lineage Stauprimide Low Conc. Activin A ESCs Pancreatic Differentiation Factors Pancreas Pancreatic Progenitor Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  14. Stauprimide Primes ESCs for Differentiation • Stauprimide – Primes undifferentiated ESCs. • Activin A - Stimulus which dictates path of differentiation to endoderm lineage. • Stauprimide-primed ESCs can differentiate into other cell fates under appropriate conditions. Neural Differentiation Factors Ectoderm Lineage Neurons Stauprimide ESCs BMP-4 Beating Cardiomyocytes Mesoderm Lineage Hematopoietic Lineage Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  15. Nonspecific Kinase Inhibitors Stauprimide UCN-01 Staurosporine Promotes Differentiation Inactive Inactive • Stauprimide is active well below toxic concentrations and does not inhibit kinases at this concentration. • Other nonspecific kinase inhibitors did not promote differentiation of ESCs at nontoxic concentrations. Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  16. Staurosporine Family UCN-01 Staurosporine Stauprimide • Staurosporine is a natural product isolated from bacterium Streptomycesstaurosporeus. • Stauprimide can be synthesized en route of the synthesis of Staurosporine. Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  17. Staurosporine R1≠R2 Regiochemically Different 2ndGlycosidation (Intramolecular) 1stGlycosidation (Intermolecular) Glycosidic bond with indolicnitrogens Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  18. First Glycosyl Donor Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  19. Aglycon Acceptor Glycosyl Donor + Ratio: 2.5:1 Aglycon Acceptor Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  20. Intermolecular Glycosidation + 47% 10% Isolated Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  21. Second Glycosyl Donor Glycosyl Donor Precursor Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  22. Second Glycosidation x E+ = PhSeCl, NBS, NIS, I2, , etc. AND Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  23. Second Glycosidation Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842. Barrett, A. G. M.; Bezuidenhoudt, B. C. B.; Gasieki, A. F.; Howell, A. R.; Russell, M. A. J. Am. Chem. Soc. 1989, 111, 1392-1396.

  24. Final Stages of Staurosporine Synthesis + Staurosporine Ratio: 1:1 Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  25. Staurosporine Family Oxidized Benzoylated Staurosporine UCN-01 Stauprimide Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842. Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  26. Final Stages of Staurosporine Synthesis Leads to UCN-01 and derivatives + Staurosporine Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.

  27. Stauprimide Synthesis Stauprimide UCN-01 Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  28. Nonspecific Kinase Inhibitors Stauprimide UCN-01 Staurosporine Promotes Differentiation Inactive Inactive • Cellular target of Stauprimide likely not a kinase which is nonspefically inhibited by kinase inhibitors. • To identify targets, a biotin-tagged analogue was used. Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  29. BiotinylatedStauprimide • Linked to proteins or molecules for use in biochemical assays. • Binds to avidin and streptavidin with high affinity and specificity. • This binding is exploited to isolate proteins. Biotin Biostauprimide (BiotinylatedStauprimide) • Similar differentiation activity as Stauprimide. Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  30. Biostauprimide Synthesis + UCN-01 Biostauprimide Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  31. Mechanism of Stauprimide • Stauprimide inhibits the transcription factor NME2 resulting in a downregulation of c-Myc expression. NME2 X X Stauprimide C-Myc Gene NME2 X Promoter Region Differentiation C-Myc Maintains ESC State Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  32. Stauprimide ESCs Stauprimide Neural Differentiation Factors Activin A BMP-4 Ectoderm Lineage Endoderm Lineage Stauprimide Mesoderm Lineage • Increased efficiency of differentiation. • Decreased ESC content in differentiated product. • Decreased contamination of unwanted cell types. Hepatocytes Neurons Cardiomyocytes Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.

  33. Small Molecules Can Induce Differentiation Fertilized Egg • Stauprimideprimed ESCs for differentiation – was not able to induce differentiation. 8 Cell Embryo Blastocyst Pluripotent • Other small molecules can induce differentiation of ESCs and ASCs. Neural Cells Blood Cells Cardiac Muscle http://www.stemcellresearchfoundation.org/WhatsNew/Pluripotent.htm

  34. Neuropathiazol Hippocampus Neuropathiazol Hippocampus Primary Neural Progenitor Cells Neurons Astrocytes Oligodendrocytes • Adult Stem Cells • (multipotent) Rat Brain Warashina, M.; Min, K.H.; Kuwabara, T.; Huynh, A.; Gage, F.H.; Schultz, P.G.; Ding, S. Angew. Chem. Int. Ed. 2006, 45, 591-593. Human Brain

  35. Differentiation into Neurons • Preferentially induces to neuronal lineage! Neuropathiazol Primary Neural Progenitor Cells Neurons Astrocytes • Large number of neuronal and astroglial cells. Retinoic Acid Warashina, M.; Min, K.H.; Kuwabara, T.; Huynh, A.; Gage, F.H.; Schultz, P.G.; Ding, S. Angew. Chem. Int. Ed. 2006, 45, 591-593.

  36. Repairing the Nervous System With Stem Cells • Parkinson’s Disease – Neurodegenerative disease caused by the death of dopaminergic neurons. • Transplantation of embryonic dopamine neurons into patients can result in an increase in dopamine function. Before Surgery After Surgery Freed, C.R.; Greene, P.E.; Breeze, R.E.; et. al. N. Engl. J. Med. 2001, 344, 710-719

  37. Mending a Broken Heart • Cardiovascular disease is a leading cause of death. • Heart attack results in deprivation of oxygen to heart muscles which causes these cells to die. • Stem cells have been investigated as possible sources for regenerating damaged myocardial cells. Left Ventricle Normal Heart Infarcted Infarcted Heart Dilated Ventricle http://stemcells.nih.gov/info/2006report/2006Chapter6.htm

  38. Cardiogenols Cardiogenol A (85%) R = Cardiogenol B (80%) • Potent differentiator of ESCs towards cardiac lineage. R = Cardiogenol C (90%) R = Cardiogenol C Cardiogenol D (75%) R = Wu, X.; Ding, S.; Ding, Q.; Gray, N.S.; Schultz, P.G. J. Am. Chem. Soc. 2004, 126, 1590-1591.

  39. Differentiation into Cardiomyocytes Cardiomyocytes Cardiogenol C Mouse ESCs • Cardiogenol C resulted in 90% of cells differentiated towards cardiac lineage without the formation of embryoid bodies (EBs). • Majority of the cell population formed beating cardiomyocytes! • Current cardiomyogenesis inducing conditions of ESCs requires aggregation and formation of embryoid bodies (EBs). • Results in only 5% of the cell population forming cardiomyocytes. Wu, X.; Ding, S.; Ding, Q.; Gray, N.S.; Schultz, P.G. J. Am. Chem. Soc. 2004, 126, 1590-1591. Winitsky, S.O.; Gopal, T.V.; Hassanzadeh, S.; et. al. PLoS Bio. 2005, 3, 662-671.

  40. Cell Reprogramming Bone Chemical Reprogramming Skeletal Muscle Adult Fibroblast Induced Pluripotent Stem Cells Heart Tissue Anastasia, L.; Pelissero, G.; Venerando, B.; Tettamanti, G. Cell Death Diff. 2010, 17, 1230-1237. Other Tissues Genetic Reprogramming

  41. Amphibian Limb Regeneration • Amphibians have the ability to regenerate amputated limbs. • A blastema forms at the amputation site. • Blastema cells can re-enter the cell cycle. • Multipotentmesenchymal progenitor cells. • Recreated mesenchymal cells can differentiate to generate cell type needed to regenerate the limb. Brockes, J.P. Science. 1997, 276, 81-87.

  42. Induced Pluripotent Stem Cells Retroviral Transfection of Pluripotency Genes hESC Culture Conditions Somatic Cell Reprogramming Pluripotent iPSC Line • Retroviruses can transfect critical transcription factors to reprogram somatic cells back to a pluripotent state. • Four transcription factors: Oct4, Sox2, Klf4 and c-Myc. • Reprogrammed fibroblasts to an embryonic stem cell-like state. • iPSCs were similar to ESCs. • Expressed stem cell proteins. • Formed embryoid bodies. • Could undergo differentiation. Takahashi, K.; Yamanaka, S.; Cell. 2006, 126, 663–676. Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Cell. 2007, 131, 861–872.

  43. Induced Pluripotent Stem Cells Retroviral Transfection of Pluripotency Genes hESC Culture Conditions Somatic Cell Reprogramming Pluripotent iPSC Line • Original methods of reprogramming were inefficient. • Less than 1% of the starting adult cell population yielded iPSCs. • Viral transfection used to genetically alter the cells can potentially trigger the expression of oncogenes. • Have a propensity to form tumors. • The c-Myc gene is an oncogene and is known to promote tumor growth . Takahashi, K.; Yamanaka, S.; Cell. 2006, 126, 663–676. Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Cell. 2007, 131, 861–872.

  44. Reversine Reversine Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.

  45. Reversine • Reversine can cause cell reprogramming. • Reversine treated myoblasts were able to form adipocytes and osteoblasts. Reversine Multinucleated Muscle Cell Myoblasts Multipotent Progenitor Cells Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.

  46. Reversine • Reversine can cause cell reprogramming. • Reversine treated myoblasts were able to form adipocytes and osteoblasts. Reversine Adipogenesis Adipocytes Reversine Multinucleated Muscle Cell Myoblasts Osteogenesis Osteoblasts Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.

  47. Reversine Reversine Reversine Dedifferentiation Multinucleated Muscle Cell Myoblasts Multipotent Progenitor Cells Osteogenic Differentiating Conditions Reversine • Reversine treatment without differentiating conditions resulted in mononucleated cells. • There was no formation of osteoblasts! Transdifferentiation Osteoblasts Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.

  48. Reversine Reversine Reversine Dedifferentiation Multinucleated Muscle Cell Myoblasts Multipotent Progenitor Cells Osteogenic Differentiating Conditions • Reversine dedifferentiates myoblasts into multipotentmesenchymal progenitor cells. Osteoblasts Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.

  49. Summary • Member of the Staurosporine family of nonspecific protein kinase inhibitors. • Primes ESCs for differentiation by downregulating expression c-Myc. • Increased efficiency of differentiation of ESCs towards various cell lineages. Stauprimide • Preferentially induced primary neural progenitor cells to neuron cells. Neuropathiazol

  50. Summary • Differentiated ESCs into functioning cardiac cells and beating cardiomyocytes. Cardiogenol C • Induced dedifferentiation. • Reprogrammed myoblasts into multipotentmesenchymal progenitor cells. • Progenitor cells could be re-differentiated into adipocytes and osteoblasts. Reversine

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