Muscle Stem Cells in Regeneration

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1 Muscle Stem Cells in Regeneration Dr. F Jeffrey Dilworth BIM6028/SMC6052 Lecture (February 11, 2016)

2 Duchenne Muscular Dystrophy is an X-linked genetic disorder that affects 1 in 3500 males Wells et al, Expert Reviews in Molecular Medicine (2002) 4: 1-23.

3 Sarcopenia is the age related loss of muscle mass that occurs due to altered capacity to regenerate Thigh muscle of young and old human subjects. With Age: - reduced muscle - Increased subcutaneous fat - Increased infiltrating fat - Increased infiltrating connective tissue Koopman R, and van Loon L J C J Appl Physiol 2009;106:

4 Stem cells and cell potency Definition: A stem cell is a clonal, self renewing entity that is multipotent and thus can generate multiple differentiated cell types. Doug Melton and Chad Cowan Handbook of Stem Cells Totipotent: The ability of a cell to give rise to all cells required for the development of a normal adult organism (fertilized egg) Pluripotent: The ability to differentiate into all the different cell types derived from the three germ layers (embryonic stem cells) Multipotent: The ability to differentiate into a limited number of cell types (hematopoietic stem cell)

5 Adult (Multipotent) Stem Cells - An adult stem cell is an undifferen(ated (or par3allydifferen3ated) cell found in 3ssues and organs - They can self-renew and differen(ate to become most or all of the specialized cell types within their specific 3ssue lineage. - Adult stem cells Maintain cell popula3ons Help you heal Play a role in aging

6 Stem cells in development, tissue homeostasis and cancer 2006 by American Association for Cancer Research Wicha M S et al. Cancer Res 2006;66:

7 How does regeneration work? Adult stem cells normally remain quiescent (non-dividing) for relatively long periods of time until they are activated by signals to maintain tissues When activated they divide through a process called asymmetric cell division Through this process they are able to maintain their populations and differentiate into the desired cell types by the creation of a progenitor cell A progenitor cell, in contrast to stem cells, is already far more specific: they are pushed to differentiate into their "target" cell.

8 Regeneration in muscle Mann et al. Skelet Muscle 1: 21, After acute muscle damage, inflammatory (macrophage) cells and fibroblasts invade the tissue. Between h after damage, first stem cells divide and begin regeneration the muscle - Regenerated muscle fibers are recognized by their nuclei being centrally localized

9 Question 1 In response to muscle injury, a satellite cell will be activated and begin to proliferate. Is the proliferating cell: a) a totipotent stem cell b) a pluripotent stem cell c) a multipotent stem cell d) a muscle progenitor cell

10 Muscle Stem Cells Major Contributor Satellite cells (α7-integrin + /CD34 + /Sca-1 - /CD31 - /CD45 - ) Other Contributors Bone marrow stem cells (CD45 + /Sca-1 + or c-kit + /CD45 + ) Muscle side populations cells (Hoechst dye) PW1+ interstitial cells (Pax7 - /PW1 + ) Mesangioblasts (CD34 + /c-kit + /Flk-1 + /Nkx2.5 - /Myf5 - /Oct4 - ) Muscle-Derived Stem Cells (Desmin + /Sca-1 + /Flk1 + /CD34 - / CD45 - /M-Cadherin - )

11 Isolation of Muscle Stem Cells: Fluorescence Assisted Cell Sorting (FACS)

12 System for demonstrating that satellite (α7 integrin+/cd34+) cells are the major contributor to muscle regeneration Tamoxifen Cre-ERT TM Cytoplasm CAG STOP DTR PAX7 promoter Nucleus Cre-ERT TM CAG DTR Tomato ON

13 Satellite cells are the major contributors to muscle regeneration Relaix and Zammit. Development 139: , 2012

14 Muscle Satellite (Stem) Cells - Mononucleated cells that reside on the muscle fiber between the plasma membrane and the basal Lamina (stained for Pax7 and Caveolin 1) - In mice, the TA myofiber is ~15 mm in length and contains an average of 10 satellite cells per fiber. The density in humans is predicted to be greater based on examination of biopsies Relaix F, Zammit P S Development 2012;139:

15 Muscle Satellite (Stem) Cells In humans, satellite cell density is higher almost 1 SC per 0.1 mm. Sartorius muscle fibers can be 500 mm, suggesting ~5000 SC per fiber

16 Satellite Cell Characteristics - Very well studied for characteristic markers, no one protein on its own can be used to identify satellite cells - Satellite cells that have never expressed Myf5 are proposed to be the true muscle stem cells. - Satellite cells can divide symmetrically or asymmetrically to repopulate the satellite cell pool or repair muscle Yin H et al. Physiol Rev 2013;93:23-67

17 Satellite cell can fuse into existing fibers to repair muscle, or generate new fibers Regenerating myofibers can be identified through positioning of the nucleus - centrally located in regeneration - peripherally located in healthy fiber New Old New

18 Satellite cell population is maintained through asymmetric cell division Relaix and Marcelle, Current Opin Cell Biol 21: , 2011

19 Dystrophic muscle is characterized by increased interstitial space that contains inflammatory cells and extensive fibrosis Lieber R L, and Ward S R Am J Physiol Cell Physiol 2013;305:C241-C252

20 Duchenne Muscular Dystrophy is an X-linked genetic disorder that affects 1 in 3500 males Wells et al, Expert Reviews in Molecular Medicine (2002) 4: 1-23.

21 Is Duchenne Muscular Dystrophy a stem cell disease? The DMD phenotype is much worse in humans compared to mdx mice. Is this due to depletion of the satellite cell pool? Alessandra Sacco et al. Cell 143: , 2010.

22 Models for Studying Muscle Regeneration Chronic Injury: - Mdx -/- mouse (mutation in dystrophin gene) - Mdx -/- /utrophin -/- - Mdx -/- /mtr -/- (telomerase deficient, need to breed for several generations) Acute Injury: - cardiotoxin or barium chloride injection directly into the muscle of interest - Cold exposure

23 Question 2 Multi-potent satellite cells are derived from: a) a population of pluripotent stem cells in the bone-marrow b) mesodermal tissues established during embryogenesis c) the directed-reprogramming of fibroblasts d) a population of multi-potent stem cells in adipose tissue

24 Origin of muscle and satellite cells - Presomitic mesoderm is segmented by a clock regulated through waves of Notch, FGF, and Wnt Signalling Yusuf and Brand-Saberi, Histochem Cell Biol 138: , 2012

25 Somites show a spatially restricted positioning in the developing embryo Day 10.5 p.c. Day 11.5 p.c. Cheng et al. Science 261: (Eric Olson)

26 Segmentation of the somites Staining for Hairy Olivier Pourquie Cell: 145: , 2011

27 Segmentation of the somites Wnt and FGF signaling originating in the precursor to the presomatic Mesoderm (posterior) Retinoic acid signaling in the anterior Moves towards the posterior. Meet at the determination front Cells in this region are responsive to Notch signaling that is released In response to the segmentation clock. Cell, Volume 145, Issue 5, 2011,

28 Muscle development Yusuf and Brand-Saberi, Histochem Cell Biol 138: , 2012

29 Transcriptional Regulation of Muscle Regeneration Kuang and Rudnicki. Trends in Molecular Medicine, 2008

30 Myf5 mrna is sequestered in quiescent satellite cells Crist et al. Cell Stem Cell, 2012

31 Stem Cell Niche: Satellite cells in skeletal muscle From Landers et al. (2012) BMC Biology 10:19

32 Question 3 In aging, satellite cells are less effective in repairing muscle because : a) the cells undergo senescence b) of changes intrinsic to the cells that affect their ability to respond to environmental cues c) of extrinsic changes to niche environment that affect their cell numbers d) all of the above

33 Satellite cells and niche Brack and Rando Cell Stem Cell 10: , 2012

34 Signaling in the satellite cell niche Brack and Rando Cell Stem Cell 10: , 2012

35 Decreased Notch signaling is observed in aging, contributing to decreased regeneration in aging Arthur ST, Cooley ID. Int J Biol Sci 2012; 8(5):

36 Skeletal muscle stem cells adopt a dormant state post mortem and retain their regenerative capacity Latil et al. Nature Communications 3: 903, 2012

37 p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice What are the signaling pathways that are disrupted in aging that lead to sarcopenia? Bernet et al. Nature Medicine (2014) 20:

38 How do satellite cells from young muscle differ from aged muscle? Bernet et al. Nature Medicine (2014) 20: Young satellite cells proliferate more and while aged satellite cells tend to be MyoD+ or MyoD+/Myog+

39 Can the young fiber restore youthful characteristics to aged satellite cells? Bernet et al. Nature Medicine (2014) 20: Aged satellite cells do not recover proliferative capacity when attached to a young myofiber

40 Does a young muscle environment restore youthful characteristics to aged satellite cells? Bernet et al. Nature Medicine (2014) 20: Aged satellite cells could fuse into damaged muscle fibers but but their capacity to repopulate the satellite cell niche was impaired

41 Is p38 MAPK signaling involved in satellite cell defect in aged muscle? Bernet et al. Nature Medicine (2014) 20: Both p38 MAPK and MK-2 kinase signaling activity is increased in aged muscle satellite cells

42 Can treatment of aged satellite cells withp38 MAPK inhibitor improve engraftment? Bernet et al. Nature Medicine (2014) 20: Pre-treatment of myofibers in culture for 12 h leads to improved engraftment of aged satellite cells, and an ability to repopulate the satellite cell niche.

43 Increased p38 signaling impairs self- renewal of aged satellite cells Nature Medicine 20, Nature Medicine 20, , 2014 Nature Medicine 20, , 2014

44 Geriatric muscle stem cells switch reversible quiescence into senescence Is the regenerative decline in aged satellite cells due to intrinsic changes or changes in the systemic environment? Used geriatric mice month old mice P Sousa-Victor et al. Nature (2014) 506,

45 How do satellite cell numbers change with age? Adult 5-6 months Old months Geriatric months Satellite cell numbers decrease in old mice, but their loss is not the cause of sarcopenia in geriatric mice P Sousa-Victor et al. Nature (2014) 506,

46 How does regenerative potential change of muscle satellite cells change with age? SAMP8=Progeria Regeneration is impaired in when geriatric satellite cells are Transplanted into young muscle P Sousa-Victor et al. Nature (2014) 506,

47 How does regenerative potential change of muscle satellite cells change with age? Injection of geriatric satellite cells into young muscle does not Permit efficient repair Therefore geriatric cells cannot be rejuvenated by a young host environment P Sousa-Victor et al. Nature (2014) 506,

48 Can geriatric satellite cells become activated after transplant into young muscle? Geriatric satellite cells injected into into mice for 24 hrs or 21 days did not allow for rejuvenation based on an inability to become activated (MyoD+) Thus, defective state is intrinsic to satellite cells P Sousa-Victor et al. Nature (2014) 506,

49 What is the intrinsic factor that leads to loss of reversible quiescence in geriatric satellite cells? Microarray analysis identified p16 ink4a as a senescence-associated gene up-regulated in the cluster G1 (up-regulated in geriatric satellite cells). p16 ink4a shows reduced levels of H2B-K119ub which is a PRC1 complex mark P Sousa-Victor et al. Nature (2014) 506,

50 Is p16ink4a responsible for the loss of reversible quiescence in geriatric satellite cells? Knock-down of p16ink4a allowed for restoration of satellite cell activation in geriatric cells. Over-expression of p16ink4a in young satellite cells blocked their activation P Sousa-Victor et al. Nature (2014) 506,

51 Is p16ink4a responsible for the loss of reversible quiescence in geriatric satellite cells? Knock-down of p16ink4a allowed for activation of satellite cells after transplantation for either 1 day or 21 days. P Sousa-Victor et al. Nature (2014) 506,

52 Does loss of p16 ink4a allow geriatric satellite cells to repopulate the satellite cell niche? Knock-down of p16 ink4a restores the ability of geriatric satellite cells to undergo reversible quiescence to repopulate The niche after multiple CTX treatments. P Sousa-Victor et al. Nature (2014) 506,

53 Does geriatric satellite cells also show a defect in proliferation? Geriatric satellite cells show a proliferative defect both in vivo and in vitro as indicated by lack of staining for Ki67+ cells P Sousa-Victor et al. Nature (2014) 506,

54 Does induction of proliferation cause geriatric satellite cells to enter senescence? Under proliferative conditions, geriatric satellite cells showed increased γh2ax, β-gal, and p16ink4a expression that could not be rescued by transplanting into young hosts P Sousa-Victor et al. Nature (2014) 506,

55 Is the effect of p16ink4a on proliferation mediated through the downstream targets Rb/E2F? Geriatric satellite cells show a decrease in phosphorylated Rb protein that prevents the expression of E2F target genes that regulate cell cycle progression. P Sousa-Victor et al. Nature (2014) 506,

56 Is the effect of p16ink4a on proliferation mediated through the downstream targets Rb/E2F? Transplantation of geriatric satellite cells that have decreased P16ink4a expression show a recovered expression of E2F target genes and decreased expression of senescence genes. P Sousa-Victor et al. Nature (2014) 506,

57 Is the overexpression of p16 ink4a also observed in geriatric satellite cells isolated from human muscle? p16 ink4a is also over expressed in human geriatric satellite cells Proliferation of human geriatric satellite cells can be rescued by knocking down p16 ink4a. P Sousa-Victor et al. Nature (2014) 506,