New approaches to bring back youthfulness to aged stem cells Aging is a nearly universal process affecting all tissues. Despite its constancy in our lives, aging remains mysterious at a fundamental level. Nevertheless, common hallmarks of aging across different species have been proposed offering an integrated view of the basic mechanisms of aging. Primary hallmarks include cell autonomous changes linked to epigenetic alterations, genomic instability, telomere attrition and loss of proteostasis (protein homeostasis), which are followed by antagonistic responses such us deregulated nutrient sensing, altered mitochondrial function and cellular senescence. Aging hallmarks converge in the exhaustion of stem cells, which provokes tissue regenerative decline. Skeletal muscle provides a stark example of this decline. Its stem cells sustain muscle regeneration throughout life but at advanced age they fail for largely undefined reasons. Several causes for this age-associated stem cell regenerative failure are emerging: decline in proteostatic quality-control mechanisms, metabolic alterations, entry into senescence and changes in the systemic (circulatory) environment. I will review our recent findings on how to improve the regenerative capacity of old stem cells by countering these age-associated alterations, with the ulterior idea that the aging process is malleable and that it is feasible to rejuvenate aged cells and tissues.

1. ICREA & Universitat Pompeu Fabra
Aging as a treatable condition:
New approaches to bring back
youthfulness to aged stem cells
ICREA Colloquium
Barcelona, October 9, 2018
Pura Muñoz-Cánoves

2. Increased risk of
tissue deterioration,
malfunction and
disease with aging
Disease, sarcopenia, frailty
Health

3. • Since 1900, lifespan has increased over 30 years
in men and women in western countries.
• So, in just 100 years, we have extended human
longevity quite significantly.
• Maybe… we should pursue not just living longer,
but better.

4. Frailty - Sarcopenia
Geriatric age

5. Geriatric age
Good mental condition
Muscle strength and
fitness
Healthy aging

6. Sarcopenia
• Sarco flesh (muscle)
• penia deficiency
Loss of skeletal muscles mass and
function (strength) with aging
Sarcopenia is associated to increased
mortality and functional decline at
advanced age

7. 40% loss of muscle mass between
20-70 years of age
6% decline in muscle mass at each
decade between 30-70 years
1.4 –2.5% decline in muscle mass
each year after 60
Changes in muscle mass with aging

8. Loss of muscle regenerative capacity is
also maximal at geriatric age
(after 75-80 years)

9. Skeletal muscle,
regeneration and
aging

10. 20000nm
Muscle fiber
Satellite cell
Pax7+Muscle fiber
ite cell
Basal lamina
10µm
Skeletal muscle and its stem cells
(satellite cells)
Quiescence

11. Adult skeletal muscle homeostasis:
regeneration after injury
INJURY
Activation
Proliferation
Differentiation
Fusion
Self-renewal
Quiescence

12. How do we study the regenerative
capacity of muscle stem cells in the lab ?

13. Injury by:
- Toxin injection
- Crushing
Formation of new fibers
In vivo model (mice): Regeneration

14. Muscle regeneration after one week

15. 1 week2 days 4 days 3 weeks
Skeletal muscle has an high regenerative capacity

16. Skeletal muscle regenerative capacity
declines with aging
Extrinsic
influences

17. Parabiosis between 2 mice of different age:
old-young

18. Adapted from Conboy and Rando, Cell Cycle 2012
YOUNG MUSCLE OLD MUSCLE
Injury:
Efficient
proliferation/differentiation
Injury:
Non-efficient proliferation
/differentiation
Aging
Rejuvenation
(Heterochronic parabiosis)
Aging and rejuvenation of skeletal muscle:
Parabiosis model

19. Major aims:
1. Understand muscle cell functions and
regenerative decline during physiological
aging, and especially at geriatric age
2. Try to combat muscle stem cell aging
and improve regeneration
• Young: 2-3 months
• Adult: 6-8 months
• Old: 20-24 months
• Geriatric: 28-32 months

20. Exacerbated muscle regeneration defect in
geriatric mice, compared to old mice
CTX injury-
induced muscle
regeneration

21. Reduced regenerative capacity of geriatric satellite cells
after transplantation into young muscle
Satellite cell-intrinsic alterations at geriatric age

22. Geriatric age induces intrinsic alterations in muscle
stem cells that affect their regenerative functions,
which cannot be fully rejuvenated by a young host
environment

23. What are the satellite cell-intrinsic
alterations at geriatric age?
Question 1:

24. p16INK4a
is expressed in geriatric satellite cells
(but not in adult or old satellite cells) in muscle
homeostatic conditions
Transcriptomic / Bioinformatic analysis:

26. “point of no-return”
p16INK4a

27. Pre-senescence
(not in quiescence)
G0
Irreversible
Arrest
p16INK4a
Proliferative pressure
Satellite cell
(Geriatric age)
Regeneration
Growth factors
Unable to proliferate
(Through p16/Rb/E2F
inhibition)
Full senescence
Sousa-Victor et al. Nature 2014
Geroconversion

28. Senescent cells
A permanent cell cycle arrest
Cellular senescence
é Size
êProliferation
é p16, é p21
é DDR
é SA-βgal activity
éSASP (secreted factors)
é ROS
êTelomere length
é Apoptosis resistance

29. How do muscle stem cells lose their
regenerative capacity with aging?
- how do they become senescent
(express p16INK4a, etc)?
Question 2:
Which cellular processes are altered in
geriatric muscle stem cells?

30. Transcriptomic/bioinformatic analysis
The quiescent state is enriched in
proteostatic pathways
Autophagy: a prevalent one

31. The process of (macro)autophagy in mammalian cells
N. Mizushima
Cold Spring Harbor Symposia on Quantitative Biology 2011

32. Questions:
Do satellite cells have autophagic activity in the
resting quiescence state?
If so, is autophagy altered in quiescent satellite
cells with aging?
GFP-LC3 transgenic mice
LC3 (punta or fluorescence levels):
as proxy for autophagosomes
LC3

33. Analysis of the autophagy flux in quiescent satellite cells
GFP-LC3
-Baf
+Baf
-Baf
+Baf
YoungOld
YoungOld
+Baf
+Baf
GFP-LC3
p<0.03
%∆GFP-LC3MFI(±Baf)
Young
Old
0
50
100
150
Bafilomycin: blocks lysosomal degradation
Autophagic activity is impaired in quiescent satellite cells with aging

34. Autophagy-lysosomal degradation
Quiescence
Senescence
Aging
Senescence
García-Prat et al., Nature 2016

35. Can autophagy be reactivated
in old satellite cells?
Rapamycin
- Rapamycin, by inhibiting mTOR, induces autophagy
Atg7 overexpression
- Atg7, a crucial protein for autophagosome formation

36. Rapamycin treatment of old GFP-LC3 mice (for 2 weeks)
restores basal autophagy in quiescent satellite cells
%∆GFP-LC3MFI(±Baf)
Rapamycin
Control
p<0.02
0
50
100
150
200 Old
Control Rapamycin
GFP-LC3 +Baf +Baf

37. Can autophagy reactivation
rescue the regenerative block
(and senescence entry) of old
(geriatric) satellite cells?

38. Reactivation of autophagy by Rapamycin treatment or Atg7
overexpression restores the expansion of geriatric satellite
cells in vivo
YoungControlRapamycin
Geriatric
LV-Atg7
GFP Pax7 Dapi/Merge
LV-Atg7 infection or
rapamycin treatment
C57BL6 mice
SCID mice
Cell Transplantation
Analysis
4 and 28 days
Young
Control
Rapamycin Geriatric
LV-Atg7
%GFP+
cells/section
p<0.008
p<0.008
p<0.007
0
50
100
150
LV-Atg7 infection or
rapamycin treatment
C57BL6 mice
SCID mice
Cell Transplantation
Analysis
4 and 28 days
Young
Control
Rapamycin Geriatric
LV-Atg7
%GFP+
cells/section
p<0.008
p<0.008
p<0.007
0
50
100
150
4 days after satellite cell transplantation

39. Reinduction of autophagy rescues the proliferative
defect and reduces senescence in geriatric satellite cells
BrdU+
cells(%)
SA-ß-gal+
cells(%)
0
2
4
6
8
10
Young
Control
Rapamycin Geriatric
LV-Atg7
p<0.001
p<0.001
p<0.001
0
10
20
30
40
p<0.005
p<0.05
p<0.05
BrdU+
cells(%)
SA-ß-gal+
cells(%)
0
2
4
6
8
10
Young
Control
Rapamycin Geria
LV-Atg7
p<0.001
p<0.001
p<0.001
0
10
20
30
40
p<0.005
p<0.05
p<0.05
p<0.001
p<0
SA-ß-gal+
cells(%)
0
20
40
60
RapamycinControl
Human

40. Conclusion:
Autophagy decline contributes to the loss
of quiescence and entry into senescence
of aged satellite cells

41. Autophagy-lysosomal degradation
Quiescence
Senescence
Aging
Senescence
García-Prat et al., Nature 2016

42. Autophagy
Quiescence
Senescence
Aging
ROS
Accumulation of damaged organelles and mitochondria
p16INK4a
/ Mitophagy
INK4a locus
derepression
García-Prat et al., Nature 2016

43. Senescence
Autophagy
Pre-senescence
Impaired autophagy
Quiescence
Stemness/Regenerative function
Aging
ProteotoxicityProteostasis
Rejuvenating strategies
Systemic (blood) / niche (local)
Autophagy reinduction
Rapamycin
Antioxidants
Summary of strategies that increase muscle stem
cell regenerative capacity

44. Autophagy / Proteostasis
Quiescence
Senescence
Aging
Caloric restriction
?

45. Senescence
Autophagy
Pre-senescence
Impaired autophagy
Quiescence
Stemness/Regenerative function
Aging
ProteotoxicityProteostasis
Rejuvenating strategies
Systemic (blood) / niche (local)
Autophagy reinduction
Rapamycin
Antioxidants
Calorie restriction
Senolytics
Exercise (?)
Summary of strategies that increase muscle stem
cell regenerative capacity

46. Anti-aging pills
today:
Science or
science fiction?
Matt Kaeberlein
Brian Kennedy
News and views - Nature 2009

47. Thank you!!!
Pedro Sousa-Victor *
Laura García-Prat *
Victoria Moiseeva
Pedro Maseres
Jessica Segalés
Sonia Alonso-Martín
Xiaotong Hong
Laura Ortet
Mercè Jardí
Marta Flández
Vera Lukesova
William Roman
Marina Raya
Antonio Serrano
Eusebio Perdiguero
Salvador Aznar-Benitah
Marco Sandri
Marta Martínez-Vicente
Esteban Ballestar
Charles Keller
Vittorio Sartorelli
Michael Rudnicki
Jun Hee Lee
Thomas Braun / Johnny Kim
Francesc Posas