Paul De SousaReader

Centre for Clinical Brain SciencesUniversity of Edinburgh

Co-founder, Chief ScientistRoslin Cells Ltd

Twenty years on from the isolation of human embryo stem cells:

Fact, Fiction and Challenges Remaining To Realize Their Therapeutic Potential

British Blood Transfusion Society Annual Conference 2017Scottish Event Campus, Glasgow, UK

Disclosure

• Full-time employee of the University of Edinburgh

• Paid-consultant for– Roslin Cells Ltd– Roslin Cell Therapies Ltd

• Shareholder & non-executive director CENSO Biotechnologies

“Embryonic” “Fetal” “Adult” “Tissue specific”or “somatic”

Etc. Etc.

renewal

differentiate

Plur

ipot

ent

Mul

tipot

ent

Prog

enito

r

Stem Cells

Why Pluripotent Stem Cells?

√ Growth

√ Differentiation

Scalable….Renewable……Genotype specific……Otherwise inaccessible or limiting cell… supply.

Viable offspring derived from fetal and adult mammalian cells. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Nature. 1997 Feb 27;385(6619):810-3.

Embryonic stem cell lines derived from human blastocysts. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998 Nov 6;282(5391):1145-7.

[Industrial Sponsors]

From discovery to clinical translation in Scotland, 20 years on

Roslin Institute(Roslin Biomed Ltd)

(Geron Biomed Ltd)

Cloned pigs Cloned transgenic sheep Cloned mice Human embryonic stem cell lines (RH1-7)

(1998)

(2005)

(2006) (2016)Roslin Cell Therapies

QA/Regulatory Authority compliance Advanced cell therapy manufacturing Research and clinical grade hESC (17) Human induced pluripotent stem cell (hiPSC) production

De Sousa et al Stem Cell Research (2016) 17: 379

Contributions to Global Effort 2017

Standardised hiPSC researchdiscovery/drug development

De Sousa et al Trends Biotech (2017) 35:573

hESC therapy development support

• Keratinocytes (Ulceration)

• Retinal pigmented epithelium (Macular degeneration)

• Neuronal committed progenitors

(Huntingtons Disease)

(Parkinsons Disease)

• Haematopoietic progenitors

• Endothelial cells

NeuralstemcellRepair

Global advances in clinical evaluation of hESC based cell therapy

Sources: www.clinicaltrials.gov ; Trounson and DeWitt 2016 Nature Reviews, Molecular 17: 195-200.

Sponsors 8 2 1 1 1

Countries (Clinical)

US, UK, China, Korea, Brazil, Japan (hiPSC)

China, Australia

US US France

Dose/patient ~106 ~107 ~107-8 ~107-8 ~109

And the journey taken…………..

Courtesy Dr Lindsay Fraser, In; Fraser, Bruce, Campbell, De Sousa. Quality assessment and production of human cells for clinical use. Methods in Molecular Biology – Huntingtons Disease (S Rowlands, S Dunnett, A Rosser Eds) Submitted.

Courtesy Dr Lindsay Fraser, In; Fraser, Bruce, Campbell, De Sousa. Quality assessment and production of human cells for clinical use. Methods in Molecular Biology – Huntingtons Disease (S Rowlands, S Dunnett, A Rosser Eds) Submitted.

Courtesy Dr Lindsay Fraser, In; Fraser, Bruce, Campbell, De Sousa. Quality assessment and production of human cells for clinical use. Methods in Molecular Biology – Huntingtons Disease (S Rowlands, S Dunnett, A Rosser Eds) Submitted.

Embryonic stem cell lines derived from human blastocysts. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998 Nov 6;282(5391):1145-7.

• (Resemblance to Mouse Embryonic Stem Cells)

• Derived from pre-/peri-implantation embryo

• Prolonged undifferentiated proliferation

• Immortal (high Telomerase)

• Karyotypic stability

• Stable developmental potential (3 germ layers, + extraembryonic)

• Non-clonal (potential variation in lineage potency)

Returning to the science of the beginning, where are we today?

Pluripotency States – naïve to primed

L Weinberger, YS Manor, JH Hanna (2015) Stem Cell TechnologiesTM www.nature.com/nrm/posters/pluripotency

Genetic (In)stability

N Lamm, U Ben-David, T Golan-Lev, Z Storchova, N Benvenisty, B Karem

Cell Stem Cell (2016) 18, 253-61 Nature (2017) 545, 229-61

• Whole exome sequence 140 hESC lines (26 GMP):

• Six TP53 mutations in 5 unrelated lines

• Data mining published RNAseq of 117 hESC lines

• Nine TP53 mutations

• Purified biologicals (variable/adventitious pathogens)

• Dynamic instability (cellular heterogeneity)

• Artefactual entity/culture (autocrine/paracrine signalling?).

• Costly (daily replenishment of bio-active factors)

• Non-scalable tools

• Immaturity (fetal equivalence) of differentiated derivatives

• Lack of predictive biomarker of potency

Pluripotent Stem Cell Production Hurdles

Production Solutions (De Sousa lab 2006-2016)

Nanoparticle mediated paracrine stimulation

Biomaterials 2012; 33 (28): 6634.

Thermomodulatable polymer substrates

Nature Comm 2013; 4: 1335 Advanced Healthcare Materials 2013; Biomaterials 2014; 35: 599Biomaterials Science 2014; 2: 1683Biomaterials Science 2015; 3: 1371

J of Biomed Eng 2011; 133: 101009Biomicrofluidics 2016; 14:014107

Scalable label free cell separation

Immunogenicity

Sources:Trounson and DeWitt (2016) Nature Reviews, Molecular 17: 195-200.English and Wood (2011) Current Opinion in Organ Transplantation. 16: 90-95.

• Undifferentiated hESC:

• Low MHC/failure to stimulate T-cell proliferation

• Suppression of T-cell cytokines (arginase I dependent)

• Inhibit Natural Killer cell mediated cytotoxicity

• Low Toll-Like-Receptor expression and responses

• IFN− γ induced upregulation of MHC I

• Differentiation induced upregulation of MHC I & II

• Cell type specific /context dependent variation

Adventitious Pathogens

Considerations:

• One “immortal” source to many recipients

• Reliance on donor sources, biological reagents, extensive bioprocessing in absence of:

• Validated screens for known/unknown pathogens

• Limitations in understanding of risk

• Eg. Prion Diseases

Prion diseases?

• Creutzfeldt-Jakob Disease (CJD) most commonin humans (0.5-1.5 cases/million per year).

• Degenerative CNS disorders caused by transmissiblepathogenic isoforms of prion protein (PRP).

Animal (Scrapie/BSE/CWD) & Human (CJD/Kuru) forms.

• CJD forms: (Different etiology /epidemiology / manifestation) Sporadic (85%) – random misfolding? Spontaneous mutation? Familial (10-15%) – mutations. Iatrogenic (1%) – inadvertent human to human. Variant (UK & France) – bovine to human.

• Global issue – NOT UK specific (!)

HESC susceptibility/manifestation of prion diseases

Factors:

Exposure to pathogenic isoforms (variant/Iatrogenic).ie. bovine/human sourced reagents in culture.

Expression of constitutive normal PRPc isoform.

Genetic susceptibility polymorphisms(eg. 177/178 vCJD cases are codon 129 MM)

Tissue/cell-specific susceptibility to infection.(uptake, amplification, transmission)

(Pathogenic mutations)

Susceptibility polymorphisms

RH3 RH5 RH1 RH4

MM MM MV MV MM

PRNP codon 129 polymorphisms x type underlie phenotype.

ie. sporadic CJD incubation period and symptoms.

Caucasians ~51% MV, ~37% MM, ~11% VV

vCJD cases 129 MM.

Recommendation:Selection of 129 MV hESC for therapy & PrP locus specific sequencing to confirm absence of pathogenic mutations..

Krejciova et al., J Pathol. 2011; 223:635-45

hESC PrP Expression

M RH3RH1 RH4 RH7 HDF W+ - + - + - + -+ -RT

PrPC

854bp

A

Dapi- Nuclei FITC- PrPc

BTr

a-1-

60 (s

tem

ness

)

PrPc

C D -ve(non-specific siRNA)

PrPc KD(siRNA )

261108

01020304050607080

MO RO Negative PrP KD

% Viable cells

pf261108.021

FL2-H

Cou

nt

100 101 102 103 1040

55111166221 alive dead

-ve

pf261108.028

FL2-H

Cou

nt

100 101 102 103 1040

112224336448

deadalive

PrP KD

PrPc: A Survival Factor?

Krejciova et al., J Pathol. 2011; 223:635-45

BSE vCJD AD

(48h)

24 h

48 h

72 h

72 h

hESC Prion Clearance

Human iPSC derived astrocytes expressing codon 129 MM genotype are susceptible to vCJD replication but MV and VV genotypes are

comparatively resistant

Courtesy of Dr James Alibhai

Krejciova Z, Alibhai J…, Head MW, Chandran S, JEM in press

129MM 129VV

Stem cells & lineage specification in principle

“Embryonic” “Fetal” “Adult” / “Tissue specific”

renewal

Etc.Etc.

differentiate

Plur

ipot

ent

Mul

tipot

ent

Prog

enito

r

In vivo

In vitro

practiceEpigenetic determinism

Epigenetic mechanisms Sequence (ACGT) not altered.

Includes:

DNA (Hydroxy)-methylation.

Histone methylation/acetylation (A.A. residue specific)

Histone variants

Restrictive (Polycomb) / Permissive (Trithorax) complexes

Global Chromatin Modifiers (Swi/Snf, Polycomb)

Micro-RNAs

Fitness

Epigenetic features of a PSC Dynamic/Permissive chromatin

DNAse I hypersensitivity

Bivalent domains

Hydroxymethylation (~1%)

Variable methylation

Fitness

Epigenomic biomarkers of pluripotency?Fitness

Which genes conservatively modified?

Functional relevance?

Environmental responsiveness?

Global CGI-methylation patterns?

• DNA rich in CpG dinucleotides (1/10 vs 1/100 bp).

• Associated with 60-70% human gene promoters.

• Normally unmethylated (vs methylated if non-CGI).

• Contexts:

•CGI-m p53 promoter → silencing → neoplasia.

• Parental imprints (ie. H19, SNRPN, MEG3 and IGF2).

• X-chromosome inactivation.

Fitness

hESC AdultTotal Common CGI-m (%) 1111 (6%) 821

Common Gene-Assoc. CGI-m 888 606Genomic Distribution Uniform More on XY, less on Ch. 1-5.

GA CGI-m only in hESC 150 -GA CGI-unmethyl in hESC 90 -

Is there an hESC specific consensus CGI-m?

hESC ga-CGI mRNA No. of Genes**

Methylated(150)

+ 96

- 34Diff. (≥1) 20

Unmethylated(90)

+ 54- 25

Diff. (≥1) 11

** Methylated – enriched for Transcriptional RepressorsUnmethylated – enriched for Transcriptional Activators.

hESC Epigenetic Signature?

Fitness

Pells et al Plos One 2015; 10(7):e0131102

HESC expressed genes with conserved & cell specific CGI-m:

Candidate epigenomic biomarkers?

TET1 Ten Eleven Translocation 1 (1 of 3 members)- 5mC → 5HMC conversion catalysed by TET oncogenes.- (Mouse ESC) depletion→ Nanog CGI-m → Silencing1,2.

→ mesendoderm/trophoblast specification. - Regulated by core pluripotency TF

KDM4C Jumonji domain 2 contain Histone Lysine Demethylase- chromatin remodelling enzyme- (Mouse ESC) depletion→differentiation.- Regulated by core pluripotency TF

Iron (Fe2+) and 2-oxoglutarate dioxygenases (O2 dependent)

1. Ito et al., 2010, Nature doi:10.1038/nature093032. Koh et al., 2011, Cell Stem Cell 8(2):200-133. Loh et al., 2007, Genes Devel 21: 2545-2557Pells et al. Plos One 2015; 10(7):e0131102

“Oxygen sensing dioxygenases”

Interference with TETs & KDM4C induces hESC differentiation & 5hmC loss

Koutsouraki et al., submitted

OC

T4

IDS-

NU

LLTE

T1TE

T3

KDM

4CYA

P1TE

T2TE

T1-3

Knockdown (KD)

0.001

0.01

0.1

1

10

Log 1

0m

RN

A Fo

ld D

iffer

ence

KDM4C KD TET1 KD TET2 KD TET3 KD TET1-3 KD

**** ******

******

******

* ******

***** ***

** ******

** * ******

******

******

****

******

***

▼ ▼▼▼ ▼ ▼ ▼ ▼ ▼▼▼▼ ▼▼ ▼ ▼ ▼ ▼

0

0.02

0.04

0.06

0.08

0.1

% o

f Gen

omic

5hm

C

Knockdown Target

******

******

** **

▼ ▼ ▼ ▼ ▼ ▼

O2 dependent trace level organometalic (NaASO2) induction of hESC differentiation, loss of dioxygenases and 5hmC

Protective Effect of Hypoxia

NANOG DAPIOCT4

NO

RM

OXI

A (2

0% O

2)

Unt

reat

ed

Con

trol

NaA

sO2

[1x1

0-9 M

]

HYP

OXI

A (0

.5%

O2)

Unt

reat

ed

Con

trol

NaA

sO2

[1x1

0-9 M

]

0.00

0.05

0.10

0.15

0.20

Untreated Control Sodium Arsenite

% o

f 5hm

C

NORMOXIA HYPOXIA

****

****

▼

* * * *

* * * *

0.01

0.1

1

10

100

1000

Log 1

0m

RN

A Fo

ld D

iffer

ence

NaAsO2

NORMOXIA HYPOXIA

****

****

****

****

****

****

****

****

****

********

****

****

****

▼▼ ▼ ▼ ▼▼

▼ ▼

Differential O2 dependent trace level organometalic (NaASO2) induced oxidative stress

0

2000

4000

6000

Rea

ctiv

e O

xyge

n Sp

ecie

s (D

CF

Fluo

resc

ence

In

tens

ity U

nits

)

NORMOXIA HYPOXIA

****

**

****

****

****

****

▼ ▼ ▼

0.1

1

10

HO-1 GPX1 GPX4 SOD1 SOD2

Log 1

0m

RN

A Fo

ld D

iffer

ence

NORMOXIA HYPOXIA

Reactive Oxygen Species Antioxidant Gene Expression

▼ ▼

0

250

500

750

1000

Untreated CTRL Sodium ArseniteMito

chon

dria

l Mem

bran

e Po

tent

ial (

Fluo

resc

ence

In

tens

ity U

nits

)

Treatments

NORMOXIA HYPOXIA

******

Mitochondrial function (uncoupling ATP/OxPhos)

Transient expression of O2 sensing dioxygenases protects against trace level organometalic (NaASO2) induced differentiation

No

Plas

mid

pCXL

E -

TET1

pCXL

E -

TET1

No

Plas

mid

pCXL

E –

KDM

4CpC

XLE

-eG

FP

pCXL

E –

KDM

4CpC

XLE

-eG

FP

- NaAsO2 + NaAsO2

0.1

1

10

Log 1

0m

RNA

Fold

Diff

eren

ce

Treatments

OCT4 NANOG SOX2

* **** ** * ***

* ** ** ***** ****

****

▼ ▼ ▼▼▼ ▼ ▼▼ ▼▼▼

0.1

1

10

100

Log 1

0m

RN

A Fo

ld D

iffer

ence

KDM4C TET1 TET2 TET3

******

******* * ****

** ** * *******

▼ ▼▼ ▼▼ ▼ ▼▼

0.00

0.04

0.08

0.12

% o

f 5-h

mC

KDM4C *** ******

***

▼

▼ ▼ ▼

0.00

0.04

0.08

0.12

% o

f 5-h

mC

TET1

******

▼

Hypoxia induced O2 sensing dioxygenases sufficient to block trace chemical oxidative stress induced differentiation

Koutsouraki et al., submitted

[Low] Ambient Oxygen [High]

TET1KDM4C

(hESC m/un-CGI)

[ROS] [ROS]

Hyp

oxia

In

duci

ble

Fact

or

++ m-histonehistone

m-Chm-C

CHROMATINCHROMATIN⊥

Trace oxidative stressors NaAsO2 ↑[ROS]

SELF-RENEWAL DIFFERENTIATION

+

Model – Environmental/Epigenetic determination of stemness

20 years on – hESC therapy facts, fiction and challenges

Long road of public/privately sponsored innovation

Further to travel before clinical practice

Vital to augment understanding of risk & cell-type validated screening for adventitious pathogents such as Prions

(Critical in light of initiated Ocular/Brain/CNS transplants recalling past history of transmission via other advanced therapies – Human Growth Hormone)

Ambient oxygen as an epigenetic mediator of stemness –implications for hESC production.

Thank you for your attention.

Acknowledgements

Dr Eirini KoutsourakiDr Steve Pells

Dr James AlibhaiDr Zuzana Krejciova

Dr Mark HeadProf. Adrian BirdProf. Siddharthan Chandran

Mr Aidan CourtneyMr Malcolm BatemanMrs Janet Downie

Dr Lindsay Fraser

Prof Marc Turner

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