1. Replicative Senescence as a Model for Osteoblast Dysfunction with Aging In vivo Andrew Rosenzweig 1, Robin K. Suda 1, F. Brad Johnson 2, and Robert J. Pignolo 1 Departments of Medicine 1, Division of Geriatric Medicine, and Pathology and Laboratory Medicine 2, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 USA

2. Age-related Bone Loss Decreased bone formation by osteoblasts Relative increase in osteoclastic resorption Uncoupling of bone formation and resorption

3. Evidence for human osteoblast dysfunction with aging Mean wall thickness of trabecular bone decreases with age Inadequate formation response to increased resorption during bone remodeling Increased bone formation that normally occurs in response to mechanical loading is diminished Inverse relationship between donor age and in vitro lifespan Lips, P Calcif Tiss Intl 26: 13-17 (1978); Kohrt, WM Intl J Sport Nutr Exer Metab 11:S137-42 (2001); Jager, A J Anat 189:257-64 (1996); Parfitt, AM Calcif Tiss Intl 36:S123-8 (1984); Clarke, BL et al J Clin Endocrinol Metab 81:2264-70 (1996); Yudoh, K et al JBMR 16:1453-64 (2001)

4. Stem Cell Mesenchymal Stem Cell OsteoprogenitorPre-osteoblastOsteoblast Chondrocytes Myocytes Fibroblasts Bone- Lining cell Osteocyte Adipocyte BMPs TGFβ BMPs Runx2 Osx PTH IGF-I, PGE2 Vitamin D Steroids Histone Collagen TGFβ1 Osteopontin Alk Phos BSP Collagen BMPs Collagen Osteocalcin Osteopontin Collagenase Other NCPs Mineralization Based on: R. Pignolo and F. Kaplan, Chapter 40: Bone Biology in Inverventional Spine (2008) Possible cellular mechanisms of age-related bone loss Osteoblast senescence MSC senescence Lineage switching Transdifferentiation

5. What is Cellular Senescence?

6. Cellular Senescence Hayflick 1961- Normal, somatic cells do not divide indefinitely but have a finite replicative lifespan. Senescent cells are characterized by an inability to progress through the cell cycle, usually with a DNA content consistent with late G1. Cells remain metabolically active but fail to initiate DNA replication. Apoptosis resistance. www2.mrc-lmb.cam.ac.uk/.../CellCycle.gif

7. Background- Cellular Senescence

8. Replicative Senescence as a Model for Osteoblast Aging Finite in vitro life span Decreased osteoblast responsiveness to extracellular signals, including 1,25 (OH)2 vitamin D3, IGF-I, PTH, and prostaglandin E2 in both primary cultures from old donors as well as in osteoblasts aged in vitro by serial passage Loss of function with in vitro and in vivo age Battmann A et al Exp Clin Endocrinol Diabeetes105:98-102 (1997); Kassem M et al Osteoporos Int 7:514-24 (1997); Kveiborg M et al Exp Gerontol 35:1061-74 (2000); Kveiborg M et al J Cell Physiol 186:298-306 (2001); Martinez ME et al Bone 24:203-9 (1999); Martinez P et al Bone 29:35-41 (2001); Yudoh, K et al JBMR 16:1453-64 (2001)

9. Limited In Vitro Life Span of Normal Human Osteoblasts

10. Replicative Senescence as a Model for Osteoblast Aging Finite in vitro life span Decreased osteoblast responsiveness to extracellular signals, including 1,25 (OH)2 vitamin D3, IGF-I, PTH, and prostaglandin E2 in both primary cultures from old donors as well as in osteoblasts aged in vitro by serial passage Loss of function with in vitro and in vivo age Battmann A et al Exp Clin Endocrinol Diabeetes105:98-102 (1997); Kassem M et al Osteoporos Int 7:514-24 (1997); Kveiborg M et al Exp Gerontol 35:1061-74 (2000); Kveiborg M et al J Cell Physiol 186:298-306 (2001); Martinez ME et al Bone 24:203-9 (1999); Martinez P et al Bone 29:35-41 (2001); Yudoh, K et al JBMR 16:1453-64 (2001)

11. Early Passage Late Passage Alk PhosMineral Impaired Differentiated Function of Human Osteoblasts with In Vitro Age

12. Markers of Osteoblast Replicative Senescence Telomere dysfunction Senescence-associated heterochromatin (HIRA-PML nuclear bodies) SA-β-galactosidase activity Nucleolar association- Acridine Orange

13. Hypothesis In vitro replicative senescence can serve as a model for osteoblast dysfunction, which may recapitulate aspects of age-related bone loss. –Osteoblast cell strains derived from young individuals have limited in vitro lifespans. –With advancing replicative age these cell strains display impairment of differentiated function. –Loss of osteoblast differentiated function occurs concomitantly with characteristics of senescence in culture.

14. Markers of Osteoblast Replicative Senescence Telomere dysfunction Senescence-associated heterochromatin (HIRA-PML nuclear bodies) SA-β-galactosidase activity Nucleolar association- Acridine Orange

15. Senescence Associated β- Galactosidase (SA β-gal) β -galactosidase is a eukaryotic hydrolase enzyme β -gal at pH 6.0 has been reported to increase during replicative senescence and may reflect replicative age of cells Limited application because not specific to senescence- –Also increased in quiescent, immortalized and serum starved cells –Reversible under other conditions –May actually be lysosomal enzyme releases at suboptimal pH (4.0)

16. SA β -gal Early PassageLate Passage

17. Acridine Orange Nucleic acid selective fluorescent cationic dye. Tips of 5 pairs of chromosomes fuse into fewer and larger fragments as they approach S phase. As cells progress through the cell cycle the fraction of cells containing 1 or 2 nucleolar fragments increase while those containing 3 or more fragments decrease. Up to 90% of senescent cells in culture may contain only 1 to 2 nucleolar fragments. Pignolo R et al Exp Geron 33:67-80 (1998)

18. Relationship of SA β-gal / Acridine Orange and Various Conditions Young cells Quiescent cells “Stressed” cells Senescent cells β-gal+β-gal- Quiescent cells “Stressed” cells Senescent cells Young cells Acridine Orange 1-2 nucleoli Quiescent cells “Stressed” cells Senescent cells ≥3 nucleoli Young cells ≥3 nucleoli

19. SA-β-gal Activity & Nucleolar Association in Senescent Human Osteoblasts Early PassageLate Passage

20. SA-β-gal Activity & Nucleolar Association in Senescent Human Osteoblasts Early passage/ToxicEarly passage/Quiescent 40x

21. HIRA Histone Regulatory homolog A Histone chaperone involved in assembly of histones onto DNA Senescent cells exhibit a specific pattern of nucleolar foci with increased heterochromatin Senescence-Associated Heterochromatin Foci (SAHF) Reorganized chromatin structure leads to a loss of transcription activity by silencing of growth-promoting genes in SAHF

22. Senescence-Associated Heterochromatin in Aging Osteoblasts Heterochromatin formation in senescent cells Early PassageLate Passage

23. HIRA-associated Nuclear Foci in Aging Osteoblasts

24. Markers of Osteoblast Replicative Senescence Telomere dysfunction Senescence-associated heterochromatin (HIRA-PML nuclear bodies) SA-β-galactosidase activity Nucleolar association- Acridine Orange

25. Telomere Dysfunction Telomere dysfunction/uncapping Persistent DNA damage Response 53BP1

26. Senescent Osteoblasts Have Dysfunctional Telomeres Red………Telomeres Green……53BP1 Arrows......53BP1-associated telomeres SenescentEarly Passage

27. Conclusions With in vitro replicative senescence human osteoblasts display a loss of differentiated phenotype concomitant with limited life span Markers of in vitro osteoblast senescence exist which can potentially be used to detect aging osteoblasts in situ Replicative senescence may serve as a model for osteoblast dysfunction that occurs with aging

28. Acknowledgements PI/Mentor –Robert J Pignolo Laboratory –Kevin Egan –Alec Richardson –Emily McMillan –Robin Suda Collaborators –F. Brad Johnson (U of PA, Phila) –Peter Adams (FCCC, Phila)

29. Questions?