adult stem cells & homeostasis
TRANSCRIPT
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Adult Stem Cells &
Homeostasis
Developmental Biology
曾 艺
生化与细胞所
2014-4-28
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Outline
• Stem cell and niche
• Tissue models for studying adult stem cells
• Experimental assays
• Adult stem cell & Cancer
• Adult stem cell & Regenerative medicine
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Adult Stem Cells
• Adult stem cells are undifferentiated cells found in many organs/tissues
• Adult stem cells self-renew and differentiate to maintain tissue homeostasis
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Stem Cell & Progenitor
• long-term self-renewal
•generate all
differentiated cells of
an organ
multipotent
progenitor unipotent
progenitor
• rapidly proliferate
•differentiate
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stem cell
Niche
Adult Stem Cell & The Niche
Niche & Microenvironment?
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stem cell
specialized
mature cell
stem cell
niche
Stem Cell Asymmetric Division
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symmetric
division stem cell expansion stem cell
Niche
Stem Cell Symmetric Division
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The Fruit Fly Male Germline Niche
(Fuller & Spradling, Science, 2007)
Niche
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Niche Decides Who Are Stem Cells
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Niche Decides Who Are Stem Cells
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Intrinsic & Extrinsic Control
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Hematopoietic Lineage
(Regenerative Medicine, 2006)
Mouse HSC : CD34lo/-, SCA-1+, Thy1.1+/lo,
CD38+, C-kit+, lin-
Human HSC : CD34+, CD59+, Thy1/CD90+,
CD38lo/-, C-kit/CD117+, lin-
(骨髓系组细胞) (淋巴系组细胞)
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(Aft et al., 2012)
Hematopoietic Stem Cell (HSC) Niche
(骨内膜)
(血管)
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(Photo: Gerd Klein team, DE)
HSCs Are Mobil in Between Niches
(骨内膜) (血管)
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(Hsu and Fuchs, 2012)
HSC Dormant & Active Niche
(dormant HSC)
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The Small Intestine Stem Cell
• a simple, highly
stereotypical layout
• the fastest self-
renewing tissue in
mammals (3-5 days)
(Crosnier et al, 2007)
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+4: Bmi1
CBC: Lgr5
(quiescent stem cell)
(active stem cell)
Long-term DNA-label retention
has tentatively located stem
cells at “position +4”
Co-exist of Quiescent and Active Stem Cell
Lgr5 is a Wnt pathway target gene
(Potten et al, Int. J. Exp. Pathol., 1997)
(Tian et al, Nature, 2011)
(Barker et al, Nature, 2007)
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Paneth Cells Are the Niche
(Sato et al, Nature, 2010)
Paneth Cell: (the niche)
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(Sato et al, Nature, 2010)
Paneth Cells Produce Wnt3
Niche
SC
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Paneth Cells Regulate the Numbers of
Intestinal Stem Cells In Vivo
(Sato et al, Nature, 2010)
Niche
SC
Paneth specific
promoter-DTA
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What We Learn from ISCs
CBC: Lgr5 (active stem cell)
+4: Bmi1(quiescent stem cell)
Paneth Cell: Wnt3 (the niche)
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The Hair Follicle Cycle
(Blanpain and Fuchs, Annu Rev Cell Dev Biol, 2006)
Anagen:
growth phase
Telogen:
the resting phase
Catagen:
regressing phase
(生长期)
(退行期)
(休止期)
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The Hair Follicle Cycle
(Blanpain and Fuchs, Annu Rev Cell Dev Biol, 2006)
Anagen:
growth phase
d4
3W
4W
6W
8W
Non
synchronized
Telogen:
the resting phase
Catagen:
regressing phase
(2%)
(13%)
(85%)
(生长期)
(退行期)
(休止期)
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The Hair Follicle
Dermal Papilla (connective tissue, capillary)
Matrix (produces cells forming
the hair fiber;melanocytes)
Inner Root Sheath
Outer Root Sheath
Arrector pili muscle
Anagen
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The Hair Follicle Bulge
Dermal Papilla (connective tissue, capillary)
Matrix(produces cells forming
the hair fiber;melanocytes)
Outer Root Sheath
Arrector pili muscle Bulge
Inner Root Sheath
Anagen
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The Bulge SCs Are Slow Cycling
(Tumbar et al, Science, 2004)
Outer bulge layer:
CD34+
tetO-H2B-GFP (tetracycline-induced GFP)
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HFSC Markers
Lgr6: HF, IFE, SG
CD34+: HF, SG
Lgr5: HF
Lgr6: (Snippert et al., Science, 2010)
Lgr5: (Jaks et al, Nature Genetics, 2008)
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Wnt and FGF Activate HFSCs
(Blanpain and Fuchs, Annu Rev Cell Dev Biol, 2006)
Dermal papilla
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BMPs Maintain HFSC Quiescence
(CD34+)
(Blanpain and Fuchs, Annu Rev Cell Dev Biol, 2006)
Dermal papilla
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Summary 1
• Stem cell and niche (Drosophila male germ line)
• Asymmetric & symmetric division
• Hematopoietic SC (dynamic niche)
• Intestine SC (quiescent and active SCs)
• Hair follicle SC (activation cycle)
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How To Assay Adult Stem Cells?
In vitro
•Colony formation (self-renewal/proliferation)
•Differentiation potential
3D: suspension/Matrigel culture
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In vitro
•Colony formation (proliferation)
•Differentiation potential
In vivo
•Transplantation (blood, hair follicle, mammary
gland)
•Injury & healing (liver, lung, trachea)
•Lineage tracing
How To Assay Adult Stem Cells?
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Hair follicle stem cell
transplantation
Hematopoietic stem cell
transplantation
Transplantation
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the mouse mammary gland
Mammary gland Transplantation
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Mammary Gland Development
Newborn Puberty Mature adult
Pregnancy
Involute
fat pad
Lactation
Unique postnatal development
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(DeOme et al, 1959)
Recipient (3-week)
fat pad clearing
MaSCs Transplantation
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fat pad clearing
Recipient (3-week) Donor
(DeOme et al, 1959)
cleared fat pad
exogenous
epithelial cells
MaSCs Transplantation
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Wild Type, GFP Wild Type, DsRed
(Lin-, CD24+, CD29hi) (Lin-, CD24+, CD29hi)
1:1
The Competitive Transplantation
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Axin2-/-, GFP Wild Type, DsRed
(Lin-, CD24+, CD29hi) (Lin-, CD24+, CD29hi)
1:1
LOF Axin2 growth advantage
The Competitive Transplantation
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Injury & Healing
• Surgical
• Hair plug
• CCl4 (liver)
• Naphthalene (lung)
Targeted-ablation:
inducible Diphtheria
toxin (DTA)
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Lineage Tracing
SC
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Intestine SC Lineage Tracing
(Barker et al, Nature, 2007)
IRES: internal ribosome entry
site
(内部核糖体进入位点序列)
Cre: site-specific recombinase
(重组酶)
CreER: Cre fused to the human
estrogen receptor (ER), can be
activated by estrogen, tamoxifen,
or 4-hydroxy-tamoxifen (4-OHT)
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Lgr5+ Cells Are CBCs
(Barker et al, Nature, 2007)
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Lineage Tracing With LacZ Expression
(Barker et al, Nature, 2007)
loxP loxP
• Spatial & temporal control
• Irreversible
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Lineage Tracing With LacZ Expression
(Barker et al, Nature, 2007)
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The ISC Lineage Tracing
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The Brainbow-2.0 constructs
– loxP sites facing each other
– Contain RFP and mCFP in head-in-head orientation
(Livet et al, 2007)
Lineage Tracing with Brainbow
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The Brainbow-2.1 construct (the Confetti)
– With 4 fluorescent protein sequences: RFP, YFP, M-CFP and nGFP; with 4 loxP sites
Lineage Tracing with Brainbow
(Livet et al, 2007)
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(Snippert et al, Cell, 2010)
Lgr5+ Cell Lineage Tracing with Brainbow
Lgr5-GFP-IRES-CreERT2, confetti
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Lgr5+ Cell Lineage Tracing with Brainbow
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Potential > Fate
In vivo
• Transplantation (blood, hair follicle, mammary
gland)
• Injury & healing (liver, lung, trachea)
• Lineage tracing
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Stem cell & Cancer
Cancer stem cell:
(Visvader, Nature, 2011)
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Stem cell & Cancer
challenging typical “cancer stem cell” model
(Visvader, Nature, 2011)
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Distinct Cell Origins in Tumors
(Visvader,
Nature, 2011)
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Intestine Cancer
(Barker et al, Nature, 2009)
Lgr5-CreER, APC fl/fl
Ah-CreER, APC fl/fl
(loxP_APC_ loxP)
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Intestine Cancer
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Implication of Adult Stem cells
- Regenerative Medicine -
(Sasai, Cell Stem Cell, 2013)
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Implication of Adult Stem cells
- Regenerative Medicine -
(Sasai, Cell Stem Cell, 2013)
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Implication of Adult Stem cells
- Regenerative Medicine -
(Sasai, Cell Stem Cell, 2013)
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Stem Cells Have Self-Organizing Abilities
(Sasai, Nature, 2013)
(共识主动性,自组织)
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(Eiraku et al, Nature, 2012)
Self-Organization of Optic Cup Formation
retinal pigment epithelium
neural retina
Mouse E11.5
Rx::GFP
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(Eiraku et al,
Nature, 2012)
Self-Organization of Optic Cup Formation
Evagination of Rx::GFP vesicles
(day 4.5-6.5)
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Self-Organization of Optic Cup Formation
Invagination of Rx::GFP vesicles
(day 6.75-9.0)
(Eiraku et al,
Nature, 2012)
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Safety of Using Self-Organizing Organoids
Sasai emphasizes that the cells in the optic cup are
“pure”, unlike those in two-dimensional aggregates,
which may still contain embryonic stem cells. This
reduces concerns that transplants of such cells might
develop cancerous growths or fragments of unrelated
tissues. “It’s like pulling an apple from a tree. You
wouldn’t expect iron to be growing inside,” says Sasai.
“You’d have no more reason to expect bone to be
growing in these eyes.”
(Cyranoski, Nature, 2012)
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(Sato et al, Nature, 2009)
Self-Organization of Crypt Formation
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A single cypt
(day 2-3)
Self-Organization of Crypt Formation
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Organoid derived from an
Lgr5-EGFP-IRES-CreERT2;Rosa26-YFP crypt
(day 7)
Self-Organization of Crypt Formation
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Summary
• Stem cell and niche (Drosophila male germ line)
• Tissue models for studying adult stem cells
(hematopoietic, intestine, hair follicle SC)
• Experimental assays
(transplantation, injury & lineage tracing)
• Adult stem cell & Cancer
• Adult stem cell & Regenerative medicine
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Take Home Messages
• What are the characteristics of adult stem
cells? (the dual jobs)
• What are the in vivo assays for examining
adult stem cell properties?
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Thank You