Stauprimide
Neuropathiazol
Controlling Stem Cell Fate with
Small Molecules - Applications in
Regenerative Medicine
Chantelle Capicciotti
Thursday, November 18, 2010
Cardiogenol C
Reversine
Stem Cells
• Differ from other cells of the body.
• Three general properties:
1. Capable of self-renewal through cell division.
2. Unspecialized.
3. Have the ability to differentiate into specialized cells.
Cell Cycle
Karp, G. Cell and Molecular Biology, 4th ed.;
Wiley: New York, 2004.
2
Embryonic Stem Cells
• ESCs are derived from blastocyst of pre-implanted embryo.
• Pluripotent – Able to differentiate into any of the three
germ layers and thus all cell types.
Blastocyst
Blastocoel
Trophectoderm
Fertilization
Zygote
Ectoderm
Mesoderm
Inner Cell Mass
Endoderm
Thomson, J.A.; Itskovitz-Eldor, J.; Shapiro, S.S.; et al. Science. 1998, 282, 1145-1147.
http://stemcells.nih.gov/info/2006report/2006Chapter1.htm
Embroynic Stem
Cells
3
Adult Stem Cells
• Found in various tissues and organs throughout the
body.
• Multipotent – Able to differentiate into several
distinct cell types of the organ or tissue they
originate.
• Maintain and repair the tissue in which they are
found.
Chen, S.; Hilcove, S.; Ding, S. Mol. BioSyst. 2006, 2, 18-24.
4
Adult Stem Cells
http://stemcells.nih.gov/info/2006report/2006Chapter2.htm
5
Regenerative Medicine
• Use of stem cells to repair, regenerate or replace
diseased or injured cells, tissues and organs.
• Treatment of cardiovascular and neurodegenerative
diseases, diabetes and spinal cord injury.
Red Blood Cells
 Bone marrow transplant –
Hematopoietic stem cell transplant
done after chemotherapy and
radiation for leukemia treatment
Marrow
White
Blood
Cells
Platelets
http://leukemiaawareness.com/
6
Maintaining Pluripotency
Proliferation
ESCs remain in
pluripotent state
ESC
Differentiation
High
Expression
Hyslop, L.A.; Armstrong, L.; Stojkovic, M.; Lako, M.
Expert Rev. Mol. Med. 2005, 7, 1-21.
Low
Expression
7
Differentiation
Proliferation
ESCs remain in
pluripotent state
ESC
 ESCs must exit pluripotent state to become more
specialized.
Differentiation
 To differentiate into any of the three germ layers,
certain pathways must be activated/deactivated.
Murry, C.E.; Keller, G. Cell. 2008, 132, 661-680
8
Differentiation
Blastocyst
Ectoderm
BMP4
Mesoderm
ESCs
Activin
Endoderm
Murry, C.E.; Keller, G. Cell. 2008, 132, 661-680
9
Culture Differentiation of ESC’s
Blastocyst
• Embryoid bodies spontaneously
differentiate into multiple cell
lineages.
Inner Cell Mass
ESCs
Feeder Layer
• Increase specificity to certain cell
types by using “cocktails” of growth
factors and signalling molecules.
 Conditions often not completely
Embryoid
Bodies
Ectoderm
defined or are non-specific.
Mesoderm
Endoderm
Hyslop, L.A.; Armstrong, L.; Stojkovic, M.; Lako, M. Expert Rev. Mol. Med. 2005, 7, 1-21.
10
Small Molecule Approach
• ESC differentiation in vitro is poorly controlled.
• Use of “cocktails” does not facilitate studies on the
molecular mechanisms involved in development.
• Small molecules offer a solution:
 Can be more specific and have a high degree of control.
 Molecular mechanisms can be studied.
Stauprimide
Primes ESCs for
differentiation
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
11
High-Throughput Screening Identification
Image-based
high-throughput
screening
Kinase-Oriented Library
Approx. 20 000 Compounds
Stauprimide
 Increased efficiency
of differentiation
towards endoderm
lineage.
Sox17
All Cells
DMSO
Stauprimide
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
12
Further Differentiation of Endoderm Cells
• Stauprimide-primed endoderm cells could be further
differentiated to hepatocytes and pancreatic precursor
cells.
Hepatocyte
Differentiation
Factors
Stauprimide
ESCs
Low Conc.
Activin A
Hepatocytes
Liver
Endoderm
Lineage
Pancreatic
Differentiation
Factors
Pancreas
Pancreatic
Progenitor
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
13
Stauprimide Primes ESCs for Differentiation
• Stauprimide – Primes undifferentiated ESCs.
• Activin A - Stimulus which dictates path of differentiation to
endoderm lineage.
• Stauprimide-primed ESCs can differentiate into other cell
fates under appropriate conditions.
Neural
Differentiation
Factors
Stauprimide
Ectoderm
Lineage
Neurons
ESCs
BMP-4
Beating Cardiomyocytes
Mesoderm Lineage
Hematopoietic
Lineage
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
14
Nonspecific Kinase Inhibitors
Stauprimide
Staurosporine
UCN-01
Promotes
Differentiation
Inactive
Inactive
 Stauprimide is active well below toxic concentrations and does
not inhibit kinases at this concentration.
 Other nonspecific kinase inhibitors did not promote
differentiation of ESCs at nontoxic concentrations.
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
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Staurosporine Family
Stauprimide
Staurosporine
UCN-01
 Staurosporine is a natural product isolated from bacterium
Streptomyces staurosporeus.
 Stauprimide can be synthesized en route of the synthesis of
Staurosporine.
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
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Staurosporine
R1≠R2
Regiochemically
Different
Glycosidic bond
with indolic nitrogens
2nd Glycosidation
(Intramolecular)
1st Glycosidation
(Intermolecular)
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
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First Glycosyl Donor
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
18
Aglycon Acceptor
Glycosyl Donor
+
Ratio: 2.5:1
Aglycon Acceptor
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
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Intermolecular Glycosidation
+
47%
10%
Isolated
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.;
Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
20
Second Glycosyl Donor
Glycosyl
Donor
Precursor
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
21
Second Glycosidation
x
E+ = PhSeCl,
NBS, NIS,
I2,
, etc.
AND
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
22
Second Glycosidation
23
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
Barrett, A. G. M.; Bezuidenhoudt, B. C. B.; Gasieki, A. F.; Howell, A. R.; Russell, M. A. J. Am. Chem. Soc. 1989, 111, 1392-1396.
Final Stages of Staurosporine Synthesis
+
Ratio: 1:1
Staurosporine
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
24
Staurosporine Family
Oxidized
Benzoylated
Staurosporine
Stauprimide
UCN-01
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
25
Final Stages of Staurosporine Synthesis
Leads to UCN-01
and derivatives
+
Staurosporine
Link, J.T.; Raghavan, S.; Gallant, M.; Danishefsky, S.J.; Chou, T.C.; Ballas, L.M. J. Am. Chem. Soc. 1996, 118, 2825-2842.
26
Stauprimide Synthesis
UCN-01
Stauprimide
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
27
Nonspecific Kinase Inhibitors
Stauprimide
Staurosporine
UCN-01
Promotes
Differentiation
Inactive
Inactive
 Cellular target of Stauprimide likely not a kinase which is
nonspefically inhibited by kinase inhibitors.
To identify targets, a biotin-tagged analogue was used.
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
28
Biotinylated Stauprimide
 Linked to proteins or molecules for use in
biochemical assays.
 Binds to avidin and streptavidin with high
affinity and specificity.
Biotin
• This binding is exploited to isolate
proteins.
Biostauprimide
(Biotinylated Stauprimide)
 Similar differentiation
activity as Stauprimide.
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
29
Biostauprimide Synthesis
+
UCN-01
Biostauprimide
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
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Mechanism of Stauprimide
 Stauprimide inhibits the transcription factor NME2 resulting in
a downregulation of c-Myc expression.
NME2
Stauprimide
X
X
C-Myc Gene
NME2
Promoter
Region
X
C-Myc
Differentiation
Maintains
ESC State
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
31
Stauprimide
ESCs
Stauprimide
Activin A
Endoderm
Lineage
Hepatocytes
Neural Differentiation
Factors
BMP-4
Mesoderm
Lineage
Cardiomyocytes
Ectoderm
Lineage
Neurons
Stauprimide
 Increased efficiency of
differentiation.
 Decreased ESC content in
differentiated product.
 Decreased contamination
of unwanted cell types.
Zhu, S.; Wurdak, H.; Wang, J.; Lyssiotis, C.A.; Peters, E.C.; Cho, C.Y; Wu, X.; Schultz, P.G. Cell Stem Cell. 2009, 4, 416-426.
32
Small Molecules Can Induce Differentiation
Fertilized
Egg
 Stauprimide primed ESCs
for differentiation – was not
able to induce differentiation.
8 Cell Embryo
Pluripotent
Blastocyst
 Other small molecules can
induce differentiation of ESCs
and ASCs.
Neural
Cells
Blood
Cells
Cardiac Muscle
http://www.stemcellresearchfoundation.org/WhatsNew/Pluripotent.htm
33
Neuropathiazol
Hippocampus
Neuropathiazol
Hippocampus
Primary
Neural
Progenitor
Cells
Neurons
Astrocytes
Oligodendrocytes
 Adult Stem Cells
(multipotent)
Rat Brain
Human Brain
Warashina, M.; Min, K.H.; Kuwabara, T.; Huynh, A.; Gage, F.H.;
Schultz, P.G.; Ding, S. Angew. Chem. Int. Ed. 2006, 45, 591-593.
34
Differentiation into Neurons
Neuropathiazol
Primary
Neural
Progenitor
Cells
 Preferentially
induces to neuronal
lineage!
Neurons
Astrocytes
Retinoic Acid
Warashina, M.; Min, K.H.; Kuwabara, T.; Huynh, A.; Gage, F.H.;
Schultz, P.G.; Ding, S. Angew. Chem. Int. Ed. 2006, 45, 591-593.
 Large number of
neuronal and astroglial
cells.
35
Repairing the Nervous System With Stem Cells
 Parkinson’s Disease – Neurodegenerative disease caused by the death of
dopaminergic neurons.
 Transplantation of embryonic dopamine neurons into patients can
result in an increase in dopamine function.
Before Surgery
Freed, C.R.; Greene, P.E.; Breeze, R.E.; et. al. N. Engl. J. Med. 2001, 344, 710-719
After Surgery
36
Mending a Broken Heart
 Cardiovascular disease is a
leading cause of death.
Left Ventricle
Normal Heart
Infarcted
Infarcted Heart
http://stemcells.nih.gov/info/2006report/2006Chapter6.htm
 Heart attack results in
deprivation of oxygen to heart
muscles which causes these
cells to die.
 Stem cells have been
investigated as possible sources
for regenerating damaged
myocardial cells.
Dilated
Ventricle
37
Cardiogenols
 Potent differentiator
of ESCs towards cardiac
lineage.
R=
Cardiogenol A
(85%)
R=
Cardiogenol B
(80%)
R=
Cardiogenol C
(90%)
R=
Cardiogenol D
(75%)
Cardiogenol C
Wu, X.; Ding, S.; Ding, Q.; Gray, N.S.; Schultz, P.G. J. Am. Chem. Soc. 2004, 126, 1590-1591.
38
Differentiation into Cardiomyocytes
Mouse
ESCs
Cardiogenol C
Cardiomyocytes
 Cardiogenol C resulted in 90% of cells differentiated towards
cardiac lineage without the formation of embryoid bodies (EBs).
• Majority of the cell population formed beating cardiomyocytes!
 Current cardiomyogenesis inducing conditions of ESCs requires
aggregation and formation of embryoid bodies (EBs).
• Results in only 5% of the cell population forming cardiomyocytes.
Wu, X.; Ding, S.; Ding, Q.; Gray, N.S.; Schultz, P.G. J. Am. Chem. Soc. 2004, 126, 1590-1591.
Winitsky, S.O.; Gopal, T.V.; Hassanzadeh, S.; et. al. PLoS Bio. 2005, 3, 662-671.
39
Cell Reprogramming
Bone
Chemical Reprogramming
Skeletal
Muscle
Adult
Fibroblast
Anastasia, L.; Pelissero, G.; Venerando, B.;
Tettamanti, G. Cell Death Diff. 2010, 17,
Genetic Reprogramming
1230-1237.
Induced Pluripotent
Stem Cells
Heart
Tissue
Other Tissues
40
Amphibian Limb Regeneration
 Amphibians have the ability to
regenerate amputated limbs.
 A blastema forms at the
amputation site.
• Blastema cells can re-enter
the cell cycle.
• Multipotent mesenchymal
progenitor cells.
 Recreated mesenchymal cells
can differentiate to generate cell
type needed to regenerate the
limb.
Brockes, J.P. Science. 1997, 276, 81-87.
41
Induced Pluripotent Stem Cells
Retroviral Transfection
of Pluripotency Genes
Somatic Cell
Reprogramming
hESC Culture
Conditions
Pluripotent
iPSC Line
 Retroviruses can transfect critical transcription factors to reprogram
somatic cells back to a pluripotent state.
• Four transcription factors: Oct4, Sox2, Klf4 and c-Myc.
 Reprogrammed fibroblasts to an embryonic stem cell-like state.
 iPSCs were similar to ESCs.
• Expressed stem cell proteins.
• Formed embryoid bodies.
• Could undergo differentiation.
Takahashi, K.; Yamanaka, S.; Cell. 2006, 126, 663–676.
Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Cell. 2007, 131, 861–872.
42
Induced Pluripotent Stem Cells
Retroviral Transfection
of Pluripotency Genes
Somatic Cell
Reprogramming
hESC Culture
Conditions
Pluripotent
iPSC Line
 Original methods of reprogramming were inefficient.
• Less than 1% of the starting adult cell population yielded iPSCs.
 Viral transfection used to genetically alter the cells can potentially
trigger the expression of oncogenes.
 Have a propensity to form tumors.
• The c-Myc gene is an oncogene and is known to promote tumor growth .
Takahashi, K.; Yamanaka, S.; Cell. 2006, 126, 663–676.
Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Cell. 2007, 131, 861–872.
43
Reversine
Reversine
Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.
44
Reversine
 Reversine can cause cell reprogramming.
 Reversine treated myoblasts were able to form
adipocytes and osteoblasts.
Multipotent
Progenitor
Cells
Myoblasts
Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.
Reversine
Multinucleated
Muscle
Cell
45
Reversine
 Reversine can cause cell reprogramming.
 Reversine treated myoblasts were able to form
adipocytes and osteoblasts.
Adipocytes
Reversine
Reversine
Myoblasts
Multinucleated
Muscle
Cell
Osteoblasts
Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.
46
Reversine
Reversine
Reversine
Dedifferentiation
Multipotent
Progenitor Cells
Osteogenic
Differentiating
Conditions
Myoblasts
Multinucleated
Muscle Cell
Reversine treatment without
differentiating conditions resulted in
mononucleated cells.
• There was no formation of
osteoblasts!
Osteoblasts
Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.
47
Reversine
Reversine
Reversine
Dedifferentiation
Multipotent
Progenitor Cells
Osteogenic
Differentiating
Conditions
Myoblasts
Multinucleated
Muscle Cell
Reversine dedifferentiates
myoblasts into multipotent
mesenchymal progenitor cells.
Osteoblasts
Chen, S.; Zhang, Q.; Wu, X.; Schultz, P.G.; Ding, S. J. Am. Chem. Soc. 2004, 126, 410-411.
48
Summary
 Member of the
Staurosporine family of
nonspecific protein kinase inhibitors.
 Primes ESCs for differentiation by
downregulating expression c-Myc.
Stauprimide
 Increased efficiency of differentiation of
ESCs towards various cell lineages.
 Preferentially induced primary neural
Neuropathiazol
progenitor cells to neuron cells.
49
Summary
 Differentiated ESCs into functioning
cardiac cells and beating cardiomyocytes.
Cardiogenol C
 Induced dedifferentiation.
 Reprogrammed myoblasts into
multipotent mesenchymal progenitor cells.
Reversine
Progenitor cells could be re-differentiated
into adipocytes and osteoblasts.
50
Conclusions
 Small molecules can have a powerful influence on stem cell
outcome and behaviour.
 While these results highlight the great potential of small
molecules for the application of regenerative medicine, they are
preliminary.
• More reliable and efficient chemical reagents that rival
endogenous factors are required.
• Need to fully establish mechanism of action to maximize the
impact of these small molecules.
51
Acknowledgments
Dr. Robert N. Ben
Mathieu Leclère
Ross Mancini
John Trant
Kathryn Davis
Anna Balcerzak
Devin Tonelli
Taz Cheema
Jacqueline Tokarew
Malay Doshi