Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7933
Supporting Information Available: Experimental procedures,
biological assays, and figures. This material is available free of
References
(1) Kim, J. H; Auerbach, J. M.; Rodriguez-Gomez, J. A.; Velasco, I.;
Gavin, D.; Lumelsky, N.; Lee, S.; Nguyen, J.; Sanchez-Pernaute,
R.; Bankiewicz, K.; McKay, R. Dopamine neurons derived from
embryonic stem cells function in an animal model of Parkinson’s
disease. Nature 2002, 418, 50–56.
(2) Ding, S.; Schultz, P. G. A role for chemistry in stem cell biology.
Nat. Biotechnol. 2004, 22, 833–840.
(3) Wichterle, H.; Lieberam, I.; Porter, J. A.; Jessell, T. M. Directed
differentiation of embryonic stem cell into motor neurons. Cell
2002, 110, 385–397.
(4) Ding, S.; Wu, T. Y. H.; Brinker, A.; Peters, E. C.; Hur, W.; Gray,
N. S.; Schultz, P. G. Synthetic small molecules that controlstem cell
fate. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7632–7637.
(5) Hwang, K. C.; Kim, J. Y.; Chang, W.; Kim, D. S.; Lim, S.; Kang, S.
M.; Song, B. W.; Ha, H. Y.; Huh, Y. J.; Choi, I. G.; Hwang, D. Y.;
Song, H.; Jang, Y.; Chung, N.; Kim, S. H.; Kim, D. W. Chemi-
cals that modulate stem cell differentiation. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 7467–7471.
(6) Warashina, M.; Min, K. H.; Kuwabara, T.; Huynh, A.; Gage,
F. H.; Schultz, P. G.; Ding, S. A synthetic small molecule that
induces neuronal differentiation of adult hippocampal neural
progenitor cells. Angew. Chem., Int. Ed. 2006, 45, 591–593.
(7) Schneider, J. W.; Gao, Z.; Li, S.; Farooqu, M; Tang, T.; Bezprozvanny,
I.; Frantz, D. E.; Hsieh, J. Small-molecule activation of neuronal cell
fate. Nat. Chem. Biol. 2008, 4, 408–410.
Figure 3. Quantitative PCR of some representative genes in 1
treated MSCs. The MSCs were incubated with 1 at 10 and 20 μM
and Ctrl (DMSO) for 2 days, and the mRNA levels were determined
by quantitative PCR analysis. The experiments were performed
three times independently.
(8) Balasubramaniyan, V.; Boddeke, E.; Bakels, R.; Kust, B.; Koois-
tra, S.; Veneman, A.; Copray, S. Effects of histone deacetylation
inhibition on neuronal differentiation of embryonic mouse neural
stem cells. Neuroscience 2006, 143, 939–951.
(9) Williams, D. R.; Lee, M. R.; Song, Y. A.; Ko, S. K.; Kim, G. H.;
Shin, I. Synthetic small molecules tha induce neruogenesis in
skeletal muscle. J. Am. Chem. Soc. 2007, 129, 9258–9259.
(10) Hall, F. L.; Fernyhough, P.; Ishii, D. N.; Vulliet, P. R. Suppression
of nerve growth factor-directed neurite outgrowth in PC12 cells by
sphingosine, an inhibitor of protein kinase C. J. Biol. Chem. 1988,
263, 4460–4466.
(11) Scheibe, R. J.; Ginty, D. D.; Wagner, J. A. Retinoic acid stimulates
the differentiation of PC12 cells that are deficient in cAMP-
dependent protein kinase. J. Cell Biol. 1991, 113, 1173–1182.
(12) Korzhevskii, D. E.; Otellin, V. A. Immunocytochemical detection
of astrocytes in brain slices in combination with Nissl staining.
Neurosci. Behav. Physiol. 2005, 35, 639–641.
(13) Yang, H.; Xia, Y.; Lu, S. Q.; Soong, T. W.; Feng, Z. W. Basic
fibroblast growth factor-induced neuronal differentiation of
mouse bone marrow stromal cells requires FGFR-1, MAPK/
ERK, and transcription factor AP-1. J. Biol. Chem. 2008, 283,
5287–5295.
Figure 4. Membrane potential assay of MSCs with acetylcholine.
MSCs were incubated with 20 μM 1 for 48 h. Membrane potential
were detected by FLIPR assay kit. Data were analyzed using Flex
station software. Open markers are control. Closed markers are 1
treatment. Arrow indicated treatment of 10 μM acetylcholine.
In the FLIPR assay, the membrane potential was changed on 1
treated MSCs (Figure 4). This result suggests that 1 can diff-
erentiate MSCs into a specific type of cholinergic neuronal cell.
In conclusion, we have developed a new synthetic small
molecule (1) that can induce neuronal differentiation in
MSCs. Compound 1 has a capacity to commit MSCs to
neuronal fate, which was confirmed by morphological change
and expression of neuronal markers (β-III tubulin and NSE).
When treated with 1, more than 95% of MSCs were converted
to neuron-like cells. Moreover, quantitative PCR results
showed that the expressions of neuropeptide, cholinergic
receptors, and Fbxo2, which are a neuronal cell marker, were
up-regulated by 1. Importantly, 1 treated MSCs showed the
neuralelectrophysiological andcholinergic neuronproperties.
Taken together, 1 will be a useful tool in studying cell therapy
and the molecular mechanisms that determine cell fate, even if
further biological evaluations and chemical derivatization to
find better small molecules are now in progress.
(14) Woodbury, D.; Schwarz, E. J.; Prockop, D. J.; Black, I. B. Adult
rat and human bone marrow stromal cells differentiate into
neurons. J. Neurosci. Res. 2000, 61, 364–370.
(15) Sanchez-Ramos, J. R. Nerual cells derived from adult bone mar-
row and umbilical cord blood. J. Neurosci. Res. 2002, 69, 880–893.
(16) Dezawa, M.; Kanno, H.; Hoshino, Ml; Cho, H.; Matsumoto, N.;
Itokazu, Y.; Tajima, N.; Yamada, H.; Sawada, H.; Ishikawa, H.;
Mimura, T.; Kitada, M.; Suzuki, Y.; Ide, C. Specific induction of
neuronal cells from bone marrow stromal cells and application for
autologous transplantation. J. Clin. Invest. 2004, 113, 1701–1710.
(17) Barnabe, G. F.; Schwindt, T. T.; Calcagnotto, M. E.; Motta, F. L.;
Martinez, G.; Oliveira, A. D.; Keim, L. M. N.; D’Almeida, V.;
Mendez-Otero, R.; Mello, L. E. Chemically-induced rat mesenchy-
mal stem cells adopt molecular properties of neuronal-like cells but
do not have basic neuronal functional properties. PLoS One 2009,
4, e5222.
(18) Cho, M. H.; Lee, J. H.; Ahn, H. H.; Lee, J. Y.; Kim, E. S.; Kang,
Y. M.; Min, B. H.; Kim, J. H.; Lee, H. B.; Kim, M. S. Induction of
neurogenesis in rat bone marrow mesenchymal stem cells using
purine structure-based compounds. Mol. BioSyst. 2009, 5, 609–
611.
(19) Erhardt, J. A.; Hynicka, W.; DiBenedetto, A.; Shen, N.; Stone, N.;
Paulson, H.; Pittman, R. N. A novel F Box protein, NFB42, is
highly enriched in neurons and induces growth arrest. J. Biol.
Chem. 1998, 273, 35222–35227.
Acknowledgment. This work was supported by the Crea-
tive Research Fund 2008, Korea Research Institute of Che-
mical Technology.
(20) Cho, G.; Lim, Y.; Zand, D.; Golden, J. A. Sizn 1 is a novel protein
that functions as a transcriptional coactivator of bone morpho-
genic protein signalling. Mol. Cell. Biol. 2008, 28, 1565–1572.