1. Asymmetric Self-Renewal By Distributed Stem Cells: Misunderstood in the Past, Important for the Future James L. Sherley, M.D., Ph.D. Director The Adult Stem Cell Technology Center, LLC Boston, MA, USA ASCTC

2. Fetal, Neonatal, Adult DevelopmentEarlyEmbryonicDevelopment Fetal SCs Germline SCs Post-natal SCs Somatic SCs Tissue SCs Adult SCs Epiblasts [embryonic SCs] Embryonic precursors precursors EmbryonicPost-Embryonic Sources of Stem Cells (SCs) During Mammalian Development Distributed Stem Cells? (DSCs)

3. Fetal SCs Germline SCs Post-natal SCs Somatic SCs Tissue SCs Adult SCs Embryonic Post-Embryonic “DISTRIBUTED STEM CELLS” DSCs (Sherley, 2008, Breast Disease 29, 37-46) For Distributed Potency Asymmetric Self-Renewal Asymmetric Self-Renewal In vivo and in vitro Embryonic stem cells Primordial germ cells Induced pluripotent SCs Symmetric Self-Renewal In vitro ASCTC

4. The Pervasive Stem Cell Definition: Self-renewal or Differentiation ASCTC SymmetricSelf-renewal

5. The Pervasive Stem Cell Definition: Self-renewal or Differentiation ASCTC SymmetricSelf-renewal An Inadequate Definition For Distributed Stem Cells

6. A Gnomonic for Distributed Stem Cells: Self-renewal AND Differentiation, continuously and simultaneously ASCTC “AsymmetricSelf-renewal”

7. Tissue Cell Turnover The mark of distributed stem cells (DSCs): Asymmetric Self-Renewal Potten et al., 2003 DeathTerminalArrestDifferentiationSymmetricKineticsAsymmetric  Self-renewal  Cell Kinetics DSC (niche) ASCTC

8. A CENTRAL QUESTION: What is the cell kinetics form of asymmetric self-renewal? ASCTC

9. Mathematical Identity, but Biological Distinctions p1-p Stochastic vs. Determined ASCTC

10. Biological Distinctions Stochastic 1.Extinction probability 2.Mutation accrual Determined 1.No DSC differentiation 2.Individual asymmetric division 3.Mutation avoidance? 4.Unique cellular programs! p1-p vs. ASCTC

11. ≠ Developmental Asymmetric Divisions Are Not Equivalent to Asymmetric Self-Renewal Divisions Asymmetric Division Sisters unequal Sisters unequal Both sisters differ Both sisters differ from parent from parent Limited Limited Asymmetric Self-Renewal Sisters unequal Sisters unequal One sister equal One sister equal to parent to parent Continuous over lifespan Continuous over lifespan ASCTC

12. Biological Distinctions Stochastic 1.Extinction probability 2.Mutation accrual Determined 1.No DSC differentiation 2.Individual asymmetric division 3.Mutation avoidance? 4.Unique cellular programs! p1-p vs. ASCTC

13. + Asymmetric Self-Renewal Associated (ASRA) Genes: Highly Specific Biomarkers for DSCs SYM ASYM [Noh et al. 2011, PLoS ONE] SYMASYM CyA CXCR6 DNA Phase [Taghizadeh et al., 2010, PLoS ONE] ASCTC

14. Stem Cell Therapy “Adult” Stem Cells (DSCs) Embryonic Stem Cells (ESCs) Induced Pluripotent Stem Cells (iPSCs) Proposed Stem Cells for Tissue Replacement ASCTC

15. Stochastic Deterministic (In Vitro) Self-Renewal OR Differentiation Stemness Preserved “AsymmetricSelf-Renewal” Embryonic and iP Stem Cells Distributed Stem Cells Mathematical Features of Cell Production with Differentiation p1-p a b d c Self-Renewal AND Differentiation Loss of Stemness a a ASCTC

16. Stem Cell Therapy “Adult” Stem Cells (DSCs) Embryonic Stem Cells (ESCs) Induced Pluripotent Stem Cells (iPSCs) Proposed Stem Cells for Tissue Replacement ASCTC

17. NormalTissue Damaged or Diseased Tissue Tissue Therapy with ESCs and iPSCs? REPAIR? ASCTC

18. NormalTissue Damaged or Diseased Tissue Short-livedRepair!!!! (Tumors!) Tissue Therapy with ESCs and iPSCs hESCs and iPSCs - ethical issues (hESCs) - epigenetic defects - genetic defects Purification - ESCs! - iPSCs! - other differentiated cells “Develop In Vitro” Differentiated Cells (Immature) ASCTC

19. NormalTissue Damaged or Diseased Tissue DSC Expansion is possible! Autologous Autologous Allogeneic Allogeneic Informed Consent Informed Consent RenewingRepairedTissue Tissue Therapy with Distributed Stem Cells - purity not required - expansion not always critical critical ASCTC

20. NormalTissue Damaged or Diseased Tissue An DSC Bottleneck for Tissue Therapy “Develop In Vitro” Isolate, stabilize, expand Distributed Stem Cells RepairedTissue? Tumor Risk Remains [Sherley, J. L. 2004, J. Biomed. Biotech. 2, 71-72.] Differentiated Cells (Immature) hESCs and iPSCs - ethical issues (hESCs) - epigenetic defects - genetic defects ASCTC

21. Distributed Stem Cell Asymmetric Self-Renewal Embryonic development hESCs? iPSCs Immature? ASCTC

22. Distributed Stem Cell Asymmetric Self-Renewal Embryonic development hESCs? iPSCs Xanthine (p53-KO mimic) Immature? Pare & Sherley, 2011, J. Biomed. Biotech. New Patent: USPTO No. 8,759,098, June 24, 2014 ASCTC DiseaseResearch