adult stem cells & homeostasis

Adult Stem Cells &

Homeostasis

Developmental Biology

曾 艺

生化与细胞所

2014-4-28

Outline

• Stem cell and niche

• Tissue models for studying adult stem cells

• Experimental assays

• Adult stem cell & Cancer

• Adult stem cell & Regenerative medicine

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

Stem Cell & Progenitor

• long-term self-renewal

•generate all

differentiated cells of

an organ

multipotent

progenitor unipotent

progenitor

• rapidly proliferate

•differentiate

stem cell

Niche

Adult Stem Cell & The Niche

Niche & Microenvironment?

stem cell

specialized

mature cell

stem cell

niche

Stem Cell Asymmetric Division

symmetric

division stem cell expansion stem cell

Niche

Stem Cell Symmetric Division

The Fruit Fly Male Germline Niche

（Fuller & Spradling, Science, 2007)

Niche

Niche Decides Who Are Stem Cells

Niche Decides Who Are Stem Cells

Intrinsic & Extrinsic Control

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）

(Aft et al., 2012)

Hematopoietic Stem Cell (HSC) Niche

(骨内膜)

(血管)

(Photo: Gerd Klein team, DE)

HSCs Are Mobil in Between Niches

(骨内膜) (血管)

(Hsu and Fuchs, 2012)

HSC Dormant & Active Niche

(dormant HSC)

The Small Intestine Stem Cell

• a simple, highly

stereotypical layout

• the fastest self-

renewing tissue in

mammals (3－5 days)

(Crosnier et al, 2007)

+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)

Paneth Cells Are the Niche

(Sato et al, Nature, 2010)

Paneth Cell: (the niche)

(Sato et al, Nature, 2010)

Paneth Cells Produce Wnt3

Niche

SC

Paneth Cells Regulate the Numbers of

Intestinal Stem Cells In Vivo

(Sato et al, Nature, 2010)

Niche

SC

Paneth specific

promoter-DTA

What We Learn from ISCs

CBC: Lgr5 (active stem cell)

+4: Bmi1(quiescent stem cell)

Paneth Cell: Wnt3 (the niche)

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）

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）

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

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

The Bulge SCs Are Slow Cycling

(Tumbar et al, Science, 2004)

Outer bulge layer:

CD34+

tetO-H2B-GFP (tetracycline-induced GFP)

HFSC Markers

Lgr6: HF, IFE, SG

CD34+: HF, SG

Lgr5: HF

Lgr6: (Snippert et al., Science, 2010)

Lgr5: (Jaks et al, Nature Genetics, 2008)

Wnt and FGF Activate HFSCs

(Blanpain and Fuchs, Annu Rev Cell Dev Biol, 2006)

Dermal papilla

BMPs Maintain HFSC Quiescence

(CD34＋)

(Blanpain and Fuchs, Annu Rev Cell Dev Biol, 2006)

Dermal papilla

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)

How To Assay Adult Stem Cells?

In vitro

•Colony formation (self-renewal/proliferation)

•Differentiation potential

3D: suspension/Matrigel culture

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?

Hair follicle stem cell

transplantation

Hematopoietic stem cell

transplantation

Transplantation

the mouse mammary gland

Mammary gland Transplantation

Mammary Gland Development

Newborn Puberty Mature adult

Pregnancy

Involute

fat pad

Lactation

Unique postnatal development

(DeOme et al, 1959)

Recipient (3-week)

fat pad clearing

MaSCs Transplantation

fat pad clearing

Recipient (3-week) Donor

(DeOme et al, 1959)

cleared fat pad

exogenous

epithelial cells

MaSCs Transplantation

Wild Type, GFP Wild Type, DsRed

(Lin-, CD24+, CD29hi) (Lin-, CD24+, CD29hi)

1:1

The Competitive Transplantation

Axin2-/-, GFP Wild Type, DsRed

(Lin-, CD24+, CD29hi) (Lin-, CD24+, CD29hi)

1:1

LOF Axin2 growth advantage

The Competitive Transplantation

Injury & Healing

• Surgical

• Hair plug

• CCl4 (liver)

• Naphthalene (lung)

Targeted-ablation:

inducible Diphtheria

toxin (DTA)

Lineage Tracing

SC

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)

Lgr5+ Cells Are CBCs

(Barker et al, Nature, 2007)

Lineage Tracing With LacZ Expression

(Barker et al, Nature, 2007)

loxP loxP

• Spatial & temporal control

• Irreversible

Lineage Tracing With LacZ Expression

(Barker et al, Nature, 2007)

The ISC Lineage Tracing

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

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)

(Snippert et al, Cell, 2010)

Lgr5+ Cell Lineage Tracing with Brainbow

Lgr5-GFP-IRES-CreERT2, confetti

Lgr5+ Cell Lineage Tracing with Brainbow

Potential > Fate

In vivo

• Transplantation (blood, hair follicle, mammary

gland)

• Injury & healing (liver, lung, trachea)

• Lineage tracing

Stem cell & Cancer

Cancer stem cell:

(Visvader, Nature, 2011)

Stem cell & Cancer

challenging typical “cancer stem cell” model

(Visvader, Nature, 2011)

Distinct Cell Origins in Tumors

(Visvader,

Nature, 2011)

Intestine Cancer

(Barker et al, Nature, 2009）

Lgr5-CreER, APC fl/fl

Ah-CreER, APC fl/fl

(loxP_APC_ loxP)

Intestine Cancer

Implication of Adult Stem cells

- Regenerative Medicine -

(Sasai, Cell Stem Cell, 2013）

Implication of Adult Stem cells

- Regenerative Medicine -

(Sasai, Cell Stem Cell, 2013）

Implication of Adult Stem cells

- Regenerative Medicine -

(Sasai, Cell Stem Cell, 2013）

Stem Cells Have Self-Organizing Abilities

(Sasai, Nature, 2013）

（共识主动性，自组织）

(Eiraku et al, Nature, 2012）

Self-Organization of Optic Cup Formation

retinal pigment epithelium

neural retina

Mouse E11.5

Rx::GFP

(Eiraku et al,

Nature, 2012）

Self-Organization of Optic Cup Formation

Evagination of Rx::GFP vesicles

(day 4.5-6.5)

Self-Organization of Optic Cup Formation

Invagination of Rx::GFP vesicles

(day 6.75-9.0)

(Eiraku et al,

Nature, 2012）

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）

(Sato et al, Nature, 2009）

Self-Organization of Crypt Formation

A single cypt

(day 2-3)

Self-Organization of Crypt Formation

Organoid derived from an

Lgr5-EGFP-IRES-CreERT2;Rosa26-YFP crypt

(day 7)

Self-Organization of Crypt Formation

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

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?

Thank You