Best Practices –Maintaining PluripotencyAnne R. Greenlee, PhDHESI-Workshop Cary, NC 27 Feb 2007

OHSU La Grande Campus

Research Directions

• Past – Fertility Risk Factor Study– Pesticide effects on embryo development– Undifferentiated mESCs for toxicity testing

• Current– Injury effects on early development – Interventions - safeguard reproductive health – Bench to bedside – translation & outreach

Today’s Talk

• Pluripotency vs Totipotency• Biomarkers of pluripotent ESC• Maintaining pluripotent ESC

– Feeders– Feeder-free– Xeno-free

• Case Study: Matrigel™ vs Gelatin

Pluripotent vs Totipotent ES Cells

• Pluripotent– Ectoderm– Mesoderm– Endoderm

• not– Trophoblast– Germ cells

• Totipotent– Ectoderm– Mesoderm– Endoderm

• and– Trophoblast– Germ cells

Hyslop et al., 2005

Blastocyst

Poueymirou et al. Nat Biotech 2007 25:91

F0 Generation Mice Fully Derived from ES Cells for Immediate Phenotypic Analysis

Trophoblast Formation

• hESC (+ BMP–4) (Xu et al., 2002)

• hESC and mESC ( Oct–4, siRNA) (Hay et al., 2004)

Hyslop et al., 2005

Pluripotent ES Cell Signaling

mESC

hESC

Pluripotent ES Cells –Surface and Molecular Markers

• hES cells– SSEA-4*– TRA-1-60*– TRA-1-81*– Β-5 tubulin*– Alk Phos– OCT-4– NANOG– SOX2– TERT

• mES cells– SSEA-1*– LIF-R*– Β-III tubulin*– Fox-D3*– Alk Phos– Oct-4– Nanog– Sox2– Tert

*Specific to hES or mES cells

Pluripotent ES cells

• Chromosomal number (modal)– mESC 40 xx, xy– hESC 46 xx, xy

• Epigenetics– Loss of H19 methylation status with prolonged culture (H9) – X-chromosome inactivation (H9 XIST+ vs H7 XIST -)

• Telomerase – high expression• Doubling time

– ≅12 h mES cells– ≅ 36-45 h hES cells

• Morphology– mESCs 4-10 cell layers on feeders– hESCs 2-4 cell layers on feeders

• Teratoma formation• Gamete – Trophoblast (?)

Maintaining Pluripotent mESC

• Feeders– Embryonic mouse fibroblasts

• No Feeders + animal proteins – Substrata

• Gelatin, Matrigel™, plastic– MEF Conditioned medium– Growth Factors and cytokines

• LIF• BMP-4

Maintaining Pluripotent hESC

• Feeders– Embryonic human fibroblasts– Fetal muscle– Skin fibroblasts– Foreskin– Fallopian, endometrial, breast

• No feeders + animal proteins– Substrata

• Matrigel™ > fibronectin > collagen>>>laminin– Conditioned medium

• Human serum• Serum replacement

– Growth Factors• TGF-β, activin A, nodal, FGF-2, Noggin

Review - Hoffman and Carpenter 2005 Nature Biotechnology 23:699-708

Xeno-Free hES Cells

• Immunogenic proteins – Neu5Gc mouse vs Neu5Ac human

• Animal pathogens– Viruses– Prions

• Separating hESC from feeder cells• Undefined conditions – replication?

Xeno-Free Culture of hES Cells

• TeSR1• Ludwig et al., Nat Biotech

2006;24:185 (+) supplemental information)– DMEM/F12– Human:

• Serum• Albumin• Growth factors

– Vitamins– Anti-oxidants– Trace minerals– Lipids

• hES Cocktail(HESCO)

• Lu et al., PNAS 2006; 103:5688 (-) supplemental information

• DMEM/F12• Wnt3a• FGF• Insulin• Transferrin• April/BAFF• Cholesterol• Albumin

Best Practices

• Safety and quality of ES cells– Records, personnel, sanitation cleanliness,

equipment, processes, reagent qc• Regenerative medicine - challenges

– Immune reactions– Chromosomal stability - oncogenesis– Epigenetic changes– Controlled differentiation

Scale-up Culture Systems - hESC

• hESC culture is labor intensive!– Manual dissection hESC colonies– Bioreactors– Perfusion systems– Matrix encapsulation - stirring

Case Study: Matrigel™ vs Gelatin

• Compared two substrata for feeder-free, short-term (P1 to P7) culture of undifferentiated mES cells

Greenlee et al. Tox in Vitro 2005;19:385

Study Objectives

• Eliminate feeder cells; unify conditions• Obtain large numbers of well-

characterized, undifferentiated mESCfor developmental tox assays

Study Methods

• Cells – D3 mES cells (CRL 1934 ATCC)

• Medium– DMEM + FBS + GF ( LIF, FGF-2, SCF)

• Substrata– Matrigel™ (GF Reduced)– 0.1% Gelatin

Biomarkers

• Growth, viability• Chromosome #• Pluripotentiality

– Alk Phos– SSEA-1

• Activated Caspase-3 (apoptosis)• EBs to beating cardiomyocytes

Population Doublings - Viability

Passage Number

Dou

blin

gs p

er 2

4 hr

% V

iabi

lity

Matrigel

Gelatin

NS

NS

Morphology - Alk Phos Staining

MatrigelP7

GelatinP7

Magnification = 100x

Alkaline Phosphatase ActivityA

lkPh

osEn

zym

e U

/ L/C

ell

mES Cell Populations P7

Both P < 0.0001

P = 0.012

SSEA-1 ExpressionSS

EA-1

Exp

r ess

ion

%

mES Cell Populations P5

NSNS

Caspase-3 Expression

Sodium arsenite ppb

% C

aspa

se-3

Exp

ress

ion

Matrigel P5

Gelatin P5

P < 0.0001

Chromosome Counts (P1 vs P4)

107

89

24

≥ 4140≤ 39

Matrigel P4

Gelatin P4

119

78

23

Matrigel P1

Gelatin P1

Chromosome # per spread (n* = 20)

35 – 45%10 – 20% 35 – 55%

*n = number of metaphase spreads

Chromosome Counts (P1 to P21)

14(67%)

3(14%)

4(19%)

≥ 4140≤ 39

Week 10 (P21)

10(66%)

8(44%)

0(0%)

Week 1 (P1)

Chromosome # per spread (n ≥ 18)

Greenlee et al. Tox in Vitro 2004;18:543

Beating Cardiomyocytes (P1 vs P7)

Days in Culture

Bea

ting

Car

diom

yocy

tes

%

Matrigel P1

Gelatin P1

Matrigel P7

Gelatin P7

P = 0.26

P = 0.01

% Cardiomyocytes P2 vs P21

P2

P21

P = 0.0002

Summary – Matrigel™ vs Gelatin

• Matrigel™ ~ 0.1% Gelatin– Viability, Growth – SSEA-1– P1 cardiomyocyte differentiation

• Matrigel™ > 0.1% Gelatin– Alkaline phosphatase activity (U/L/cell)– Dose-responsive Caspase-3 – P7 cardiomyocyte differentiation

Conclusion

• Matrigel™ shows promise as a substratum for short-term (~P1 - P7) feeder-free propagation of undifferentiated mES cells

Where to?

• Perla et al. Paraquat toxicity in a mouse embryonic stem cell model. 2007 Summit on Environmental Challenges to Reproductive Health and Fertility. UCSF Mission Bay Campus, San Francisco.

• Greenlee et al. Mouse embryonic stem cell model to predict risk of neural tube birth defects. Colgate Palmolive - SOT

Summary

• Pluripotency vs Totipotency – blurry• Substrata, niche, culture conditions -

role in genome-proteome-metabolome• Nanog – Oct 4 – Sall4 pluripotency• mES ≠ hES cells• Xeno-Free – uncharted effects

Acknowledgements

• Marshfield Clinic Research Foundation

• TA Koepel, BS• SJ Kaiser, BS• TM Ellis, BS• K Liu, PhD• MCRF - NIOSH

• OHSU• Venu Perla, PhD• Gabriela Oro, BS, RN• Solyssa Visalli, BS• School of Nursing• Betty Gray Foundation• Colgate-Palmolive

ILSI Health and Environmental Sciences InstituteDavid Sandler, Organizing committee

Thank You - Questions?