A novel small molecule facilitates the reprogramming of human somatic cells into a pluripotent state and supports the maintenance of an undifferentiated state of human pluripotent stem cells

Article abstract of DOI:10.1002/anie.201206691

Booster of pluripotency: RSC133, a new synthetic derivative of indoleacrylic acid/indolepropionic acid, exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic

Full text of DOI:10.1002/anie.201206691

Angewandte
Chemie
DOI: 10.1002/anie.201206691
Pluripotency
A Novel Small Molecule Facilitates the Reprogramming of Human
Somatic Cells into a Pluripotent State and Supports the Maintenance of
an Undifferentiated State of Human Pluripotent Stem Cells**
Jungwoon Lee, Yan Xia, Mi-Young Son, Guanghai Jin, Binna Seol, Min-Jeong Kim, Myung
Jin Son, Misol Do, Minho Lee, Dongsup Kim, Kyeong Lee,* and Yee Sook Cho*
Human somatic cells can be reprogrammed to a pluripotent
state by forced expression of a combination of four tran-
scription factors: Oct-4 and Sox-2 with either Klf-4 and c-Myc
(OSKM); or Nanog and LIN28 (OSNL).[1] Because of the
groundbreaking cellular reprogramming discovery that pluri-
potent cells have the ability to differentiate into all cell types
found in adults,[1–2] human induced pluripotent stem cells
(hiPSCs) generated from a patientꢀs own cells have been
considered a highly promising cellular source for basic
research, drug development, and toxicity testing, as well as
regenerative medicine.[3] However, current reprogramming
methods have some known limitations including a low
efficiency and slow kinetics of the reprogramming process,
and a safety concern needs to be addressed before they can be
widely applied in different settings including clinical uses.
Currently, small molecules that modulate specific signal-
ing pathways and epigenetic modifications have been a major
focus to control stem cell fate and function, especially for the
maintenance and/or reacquisition of pluripotency and differ-
entiation.[4] The various small-molecule inhibitors targeting
either DNA methyltransferase (AZA),[4d,5] G9a histone
methyltransferase (BIX-01294),[5] histone deacetylase (VPA,
TSA, and SAHA),[4d,6] TGF-b (A-83-01, SB431542, and E-
616452),[7] MEK (PD0325901),[8] GSK-3 (CHIR99021),[8]
mTOR (rapamycin),[4f] or Pan-Src family kinase[9] have been
reported to enhance iPSC generation; some of these inhib-
itors have even been shown to compensate for reprogram-
ming factors. However, only a limited number of small
molecules are shown to be effective in the human cell system.
Herein, we report a novel synthetic chemical, RSC133, which
was found to effectively enhance reprogramming of human
somatic cells and maintenance of human stem cell pluripo-
tency.
To identify small molecules that can promote the reprog-
ramming process, our in-house chemical library of hetero-
cyclic compounds (MW< 500) was initially screened using
mouse embryonic fibroblasts (MEFs) that are hemizygous for
the Oct4-GFP transgene (OG-MEFs).[10] The MEFs were
reprogrammed by transducing retroviral vectors that encoded
the standard four reprogramming factors (OSKM) and then
either not treated or treated with chemical compounds
(Figure S1 in the Supporting Information). Based on primary
screening results, we further synthesized new series of
indoleacrylic acid and indolepropionic acid derivatives
(Scheme S1 in the Supporting Information) and evaluated
their structure–activity relationship (SAR) for reprogram-
ming efficiency (Table S1 in the Supporting Information).
Among the candidate compounds examined, indoleacrylic
acid analogues ID133 and ID558, which contain an indole ring
with a free NH group and a benzoic acid analogue connected
through a double bond, were the most potent in enhancing
reprogramming efficiency; the enhanced efficiency was con-
firmed by a substantial increase in the number of GFP-
positive colonies compared to the untreated control (Fig-
ure S1 and Table S1 in the Supporting Information). Of note,
compound ID133 features a simple benzamide in the benzoic
acid part, while ID558 contains 4-hydroxy-N,N-dimethyl-
benzamide. Because of its ability to promote iPSC generation
in both mouse and human (see below), we renamed the novel
small molecule ID133 as the reprogramming stimulating
compound (RSC) 133 (Figure 1a). The full characterization
of the identified RSC133 is shown in Figures S2–S5 in the
Supporting Information.
[*] Dr. J. Lee, M.-Y. Son, B. Seol, M.-J. Kim, Dr. M. J. Son, M. Do,
Dr. Y. S. Cho[+]
Regenerative Medicine Research Center
Korea Research Institute of Bioscience and Biotechnology
125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (S. Korea)
E-mail: june@kribb.re.kr
M.-J. Kim, M. Do, Dr. Y. S. Cho[+]
University of Science & Technology
113 Gwahank-ro, Yuseong-gu, Daejeon 305-333 (S. Korea)
Y. Xia, G. Jin, Prof. K. Lee[+]
College of Pharmacy
Dongguk University-Seoul
26 Pildong-3-ga, Junggu, Seoul 100-715 (S. Korea)
E-mail: kaylee@dongguk.edu
M. Lee, Prof. D. Kim
Department of Bio and Brain Engineering
Korea Advanced Institute of Science and Technology
291 Daehak-ro, Yuseong-gu, Daejeon 305-701 (S. Korea)
We selected 35 candidates including indole analogues and
further explored whether those chemicals could enhance the
reprogramming process of human foreskin fibroblasts (hFFs,
ATCC# CRL-2097) into hiPSCs. The overall reprogramming
procedure we used in this study is a slightly modified version
of the published protocols.[1a,6] The OSKM-transduced hFFs
not treated or treated with chemical compounds were
[+] These authors contributed equally to this work.
[**] This work was supported by the NRF grant (2010-020272(3) and
2012M3A9C7050224) and the KRIBB/KRCF stem cell research
initiative program (NAP).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201206691.
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To optimize the sequence and duration of RSC133
treatment, we tested 11 different reprogramming culture
conditions (Figure 1c) and found that the addition of RSC133
(10 mm) to the culture soon after OSKM transduction (Fig-
ure 1c, condition 1–4) was more effective than other con-
ditions, where RSC133 was added after reseeding (Figure 1c,
condition 5–10). Out of all the various conditions, the
continuous treatment with RSC133 throughout reprogram-
ming was the most effective way to increase reprogramming
efficiency (approximately 3- to 4-fold; Figure 1c, condition 1;
days À5 to 15). In combination with hypoxic conditions (5%
O2), RSC133 synergistically increased the reprogramming
efficiency by approximately 5- to 6-fold compared to the
untreated control (data not shown). Under our conditions, the
stimulatory effect of RSC133 on reprogramming was 1.5- to
3.3-fold higher than that of well-known chemicals (AZA,
VPA, TSA, and SB431542) that enhance iPSC generation
(Figure S7 in the Supporting Information). The hFFs reprog-
rammed with RSC133 displayed a rapid activation of
pluripotency-associated genes Nanog, Oct4, and Rex1 up to
2- to 2.5-fold (Figure 2c) and highly proliferative (bromo-
deoxyuridine (BrdU)-positive) hESC-like colonies that
showed positive immunostaining for pluripotency markers
Nanog or Tra-1-81 (Figure 2d) on day 10 after treatment
compared to the untreated control. These results suggest that
RSC133 significantly enhanced both the efficiency and
kinetics of the reprogramming process.
Reports have shown that the ectopic expression of four
reprogramming factors OSKM inhibited cell proliferation
and caused cell-cycle arrest, senescence, and apoptosis during
reprogramming and that is significantly related with the up-
regulation of p53, p21, and p16/INK4A, which are negative
regulators of the reprogramming process.[4c,11] RSC133 con-
stituted a favorable condition for human somatic cell
reprogramming through affecting multiple cellular responses.
Firstly, RSC133 exerted positive effects on cell proliferation.
RSC133 significantly increased the total number of cells,
counted by trypan blue exclusion, by approximately 1.7-fold
(Figure 2a) and the percentage of proliferating, BrdU-
positive cells by approximately 2.5-fold during reprogram-
ming on day 10 compared to the untreated control (Fig-
ure 2b). Positive effects of RSC133 on cell proliferation were
also observed in normal culture condition of hFFs (Fig-
ure S6b,c in the Supporting Information). Cell proliferation is
tightly associated with supporting self-renewal and pluripo-
tent properties in ESCs,[12] and the induction of cell prolifer-
ation notably contributes to efficient somatic cell reprogram-
ming.[4f,12a]
Secondly, RSC133 ablated pro-senescence phenotypes
through down-regulating critical inducers of cellular senes-
cence, such as p53, p21, and p16/INK4A. The up-regulation of
p53, p21, and p16/INK4A genes, mediated by OSKM
induction, was markedly inhibited by RSC133 up to 2.13-,
3.09- and 2.79-fold, respectively during reprogramming on
day 10 compared to the untreated control (Figure 2c). In
parallel, the proportion of senescence-associated b-galactosi-
dase (SA-b-gal)-positive cells was decreased by approxi-
mately 37% during reprogramming on day 10 compared to
Figure 1. A new indole compound, RSC133, promotes the reprogram-
ming of human somatic cells to pluripotent state. a) Chemical
structure of RSC133. b) Dose-dependent enhancement of reprogram-
ming efficiency by RSC133. The OSKM-transduced hFFs were incu-
bated with or without RSC133 at the indicated concentrations. c) Time
course study of RSC133 treatment on reprogramming. The OSKM-
transduced hFFs were incubated with or without RSC133 (10 mm) at
the indicated time periods. The hESC-like colonies were stained and
quantified for AP to determine reprogramming efficiency. The relative
fold changes in AP+ colonies compared to the untreated control
(designated as 1) were shown (orange bars indicate initial treatment
with RSC133 on days À5 to 0, and white bars indicate the treatment
started on day 0, 5, or 10 as shown in the scheme on the left). Results
(b and c) are shown as the mean Æstandard deviation (SD; n=3).
The statistical significance was determined using Student’s t-test.
*P<0.05, **P<0.005.
monitored daily for morphologic changes culminating in
human embryonic stem cell (hESC)-like colonies, and the
resulting hiPSCs were verified by alkaline phosphatase (AP)
staining on day 15 after treatment. Under our conditions, only
compound RSC133 significantly increased the reprogram-
ming efficiency of hFFs in a dose-dependent manner up to
10 mm compared to the untreated control (Figure 1b,c and
Table S1 in the Supporting Information). An increase of
RSC133 concentration to 20 mm, with no changes in cell
viability, did not further enhance reprogramming efficiency
(Figure 1b and Figure S6a in the Supporting Information).
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a particular ligand.[13] Using AutoDock Vina,[14] one of the
most widely used molecular docking programs, we calculated
the protein–ligand docking scores between RSC133 and
nearly all human protein structures currently available in
the Protein Data Bank (total 10886 protein structures), and
found that RSC133 most strongly binds to Dnmt1 with the
docking score of À9.7. Considering that the second best
docking score is À8.8 and the docking score of Dnmt1
(= À9.7) is well separated from the background score
distribution (Figure 3a), we concluded that Dnmt1 is indeed
a specific binding target of RSC133. Visual inspection
revealed that RSC133 is docked into the site corresponding
to cofactor S-adenosyl methionine binding pocket and
stabilized mainly through multiple hydrogen bonds (Fig-
ure 3b). In correlation, RSC133 significantly reduced Dnmt1
enzymatic activity by approximately 38% during reprogram-
ming on day 10 compared to the untreated control (Fig-
ure 3c).
Epigenetic chromatin remodeling modulated by DNA
methylation and/or histone modification plays a critical role
in the global transcriptional regulation during the reprogram-
ming process.[4d,6] The inhibition of DNA methylation by
treatment with inhibitor AZA or knockdown of Dnmt1
expression induces rapid conversion from a partially to a fully
reprogrammed state.[4d] An increased level of H3K9 histone
acetylation (ace) is specifically correlated with restoring
pluripotency and reprogramming capacity.[15] A report also
demonstrated that DNA methylation, mediated by Dnmt1, is
closely associated with altered chromatin state through
HDAC activity.[16] Interestingly, the total levels of H3K9ace
were significantly up-regulated in RSC133-reprogrammed
cells compared to the untreated control (Figure 3d). Further-
more, RSC133 reduced HDAC enzyme activity by approx-
imately 13.7% during reprogramming on day 10 compared to
the untreated control (Figure 3e). The expression level of
HDAC1 proteins but not of HDAC2 was down-regulated by
RSC133 on day 10 after treatment, compared to the untreated
control, although they share an almost identical catalytic core
domain and an extensively similar amino acid sequence
(Figure S9 in the Supporting Information). Reports have
demonstrated that HDAC1 occupied the gene loci for the four
factors: Oct4, Sox2, Klf4, and c-Myc in ESCs, and the
pluripotency-associated genes were up-regulated in the
HDAC1 null ES cells.[17] These results collectively indicate
that RSC133-mediated inhibition of Dnmt1 and subsequent
decrease of HDAC1 activity enhances the reprogramming
process by indirectly affecting gene transcriptional modula-
tion through the changed DNA methylation and chromatin
states, and increased accessibility of the gene loci for the four
defined reprogramming factors.
Figure 2. RSC133 ablated reprogramming barriers and resulted in
accelerated kinetics of reprogramming. The OSKM-transduced hFFs
were incubated with 10 mm RSC133 (brown in (a–c)) or without
RSC133 (white in (a–c)) for 10 days unless otherwise indicated.
a) RSC133 promoted the cell growth rate during reprogramming. The
growth curves were plotted using total cell counts per well. The viable
cells were counted by using a trypan blue exclusion assay. b) RSC133
increased the cell proliferation rate during reprogramming. The prolif-
eration rate of the reprogramming cells, determined by the number of
BrdU+ cells, was shown as the mean ÆSD of three independent
experiments. The percentage of BrdU+ cells in each condition (treated
with RSC133) was compared with the untreated control. The statistical
significance was determined using Student’s t-test. *P<0.05. c) Quan-
titative real-time RT-PCR analysis for pluripotency (Nanog, Oct4, and
Rex1)- and cell cycle arrest (p53, p21, and p16)-associated genes. All
values were calculated with respect to the hFFs control, which was set
to 1. hiPS (gray bars) and hES (black bars) indicate the positive
control. d) Immunocytochemical analysis of BrdU and pluripotency-
associated markers (Nanog and Tra-1-81). Scale bars represent
200 mm. GAPDH=glyceraldehyde-3-phosphate dehydrogenase,
DAPI=4’,6-diamidino-2-phenylindole.
the untreated control (Figure S8 in the Supporting Informa-
tion).
The RSC133-generated hiPSC lines (1 and 2, chosen
randomly) picked up after three weeks post-infection showed
silencing of exogenous retroviral four factors (OSKM),
expression of the pluripotency markers, normal karyotype,
and pluripotency both in vitro and in vivo (Figure S10 in the
Supporting Information).
We also confirmed that RSC133 supported the mainte-
nance of human pluripotent stem cells (hESCs and hiPSCs) in
an undifferentiated state. H9 hESCs cultured in uncondi-
Thirdly, RSC133 could also regulate the epigenetic
modulators, such as DNA methyltransferase (Dnmt) and
histone deacetylase (HDAC). To identify specific molecular
targets of RSC133, we performed in silico reverse docking
calculations. In reverse docking, in contrast to the conven-
tional virtual screening in which one searches chemical space
for chemicals that can specifically bind to a specific protein
target, one tries to find the protein targets that can bind to
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RSC133 to UM significantly protected hESCs from the loss
of self-renewing undifferentiated states, which was confirmed
by hESC-typical morphology and pluripotency marker
expressions (Figure S11a,b in the Supporting Information).
The undifferentiated hESCs maintained in the UM plus
RSC133 displayed significant amounts of H3K9ace on par
with the levels in hESCs cultured with the conditioned
medium (CM; Figure S11c in the Supporting Information),
thus indicating that RSC133 can restore pluripotency of
inferior ESCs by increasing the level of H3K9ace.
In conclusion, we present a novel and effective small
molecule, RSC133, which facilitates the maintenance of
pluripotent hESCs and hiPSCs as well as functions as
a potent chemical booster for human somatic cell reprogram-
ming by improving reprogramming efficiency and kinetics
(Figure 3 f). The reprogramming of human somatic cells is
a comparatively slow and gradual process, and the funda-
mental molecular mechanisms regulating the reprogramming
process are still largely unknown. The identification of small
molecules that are effective for somatic cell reprogramming
and/or supporting hESC/hiPSC pluripotency would provide
a better understanding of pluripotency and improve reprog-
ramming technology that, if coupled with scalable, defined,
and safe culture conditions, would result in a higher quality of
hiPSCs.
Received: August 18, 2012
Published online: November 4, 2012
Keywords: gene expression · pluripotency · reprogramming ·
.
small molecules · stem cells
Figure 3. The beneficial effect of RSC133 on reprogramming was
closely related to its inhibitory effects on Dnmt1 and HDAC1 enzyme
activity. The OSKM-transduced hFFs were incubated with or without
RSC133 treatment for 10 days unless otherwise indicated. a) Distribu-
tion of docking scores between RSC133 and all human protein
structures calculated by AutoDock Vina. Docking score of Dnmt1
(À9.7, arrow) is clearly separated from background score distribution
(blue curve), thus indicating that Dnmt1 is a specific molecular target
of RSC133. b) RSC133–Dnmt1 docking model. RSC133 is shown in
light blue. c) Repression of Dnmt1 enzyme activity by RSC133. The
relative activity of Dnmt1 was shown with the value obtained without
RSC133 for 10 days taken as 100%. d) Immunocytochemical (left
panel) and Western blot (right panel) analysis of H3K9ace and Nanog
proteins. The cells were stained with antibodies against H3K9ace (red)
and Nanog (green). The DNA was stained with DAPI (blue). H3 was
used as an internal control for western blot analysis. e) Repression of
HDAC enzyme activity by RSC133. The HDAC activity was determined
by using a colorimetric assay. All of the values were calculated with
respect to the value for the hFFs (control), which was set to 1.
f) Overview of the RSC133-mediated reprogramming process. The
RSC133 treatment during the reprogramming process improved the
reprogramming efficiency and kinetics (green arrow). The timing of
gene activation of the pluripotency markers (Oct4 and Nanog) and of
H3K9ace is indicated by orange bars. The timing of gene repression of
Dnmt1 is indicated by vanishing blue bars. Results in (c) and (e) are
shown as the mean ÆSD (n=3). The statistical significance was
determined using Student’s t-test. *P<0.01, **P<0.005.
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