SEMINAR 5:

SENESCENCE

Dra. Laura Ortet

ÍNDEX
• Introduction
• Aging
• Hayflick´s limit
• Senescence
• Causes
• In tissue formation, ageing and disease
• Identification of senescent cells
• Genes regulating entry into senescence
• Senescence markers
ÍNDEX
• Senescence
• SASP: Senescence Associated Secretory
Phenotype
• Senescence is not quiescence
• Skeletal muscle tissue
• Senescence atlas reveals an aged-like
inflamed niche that blunts muscle
regeneration
INTRODUCTION
How to Cure Aging – During Your Lifetime?
INTRODUCTION
The time in the organisms: aging

Progressive loss of functionality with impairment of the fertility and increment of

the mortality associated to increasing age

Cellular

Organisms
AGING
The hallmarks of aging

López-Otín 2013
AGING
The hallmarks of aging

2022
AGING
Who gets old?
- NO evidence of aging in prokariots

- YES in S. cerevisiae whith the appearance of the assymetric division: sexual

reproduction

- YES in multicellular organisms

STRONG ASSOCIATION BETWEEN THE EXISTENCE

OF GERM LINE/SOMATIC CELLS
AND AGING
 HAYFLICK’S LIMIT

Hayflick’s limit 1961 HMF: Human Diploid Fibroblasts

senescent cells

Days in culture
HAYFLICK’S LIMIT
SENESCENCE: causes
SENESCENCE: in tissue formation, ageing and disease

Beneficial senescence
Detrimental senescence
SENESCENCE: Identification of senescent cells

Flat morphology

HMF: human normal fibroblasts (phase a) and senescent (phase b)

SENESCENCE: Identification of senescent cells
SA-bGal expression: Senescence-Associated b-galactosidase (measured at acidic pH)

X-Galactose b-galactosidase
(X-Gal) Blue precipitate
pH=6.0
SENESCENCE: Identification of senescent cells
Senescent fibroblasts

p16INK4a
p19ARF
p53
p21Cip1

Immortalized fibroblasts

p16INK4a
p19ARF
p53 mutated
p21Cip1
SENESCENCE: Senescence markers

• Increased SA-B-gal activity

• Large cell size
• Decreased proliferation
• Reduced lamin B1
• Lack of apoptotic programmed death
• High Cdk2a (p16INK4a) expression
• 8-oxoguanine (ROS-induced DNA damage) increased
• Telomeric DDR increased
SENESCENCE: Senescence markers
SENESCENCE
SASP: Senescence Associated Secretory Phenotype

Pro-tumorigenic

SASP

- Proinflammatory cytokines
- Proteases
- Growth factors
- Immunomodulatory cytokines
- Exosomes
- DNA
anti-tumorigenic - microRNAs….
SENESCENCE:
Senescence is not quiescence

ARE NOT THE SAME

Both quiescent and senescent cells are out of the cell cycle:
they do not divide (=are in G0)

however,

Senescent cells have accumulated DNA damage and they

cannot
go back into cell cycle= they can not proliferate again

Quiescent cells do not proliferate but with the right

combination of
mitogens, nutrients, etc. they can come back into cell cycle
again
SENESCENCE:
Senescence is not quiescence
Skeletal muscle tissue:
Skeletal muscle tissue:
SCs

FAPs
Skeletal muscle tissue:
Skeletal muscle tissue:
Impaired regeneration in aged muscle and chronic
injury
What is the role of senescence in muscle regeneration and during
ageing?

• Current understanding of senescent cells comes from in vitro studies.

• Which is the identity of senescent cells in vivo (i.e. those that emerge after
tissue injury)?
• How can we isolate senescent cells without reporter mice?
Kinetics of cellular senescence in muscle regeneration

Senescent cells:
- emerge after injury
- are more prominent and persistent in geriatric m
Senescent cell isolation protocol with SPiDER-b-gal (in vitro)
Senescent cell isolation protocol with SPiDER-b-gal (in vitro)
Senescent cell isolation protocol with SPiDER-b-gal ( in vivo)
Senescent cells emerge from distinct populations upon injury
Three major sources of senescent cells: MCs, FAPs and SCs
Simultaneous isolation of senescent SCs, FAPs and MCs from
regenerating muscle tissue in vivo
Simultaneous isolation of senescent SCs, FAPs and MCs from
regenerating muscle tissue in vivo
Main messages so far
Main messages so far

• Emerge from distinct populations upon

injury.
Do senescent cells participate in muscle
regeneration?
Genetic reduction of senescent cells accelerates muscle regeneration in
geriatric mice
Genetic reduction of senescent cells restores muscle regeneration in
young mice
Senolytics: drugs that selectively kill senescent cells
Pharmacological reduction of senescent cells accelerates muscle
regeneration in young and geriatric mice
Transplantation of senescent cells delays muscle regeneration in receptor
mice
 DMD in skeletal muscle tissue

Healthy donor DMD patient

SA-b-gal Presence of senescent cells in
Dystrophin H&E

mdx-2/DBA
skeletal muscle tissue of mdx mice

Kamdar and Garry, 2016

What is the role of senescent cells in

the context of DMD?  senolytics
treatment
Reduction of senescent cells ameliorates disease progression in
mdx-2/DBA mice
Reduction of senescent cells ameliorates disease progression in
mdx-2/DBA mice