Paul De Sousa Reader Centre for Clinical Brain Sciences University of Edinburgh Co-founder, Chief Scientist Roslin 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 2017 Scottish Event Campus, Glasgow, UK
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Twenty years on from the isolation of human embryo stem cells · Pells et al. Plos One 2015; 10(7):e0131102. 3. Loh et al., 2007, Genes Devel 21: 2545-2557 “Oxygen sensing dioxygenases”
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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
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
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.
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
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