1. Regulation of Embryonic Stem
Cell Pluripotency by Heat Shock
Protein 90
ERIC BRADLEY, ERHARD BIEBERICH, NAHID F. MIVECHI, DANTERA
TANGPISUTHIPONGSA, GUANGHU WANG
STEM CELLS 2012
Speaker: Abelrasoul, Mahmoud Shaaban
2013-1-08

2. Origin of embryonic stem cells
Sperm & Ovum
fertilization
zygote
Equal Divisions
two, four, eight… blastomeres
Reararrangements
Blastocyst
Inner cell mass
Pluripotent ESCs

3. Why embryonic stem cells?
• ESCs have self-
renewal and
pluripotency.
• Undifferentiated
ESCs can form any
adult cell.

4. Factors maintaining ESCs
pluripotency
• Nanog
• Oct 4
• Sox 2
• LIF
• c-Myc
• Klf4
• Stat3 …..

5. Molecular Chaperone
• Proteins that assist the non-covalent folding or unfolding of
other macromolecules.
• Prevent newly synthesized ploypeptides from aggregation.
• Alzheimer's disease, amyloid fibrils.
Nature 416, 483-484 (4 April 2002) | doi:10.1038/416483a

6. Heat shock protein 90(Hsp90)
• Important molecular chaperon
participates in stabilizing and
activating more than 100
proteins referred to as Hsp90
‘‘client proteins’’.
• Regulate proteostasis
(eukaryotic protein homeostasis)
• Hsp90β: highly expressed.
• Hsp90α: stress-inducible.
L. Neckers, CCR

7. Chaperone Machinery
• Hsp90, Hsp70, and other co-chaperones, such
as Hop, form a dynamic complex known as the
Hsp90 chaperone machinery.
• Cancer cells use this machinery to prevent
degradation and misfolding of mutated and
overexpressed oncoproteins .
• So, Hsp90 is an oncogene tolerance and cancer
cell survival.

8. Leukemia inhibitory factor(LIF)
• LIF is an interleukin 6 class cytokine
affects cell growth by inhibiting
differentiation.
• When LIF levels drop, the cells differentiate.
• LIF can maintain pluripotency of ESCs by
the phosphorylation and subsequent nuclear
translocation of Stat3 via an Interleukin-6
pathway.

9. •Stat3 is physically
associated with Hsp90,
Song H et al 2004.
•LIF promotes Hsp90
association with Stat3 in
mouse ESCs, Setati MM
et al 2010.
Mol Cancer Ther 2007;6:2386-2390

10. The purpose of the work
• To give an evidence that Hsp90 is required for
pluripotency in mouse ESCs by using multiple
approaches.
• To show that Oct4 and Nanog are potential
Hsp90 client proteins.

11. Materials and Methods
• Hsp90 inhibited by 17-AAG in J1 mouse ESCs.
• To determine ESCs pluripotency after
treatment we examined:
1. Morphology.
2. AP staining.
3. Teratoma formation.
4. Pluripotency protein markers (Oct4, Nanog,
and phosphorylated Stat3).

12. Results and Discussion
• Figure 1. A. showing dome-shaped control ECSs
and extended flattened 17-AAG treated ESCs.
• Dose: 250 µM 17-AAG for 48 hours. Scale bar= 30 µm.
Morphology changed

13. •Alkaline phosphatase (AP) staining of the mouse ESC colonies.
•Red color refers to AP +ve stained undifferentiated ESCs (pluripotent).ECSs loosed
pluripotency by Hsp90
inhibition

14. • Teratoma formation 17-AAG
treated or untreated cells were
injected intracranially into P3
mouse brains. Mice were
sacrificed, and relative teratoma
weight was measured 4 weeks
later.
Teratoma size decreased
by more than 50% by 17-
AAG treatment

15. • Western blot analysis of
pluripotency factors. pStat3,
tStat3, Nanog, and Oct4
were detected using 250 nM
17-AAG-treated mouse ESC
samples
levels of the
pluripotency factors Oct4,
Nanog, and pStat3 were
greatly
diminished in the 17-AAG-
treated samples
Similar phenotypes were observed when the ESCs were treated by
another Hsp90 inhibitor GA

16. Hsp90 levels during differentiation into
embryoid bodies (EBs).
• Figure S2 Hsp90 proteins were down-regulated during ES differentiation into
EBs. Cell lysates were collected at stages indicated and Westernblot analysis
performed for Hsp90α and Hsp90β. ES, feeder free ESCs; EB1-1, floating EBs
24 hours after plating; EB1-2, floating EBs 48 hours after plating; EB2-1,
attached EBs 24 hours after attached to the tissue culture dishes; EB2-2,
attached EBs 48 hours after attached to the tissue culture dishes.

17. Hsp90 Inhibition by MicroRNA
• Western blot analysis showed the Characterization of
miRNA against Hsp90α and Hsp90β in NIH3T3 cells.
miRNAs A2139 and A2161 against Hsp90α, and B1201,
B1626 against Hsp90β were transfected into NIH3T3 cells.

18. Hsp90 Inhibition by MicroRNA
• When miRNA A2139 and B1201 were electroporated into mouse
ESCs, we found that these miRNAs effectively
reduced the protein levels of Hsp90α and Hsp90β,
and a mixture of A2139 and B1201 reduced both.
Oct4 and Nanog
reduced by Hsp90
miRNAs

19. Hsp90 Inhibition by MicroRNA
• Hsp90 miRNAs significantly reduced the number of AP posi-
tive colonies, with the double transfection of both Hsp90α
and Hsp90β miRNA being the most potent.

20. • Rescue assay, Overexpression of Hsp90β by a lentiviral vector
partially restored the loss of the ESC pluripotency as determined
by AP staining and western blot analysis.

21. Is Hsp90 Associated with Oct4 and Nanog ?
and Can Protects them from degradation
through Ubiquitin proteasomal degradation
pathway ?????

22. • both Hsp90α and Hsp90 β pulled down endogenous Oct4 from ESC lysates.
In the reciprocal immunoprecipation reaction, Oct4 antibody pulled down
both endogenous Hsp90α and Hsp90β.
• Similarly, endogenous Nanog and Hsp90 were found to be pulled down by
each other.

23. • Inhibition of the ubiquitin
proteasomal degradation pathway by
MG132 in 17-AAG-treated ESCs.
• MG132 treatment prevented the Nanog
and Oct4 downregulation mediated by
Hsp90 inhibition.
Oct4 and Nanog are potential
novel Hsp90 client proteins, through
which Hsp90 participates in the
maintenance of ESC pluripotency.

24. Hsp90 Inhibition Reduced Oct4 mRNA Level
in Both Mouse and Human ESCs
• Oct4 mRNA level was reduced by 17-AAG in mouse ESCs in
a dose-dependent manner, while Nanog, Sox2, c-Myc, and
Klf-4 mRNA levels remained intact.

25. • Quantitative real-time PCR indicated that Hsp90 inhibition
by 17-AAG reduced the Oct4 mRNA level in mouse and
human ESCs.
• These data indicate that Hsp90 regulates Oct4 mRNA
transcription, maturation, or stability in ESCs.
Mouse
ESCs Human
ESCs

26. • Oct4 mRNA level was downregulated by 17-AAG in EBs.
• The primitive ectoderm marker Fgf5 was reduced, while the
primitive endoderm marker GATA4 remained unchanged.

27. Hsp90 inhibition in Dose-dependent manner
• (A): Feeder-free mouse
ESCs were treated for 48
hours with 17-AAG of
concentrations indicated.
Cell lysates were collected,
and Western blot analysis
was performed for proteins
indicated.
HOP co-chaperone is
increased as a potential
compensatory
response.

28. • Figure S5. Hsp90 inhibition induced apoptosis.

29. Was the pluripotency loss mediated by
Hsp90 inhibition as a result of increased cell death ?
No, it is not a
secondary
effect of
apoptosis.

30. • Figure 6. Heat shock protein 90 (Hsp90) inhibition increases markers for
mesoderm differentiation.
• AAG, allylamino-17-demethoxygeldanamycin; AFP, a-fetoprotein; Con,
vehicle treated; Hoe, nuclei staining with Hoechst; SM, smooth muscle.

31. Conclusions
• Hsp90 can maintain the ESCs pluripotency.
• Pluripotency loss is prior to apoptosis
activation. Thus, Hsp90 is required for cell
survival too.
• Oct 4 and Nonog are potential Hsp90 client
proteins.

32. • After Hsp90 inhibition by 17-AAG, the ESCs
increased the expression of mesoderm markers
desmin and protein T, implying that they
might preferentially differentiate into
mesoderm lineages.
• Hsp90 reduced the mRNA level of Oct4. This may
resulted from ESCs differentiation into mesoderm
lineages.