Sponsored by:

Participating Experts:Ronald D.G. McKay, Ph.D.National Institutes of HealthBethesda, MD

Brought to you by the Science/AAAS Business Office

Amy Wagers, Ph.D.Harvard UniversityBoston, MA

Mark D. Noble, Ph.D.University of Rochester Medical CenterRochester, NY

28 January, 201028 January, 2010

Moving Stem Cell Research ForwardMoving Stem Cell Research ForwardThe Need for Standardization

Webinar SeriesWebinar SeriesScienceScience

Dr. Ron McKay

National Institute of NeurologicalDisorders and Stoke (NINDS)National Institutes of Health

Bethesda, MD

Behaviour

Kim et al., Nature, 2002

DunnettDunnett et al. et al. Exp Brain Exp Brain ResRes19881988

Ipsi

alat

eral

rota

tions

pe

r 90

min

0

500

1000

1500

2000

Graft

6-OHDA in graft

Lesion alone

months1 2 3 4 4.5

You can tell the difference between good and bad colonies but we need simple tests for the pluripotent state that can make any cell type and we need to develop standard conditions to grow the cells for long periods.

The problem - hES cells are hard to grow because they grow as colonies that are heterogeneous

A good colony A bad colony

The promise - human ES cells make islets that function in a mouse model of diabetes

Shim et al., Diabetologia 2007

days

Blood Glucose ng/ml

-6 0 4 8 12 16 20 24 28 32

33

22

11

lesion

transplant

graftremoval

There are also differences between hESlines express different levels of growth regulators

BG01 H1

MDM2lo - hard to grow MDM2hi - easy to grow

BG01acquires chromosome duplications found in teratocarcinoma

BG01.1 and BG01.8 subclones

Lines that are hard to grow acquire genetic change more readily

For example,

BG01 and H1

express different

levels of the growth

regulator Mdm2

A good colony

A bad colony H1 hES cells

NANOG PO4 p53S15

Even within one line different levels of growth regulators are seen in different colonies

Cell types in early mouse development

Days after Fertilization

E2.5

E3.5

E4.5

E5.5

Zygote

Morula

TrophectodermInner Cell Mass

EndodermEctoderm

Mouse ES

E6.5

Epiblast

Mesoderm

ImplantExtra-emb. Endoderm

EpiS cells

Embryoid Body

Mes-endoderm

E7.5

Tesar et al.,Nature, 2007

Epiblast E5.5

Mdm2 Cancer risk factor Mdm2

Epiblast+ 3 days in vitro

DissectEpiblast

200 m

Cell lines

G l o b a l g e n e e x p r e s s i o n

Tesar et al., Nature 2007

Controlling renewal & differentiation in mouse ES and EpiSCs

T e r a t o m a s

EpiS

Epi S

EpiS

9

BG

02 p

50

BG

02 p

54

WA

01 p

52

WA

01 p

56

ES04

p72

ES04

p76

TE06

p60

TE06

p64

TE03

p66

TE03

p70

BG

03 p

50

BG

03 p

54

ES03

p84

ES03

p88

UC

01 p

56

UC

01 p

60

WA

07 p

43

WA

07 p

47

WA

13 p

76

WA

13 p

80

Cell Line SNP 309

BG02 T/T

BG01 T/T

BG03 T/T

WA13 T/T

WA09 T/T

WA07 T/T

UC06 G/T

UC01 G/T

TE06 G/T

ES03 G/T

WA01 G/T

TE03 G/G

ES03

The “G” allele at 309 results in higher levels of Mdm2 due to increased SP1 binding.

MDM2 SNPs

Cluster Analysis of whole genome expression in independent passages of hES Cells.

TE03

Controlling growth of human ES cells and cancer risk

ES cells grown at NIH are consistent

Other labs

Whole transcriptome characterization of undifferentiated hES cells

the unpublished work presented here is from:

the NIH Stem Cell Facility

Josh Chenoweth & Paul Tesar

Table 1. hESCs Acquired and AnalyzedLine Source Provider ID Passages AnalyzedBG01 Bresagen BG01 p63, p67BG02 Bresagen BG02 p50, p54BG03 Bresagen BG03 p50, p54ES01 ES Cell Int. hES 1 p67, p71ES02 ES Cell Int. hES 2 p45, p49ES03 ES Cell Int. hES 3 p84, p88ES04 ES Cell Int. hES 4 p72, p76ES05 ES Cell Int. hES 5 p55, p59 ES06 ES Cell Int. hES 6 p58, p62SA01 Cellartis AB SA01 p28, p32SA02 Cellartis AB SA02 p35, p39TE03 Technion I-3 p66, p70TE04 Technion I-4 p32, p36TE06 Technion I-6 p60, p64UC01 UCSF HSF1 p56, p60UC06 UCSF HSF6 p55, p59WA01 WiCell H1 p52, p56WA07 WiCell H7 p43, p47WA09 WiCell H9 p41, p45WA13 WiCell H13 p76, p80WA14 WiCell H14 p35, p39

hESC

hESC+p4

EB FBS

EB FBS

EB KSR

EB KSR

FISH Flow Cytometry

Karyotype

STR GenotypeWhole Genome 

Expression

Mycoplasma

aCGH

Tissue-specific stem cells

Amy J. Wagers, Ph.D.Associate Professor of Stem Cell and 

Regenerative Biology, Harvard University

Joslin

Diabetes Center

Harvard Stem Cell Institute

The problem•

Multicellularity

requires specialization of 

cell types.

The problem•

Multicellularity

requires specialization of 

cell types.•

Specialized cells often lose the capacity to 

reproduce themselves.

The problem•

Multicellularity

requires specialization of 

cell types.•

Specialized cells often lose the capacity to 

reproduce themselves.•

When these specialized cells become 

exhausted or are injured, they must be  replaced from unspecialized precursors.

The problem•

Multicellularity

requires specialization of 

cell types.•

Specialized cells often lose the capacity to 

reproduce themselves.•

When these specialized cells become 

exhausted or are injured, they must be  replaced from unspecialized precursors.

STEM CELLS

Stem Cells•

Are themselves undifferentiated

but can 

generate specialized cells.

Stem Cells•

Are themselves undifferentiated

but can 

generate specialized cells.

•

Self‐renew

and thus maintain cell  replacement potential for long periods of 

time.

Stem Cells•

Are themselves undifferentiated

but can 

generate specialized cells.

•

Self‐renew

and thus maintain cell  replacement potential for long periods of 

time.

•

Promise for experimental and regenerative  medicine: 

–

Manipulation of endogenous cells

–

Cell transplantation

–

Laboratory studies of development and disease

Stem Cell Relationships•Embryonic•Fetal•Adult

Stem Cell RelationshipsES cells •Embryonic

•Fetal•Adult

Stem Cell RelationshipsES cells •Embryonic

•Fetal•Adult

Stem Cell Relationships

• Stem cells in the embryo/fetus function to generate tissues.

ES cells •Embryonic•Fetal•Adult

Stem Cell Relationships

• Stem cells in the embryo/fetus function to generate tissues.

• Some of these stem cells may be retained after birth to replenish or regenerate adult cells and tissues.

ES cells •Embryonic•Fetal•Adult

Tissue-specific stem cellsTissue Stem cell Differentiated progenyBlood HSC All lineages of blood cells Brain NSC Neurons, glia

Intestine ISC Intestinal epitheliumSkin Bulge cell Hair, sebaceous gland, epidermisMuscle Satellite cell Myoblasts, myofibersGermline Germ cell Oocyte, spermLiver Oval cell Hepatocyte, bile ductHeart Cardiac progenitor Cardiomyocytes, smooth muscle, Blood vessels EPC EndotheliumLung BASC Alveoli, pneumocytesFat Adipose stem cell Adipocytes

Mammary gland MaSC luminal and myoepithelial cells, lobuloalveolar units (pregnancy)

Kidney ? Renal tubulePancreas ? Exocrine/endocrine cells

Tissue-specific stem cellsTissue Stem cell Differentiated progenyBlood HSC All lineages of blood cells Brain NSC Neurons, glia

Intestine ISC Intestinal epitheliumSkin Bulge cell Hair, sebaceous gland, epidermisMuscle Satellite cell Myoblasts, myofibersGermline Germ cell Oocyte, spermLiver Oval cell Hepatocyte, bile ductHeart Cardiac progenitor Cardiomyocytes, smooth muscle, Blood vessels EPC EndotheliumLung BASC Alveoli, pneumocytesFat Adipose stem cell Adipocytes

Mammary gland MaSC luminal and myoepithelial cells, lobuloalveolar units (pregnancy)

Kidney ? Renal tubulePancreas ? Exocrine/endocrine cells

Tissue‐specific (“adult”) stem cells  give rise to a more limited subset  of cell types than ES cells, typically  they can differentiate to produce 

the specialized cell types of the  tissue from which they originate. 

Sources of Stem Cells•

Embryonic

•

Fetal•

Adult

•

The best source of stem cells for the study  or treatment of a particular disease will 

likely vary depending on the tissue and/or  cell type that is targeted.

Sources of Stem Cells•

Embryonic

•

Fetal•

Adult

•

The best source of stem cells for the study  or treatment of a particular disease will 

likely vary depending on the tissue and/or  cell type that is targeted.

•

Critical to be able to identify

and isolate these stem cells in tissues and in culture.

Identifying stem cells

•

Retrospective

‐

lineage tracing (chromosomal  translocations, viral insertion, Cre/lox 

recombination)

Identifying stem cells

•

Retrospective

‐

lineage tracing (chromosomal  translocations, viral insertion, Cre/lox 

recombination)

•

Prospective

– in vitro differentiation;  transplantation, marker expression

Identifying stem cells

•

Retrospective

‐

lineage tracing (chromosomal  translocations, viral insertion, Cre/lox 

recombination)

•

Prospective

– in vitro differentiation;  transplantation, marker expression

Functional tests are critical!

Identifying stem cells

•

Retrospective

‐

lineage tracing (chromosomal  translocations, viral insertion, Cre/lox 

recombination)

•

Prospective

– in vitro differentiation;  transplantation, marker expression

In vitro recapitulation of stem cell function  may require complex environments

Stem cell

Niche

Signals from the niche regulate stem cell proliferation, differentiation, and survival.

Signals from the environment regulate stem cell fate

Tissue Maintenance and Regeneration

Blood system – HSCs Skeletal muscle – SMPs

Self-renewalDifferentiation

• Direct isolation• Functional assays

Targeting stem cells and their niches for therapy

•

Regulate stem cell  number.

Stem cell

Niche

Targeting stem cells and their niches for therapy

•

Regulate stem cell  number.

•

Regulate stem cell  activity.

Stem cell

Niche

Targeting stem cells and their niches for therapy

•

Regulate stem cell  number.

•

Regulate stem cell  activity.

–

To expand

stem cells 

outside the bodyStem cell

Niche

Targeting stem cells and their niches for therapy

•

Regulate stem cell  number.

•

Regulate stem cell  activity.

–

To expand

stem cells 

outside the body

–

To manipulate

them 

within the bodyStem cell

Niche

Targeting stem cells and their niches for therapy

•

Regulate stem cell  number.

•

Regulate stem cell  activity.

–

To expand

stem cells 

outside the body

–

To manipulate

them 

within the body

–

To transplant

them 

into complex tissues.

Stem cell

Niche

Targeting stem cells and their niches for therapy

•

To combat  degenerative 

diseases (genetic  and age‐related)  and malignancy.

Stem cell

Niche

Summary•

Stem cells are unique cells capable of both 

self‐renewal

and production of at least one  specialized

cell type.

Summary•

Stem cells are unique cells capable of both 

self‐renewal

and production of at least one  specialized

cell type.

• There are important differences

between 

embryonic and adult stem cells, but both  provide significant therapeutic

opportunities.

Summary•

Stem cells are unique cells capable of both 

self‐renewal

and production of at least one  specialized

cell type.

• There are important differences

between 

embryonic and adult stem cells, but both  provide significant therapeutic

opportunities.

• Stem cells may provide a means for 

generating replacement

cells

for multiple  disorders and may provide insights into the 

fundamental mechanisms behind normal

cell formation

and diseases

such as cancer. 

Challenges for the future of stem cell research

•

Do stem cells exist for all adult tissues? •

Where they do exist, how can we identify 

and expand them?•

Will therapeutic screening using patient‐

specific stem cells (iPS

or tissue‐specific)  allow development of more effective drugs?

•

How do we productively engraft stem cells  into complex tissues?

M. Chemiakin

Precursor cell standardization:

lineage, differentiation,

metabolism

Mark Noble

University of Rochester Stem Cell and

Regenerative Medicine Institute

Margot Mayer-Pröschel

Chris Pröschel

Stephen and Jeannette Davies

Oligodendrocyte/type-2 astrocyte progenitor cells (O-2A/OPCs)

Oligodendrocytes Type-2 astrocytes

Type-1 astrocytes

Glial-restricted precursor (GRP) cells

Neuroepithelial stem cells (NSCs)

NRP cells

Neurons

AdultO-2A/OPCs

Noble et al. Dev. Biol. (2004) 265:33-52

Developmental maladies are diseases of precursor cells

O-2A/OPCs

Type-2 astrocytes

Type-1 astrocytes

GRP cells

Type-2 astrocytes

+ BMP

+ BMP+ CNTF

Optimizing SCI repair by pre-differentiation of specific progenitor cells into specific populations of astrocytes

J. Davies et al., 2008 Journal of Biology

GDAgp130GDABMPLesion Control

GRP cells & GDAsCNTF promote allodynia: ALL pain (NO gain)

GDAsBMP: NO pain (all gain)

CGRP+ c-fibers

Davies et al., 2008 Journal of Biology

The wrong cells cause harm

GRP cell

GDABMP GDAgp130

BMP CNTF, LIF, IL-6

What’s critical? Is it the signaling pathway or the cell of origin? Or both?

O-2A progenitor cell

BMP

The contribution of lineage to tumor phenotype

Transcription Translation

Metabolic Status Protein Activation Status

Cells do something

0

50

100

150

200

250

CXOCON

Uni

ts o

f flu

ores

cenc

eCortical O-2As undergo more self-renewal and generate fewer oligodendrocytes than do optic nerve-derived O-2As.

The redox state of freshly isolated O-2A/OPCs is in agreement with their self-renewal potential. In other words, redox modulationagain appears to be partof in vivo regulation.

Power et al. (2002) Dev Biol 245:362-375

The challenge of toxicologyWorld Health Organization estimates 30-40% of the burden of childhood disease is due to environmental factors

There are 80-150 thousand registered chemicals for which we have no information - which means they are unregulated (an assumption of safety)

We each have hundreds of these chemicals in our bodies - and we know nothing about combined activities

c-Cbl targets

PDGFR

EGFR

c-Met

c-Kit

IGF-IR

etc

The redox/Fyn/c-Cbl pathway: Oxidative status converges on c-Cbl-mediated degradation of specific RTKs

Li et al. (2007) PLoS Biology 5:e35

ERK1/2 Akt

SRE NF-kB

Fyn

c-CblIncreased oxidative

status

B

UbUb

P P

P

P

P P

ERK1/2 Akt

SRE

c-Cbl targeted RTK

NF-kB

AReceptor ligand

P P

P P

ERK1/2 Akt

SRE NF-kB

Fyn

c-CblIncreased oxidative

status

C

UbUb

P P

P

P

P P

ERK1/2 Akt

SRE NF-kB

Fyn

c-CblIncreased oxidative

status

D

P

P

Increased oxidative

status

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28 January, 201028 January, 2010

Moving Stem Cell Research ForwardMoving Stem Cell Research ForwardThe Need for Standardization

Webinar SeriesWebinar SeriesScienceScience