Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds

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DOI: 10.1126/science.1239278 · Source: PubMed
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Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource, with potential for studying disease and use in regenerative medicine. However, genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here, we show that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells resemble embryonic stem cells in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. By using small molecules, exogenous “master genes” are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.
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Pluripotent stem cells, such as embryonic stem cells (ESCs), can
self-renew and differentiate into all somatic cell types. Somatic cells can
be reprogrammed to become pluripotent via nuclear transfer into oocytes
or through the ectopic expression of defined factors (14). However,
exogenous pluripotency-associated factors, especially Oct4, are indis-
pensable for establishing pluripotency (57), and previous reprogram-
ming strategies have raised concerns regarding the clinical applications
(8, 9). Small molecules have advantages because they can be cell per-
meable, non-immunogenic, more cost-effective, and can be more easily
synthesized, preserved, and standardized. Moreover, their effects on
inhibiting and activating the function of specific proteins are often re-
versible and can be finely tuned by varying the concentrations. Here, we
identified small-molecule combinations that were able to drive the re-
programming of mouse somatic cells toward pluripotent cells.
To identify small molecules that facilitate cell reprogramming, we
searched for small molecules that enable reprogramming in the absence of
Oct4 using Oct4 promoter-driven GFP expression (OG) mouse embryonic
fibroblasts (MEFs), with viral expression of Sox2, Klf4, and c-Myc. After
screening up to 10,000 small molecules (table S1A), we identified For-
skolin (FSK), 2-Methyl-5-hydroxytryptamine (2-Me-5HT), and D4476
(table S1B) as chemical “substitutes” for Oct4 (Fig. 1, A and B, and figs.
S1 and S2). Previously, we had developed a small-molecule combination
“VC6T” [VPA, CHIR99021 (CHIR), 616452, Tranylcypromine], that
enables reprogramming with a single gene, Oct4 (6). We next treated
OG-MEFs with VC6T plus the chemical substitutes of Oct4 in the ab-
sence of transgenes. We found VC6T plus FSK (VC6TF) induced some
GFP-positive clusters expressing E-cadherin, a mesen-
chyme-to-epithelium transition marker, reminiscent of early reprogram-
ming by transcription factors (10, 11)
(Fig. 1C and fig. S3). However, the
expression of Oct4 and Nanog was not
detectable and their promoters remained
hypermethylated, suggesting a repressed
epigenetic state (fig. S3).
To identify small molecules that fa-
cilitate late reprogramming, we used a
doxycycline (DOX)inducible Oct4
expression screening system, adding
DOX only in the first 4-8 days (6).
Small-molecule hits, including several
cAMP agonists (FSK, Prostaglandin E2,
and Rolipram) and epigenetic modula-
tors (3-deazaneplanocin A (DZNep),
5-Azacytidine, Sodium butyrate, and
RG108), were identified in this screen
(fig. S4 and table S1B).
To achieve complete chemical re-
programming without the
Oct4-inducible system, these small
molecules were further tested in the
chemical reprogramming of OG-MEFs
without transgenes. When DZNep was
added 16 days after treatment with
VC6TF (VC6TFZ), GFP-positive cells
were obtained up to 65-fold more fre-
quently than those treated with VC6TF,
forming compact, epithelioid,
GFP-positive colonies without clear-cut
edges (Fig. 1, D and E, and fig. S5). In
these cells, the expression levels of most
pluripotency marker genes were ele-
vated but still lower than in ESCs, sug-
gesting an incomplete reprogramming
state (fig. S6). After switching to 2i-medium with dual inhibition (2i) of
glycogen synthase kinase-3 and mitogen-activated protein kinase sig-
naling after day 28 post-treatment, certain GFP-positive colonies devel-
oped an ESC-like morphology (domed, phase-bright, homogeneous with
clear-cut edges) (Fig. 1F) (12, 13). These colonies could be further cul-
tured for more than 30 passages, maintaining an ESC-like morphology
(Fig. 1, G and H). We refer to these 2i-competent, ESC-like, and
GFP-positive cells as chemically induced pluripotent stem cells (CiPSCs).
Next, we optimized the dosages and treatment duration of the small
molecules, and were able to generate 1-20 CiPSC colonies from 50,000
initially plated MEFs (fig. S7). After an additional screen, we identified
some small-molecule boosters of chemical reprogramming, among
which, a synthetic retinoic acid receptor ligand, TTNPB, enhanced
chemical reprogramming efficiency up to 40-fold, to a frequency com-
parable to transcription factor-induced reprogramming (up to 0.2%) (fig.
S8 and table S1B). Furthermore, using the small-molecule combination
VC6TFZ, we obtained CiPSC lines from mouse neonatal fibroblasts
(MNFs), mouse adult fibroblasts (MAFs), and adipose-derived stem cells
(ADSCs) with OG cassettes by an efficiency approximately 10 times
lower than that obtained from MEFs (fig. S9 and table S3). Moreover, we
induced CiPSCs from wild-type MEFs without OG cassettes or any other
genetic modifications by a comparable efficiency to that achieved from
MEFs with OG cassettes (fig. S9). The CiPSCs were also confirmed to be
viral-vector free by genomic PCR and Southern blot analysis (fig. S10).
The established CiPSC lines were then further characterized. They
grew with a doubling time (14.1-15.1 hours) similar to that of ESCs (14.7
hours), maintained alkaline phosphatase activity, and expressed pluripo-
tency markers, as detected by immunofluorescence and RT-PCR (Fig. 2,
Pluripotent Stem Cells Induced from
Mouse Somatic Cells by
Small-Molecule Compounds
Pingping Hou,
* Yanqin Li,
* Xu Zhang,
* Chun Liu,
* Jingyang
* Honggang Li,
* Ting Zhao,
Junqing Ye,
Weifeng Yang,
Kang Liu,
Jian Ge,
Jun Xu,
Qiang Zhang,
Yang Zhao,
College of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871,
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School,
Shenzhen 518055, China.
Beijing Vitalstar Biotechnology Co., Ltd., Beijing 100012, China.
*These authors contributed equally to this work.
These authors contributed equally to this work.
Corresponding author. E-mail: (H.D.); (Y.Z.).
Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell
resource, with potential for studying disease and use in regenerative medicine.
However, genetic manipulation and technically challenging strategies such as
nuclear transfer used in reprogramming limit their clinical applications. Here, we
show that pluripotent stem cells can be generated from mouse somatic cells at a
frequency up to 0.2% using a combination of seven small-molecule compounds. The
chemically induced pluripotent stem cells (CiPSCs) resemble embryonic stem cells
(ESCs) in terms of their gene expression profiles, epigenetic status, and potential for
differentiation and germline transmission. By using small molecules, exogenous
“master genes” are dispensable for cell fate reprogramming. This chemical
reprogramming strategy has potential use in generating functional desirable cell
types for clinical applications.
on July 18, 2013www.sciencemag.orgDownloaded from
/ / 18 July 2013 / Page 2/ 10.1126/science.1239278
A and B, and fig. S11). The gene expression profiles were similar in
CiPSCs, ESCs, and OSKM-iPSCs (iPSCs induced by Oct4, Sox2, Klf4
and c-Myc) (Fig. 2C and fig. S12). DNA methylation state and histone
modifications at Oct4 and Nanog promoters in CiPSCs were similar to
that in ESCs (Fig. 2D and fig. S13). In addition, CiPSCs maintained a
normal karyotype and genetic integrity for up to 13 passages (fig. S14 and
table S2).
To characterize their differentiation potential, we injected CiPSCs
into immunodeficient (SCID) mice. The cells were able to differentiate
into tissues of all three germ layers (Fig. 3A and fig. S15). When injected
into eight-cell embryos or blastocysts, CiPSCs were capable of integra-
tion into organs of all three germ layers including gonads and transmis-
sion to subsequent generations (Fig. 3, B to E, and fig. S16). Unlike
chimeric mice generated from iPSCs induced by transcription factors
including c-Myc (14), the chimeric mice generated from CiPSCs were
100% viable and apparently healthy for up to 6 months (Fig. 3F). These
observations suggest that the CiPSCs were fully reprogrammed into
pluripotency (table S3).
We next determined which of these small molecules were critical in
inducing CiPSCs. We found four essential small molecules whose indi-
vidual withdrawal from the cocktails generated significantly reduced
GFP-positive colonies and no CiPSCs (Fig. 4, A to C). These small
molecules (C6FZ) include: CHIR (C), a glycogen synthase kinase 3
inhibitor (15); 616452 (6), a transforming growth factor-beta inhibitor
(16); FSK (F), a cAMP agonist (fig. S17) (17); and DZNep (Z), an
S-adenosylhomocysteine (SAH) hydrolase inhibitor (figs. S18 and S19)
(18, 19). Moreover, C6FZ was able to induce CiPSCs from both MEFs
and MAFs, albeit by a 10 times lower efficiency than that induced by
VC6TFZ (fig. S20 and table S3).
To better understand the pluripotency-inducing properties of these
small molecules, we profiled the global gene expression during chemical
reprogramming and observed the sequential activation of certain key
pluripotency genes, which was validated by real-time PCR and immuno-
fluorescence (fig. S21). The expression levels of two pluripotency-related
genes, Sall4 and Sox2, were most significantly induced in the early phase
in response to VC6TF, as was the expression of several extra-embryonic
endoderm (XEN) markers Gata4, Gata6, and Sox17 (Fig. 4, D to F, and
figs. S22 to S24). The expression of Sall4 was enhanced most signifi-
cantly as early as 12 hours after small-molecule treatment, suggesting that
Sall4 may be involved in the first step toward pluripotency in chemical
reprogramming (fig. S22B). We further examined the roles of the en-
dogenous expression of these genes in chemical reprogramming, using
gene overexpression and knockdown strategies. We found the concomi-
tant overexpression of Sall4 and Sox2 was able to activate an Oct4 pro-
moter-driven luciferase reporter (fig. S25) and was sufficient to replace
C6F in inducing Oct4 expression and generating iPSCs (Fig. 4, G and H,
and fig. S26). The endogenous expression of Sall4, but not Sox2, requires
the activation of the XEN genes, and vice versa (fig. S27). This suggests a
positive feedback network formed by Sall4, Gata4, Gata6, and Sox17,
similar to which was previously described in mouse XEN formation (20).
Moreover, knockdown of Sall4 or these XEN genes impaired Oct4 acti-
vation and the subsequent establishment of pluripotency (fig. S28), in
consistent with our previous finding that Gata4 and Gata6 can contribute
to inducing pluripotency (21). Taken together, these findings revealed a
Sall4-mediated molecular pathway that acts in the early phase of chemical
reprogramming (Fig. 4L). This step resembles a Sall4-mediated dedif-
ferentiation process in vivo during amphibian limb regeneration (22).
We next investigated the role of DZNep, which was added in the late
phase of chemical reprogramming. We found that Oct4 expression was
enhanced significantly after the addition of DZNep in chemical repro-
gramming (Fig. 4D), and DZNep was critical for stimulating the expres-
sion of Oct4 but not the other pluripotency genes (Fig. 4I). As an SAH
hydrolase inhibitor, DZNep elevates the concentration ratio of SAH to
S-adenosylmethionine (SAM) and may thereby repress the
SAM-dependent cellular methylation process (fig. S18) (18, 19). Con-
sistently, DZNep significantly decreased DNA and H3K9 methylation at
the Oct4 promoter, which may account for its role in Oct4 activation (Fig.
4, J and K) (23, 24). As master pluripotency genes, Oct4 and Sox2 may
thereby activate other pluripotency-related genes, and fulfill the chemical
reprogramming process, along with the activation of Nanog and silencing
of Gata6, in the presence of 2i (12, 13, 25, 26) (Fig. 4F and fig. S29). In
summary, as a master switch governing pluripotency, Oct4 expression,
which is kept repressed in somatic cells by multiple epigenetic modifica-
tions, is unlocked in chemical reprogramming by the epigenetic modu-
lator DZNep, and stimulated by C6F-induced expression of Sox2 and
Sall4 (Fig. 4L).
Our proof-of-principle study demonstrates that somatic reprogram-
ming toward pluripotency can be manipulated using only small-molecule
compounds (fig. S30). It reveals that the endogenous pluripotency pro-
gram can be established by the modulation of molecular pathways non-
specific to pluripotency via small molecules rather than by exogenously
provided “master genes.” These findings increase our understanding
about the establishment of cell identities and open up the possibility of
generating functionally desirable cell types in regenerative medicine by
cell fate reprogramming using specific chemicals or drugs, instead of
genetic manipulation and difficult-to-manufacture biologics. To date, the
complete chemical reprogramming approach remains to be further im-
proved to reprogram human somatic cells and ultimately meet the needs
of regenerative medicine.
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Acknowledgments: We thank X. Zhang, J. Wang, C. Han, Z. Hou, J. Liu,
and L. Ai for technical assistance. This work was supported by grants
from the National 973 Basic Research Program of China
(2012CD966401, 2010CB945204), the Key New Drug Creation and
Manufacturing Program (2011ZX09102-010-03), the National
Natural Science Foundation of China (90919031), the Ministry of
Science and Technology (2011DFA30730, 2013DFG30680), the
Beijing Science and Technology Plan (Z121100005212001), the
Ministry of Education of China (111 project), and a Postdoctoral
Fellowship of Peking-Tsinghua Center for Life Sciences. Microarray
and RNA-seq data are deposited in the Gene Expression Omnibus
(GEO) database (accession number GSE48243). The authors have
filed a patent for the small-molecule combinations used in the
chemical reprogramming reported in this paper.
Supplementary Materials
Materials and Methods
Figs. S1 to S30
Tables S1 to S5
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17 April 2013; accepted 11 July 2013
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Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B)
plus chemicals or Oct4. Error bars indicate standard deviation (s.d.) (n = 3). (C)
GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical
treatment. (D) Numbers of GFP-
treatment on day 36. Error bars indicate the s.d. (n = 2). (E to G) Morphology of
a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment
(E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged
CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC
generation. For (C) and (E to G), scale bars, 100 μm. For (D), cells for
reprogramming were replated on day 12.
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Fig. 2. Characterization of CiPSCs. (A and B) Pluripotency marker expression as
illustrated by immunofluorescence (A, clone CiPS-25) and RT-PCR (B). Scale bars,
100 μm. (C) Hierarchical clustering of global transcriptional profiles. 1-PCC,
Pearson correlation coefficient. (D) Bisulfite genomic sequencing of the Oct4 and
Nanog promoter regions. MNF-CiPS-7, MNF-derived CiPSC line #7; MAF-CiPS-1,
MAF-derived CiPSC line #1.
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Fig. 3. Pluripotency of CiPSCs. (A) Hematoxylin and eosin staining of
CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone
CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2
offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing
pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n,
total numbers of chimeras studied.
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Fig. 4. Step-wise establishment of the pluripotency circuitry during chemical
reprogramming. (A and B) Numbers of GFP-positive (A) and CiPSC (B) colonies
induced by removing individual chemicals from VC6TFZ. The results of three
independent experiments are shown with different colors (white, gray and black).
(C) Structures of the essential chemicals. (D and E) The expression of
pluripotency-related genes (D) and Gata6, Gata4 and Sox17 (E) as measured by
real-time PCR. (F) Gene expression heat map at the single colony level. The value
indicates the log
-transformed fold change (relative to Gapdh and normalized to
the highest value). (G and H) Oct4 activation (G) and numbers of GFP-positive and
iPSC colonies (H) induced by the overexpression of Sall4 and Sox2, with removing
C6F from VC6TFZ. (I to K) The expression of pluripotency-related genes (I), DNA
methylation (J) and H3K9 dimethylation (K) states of the Oct4 promoter in the
presence and absence of DZNep on day 32. (L) Schematic diagram illustrating the
wise establishment of the pluripotency circuitry during chemical
reprogramming. Error bars indicate the s.d. (n ≥ 2).
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  • ... Originally, the reprogramming factor cocktail contained SOX2, KLF4, OCT4 (encoded by Pou5f1 or POU5F1 gene in mice or humans, respectively), and c-MYC (SKOM), but other combinations of exogenous factors are also effective [26,27]. More recently, mouse reprogramming has been achieved using only chemicals [28]. Remarkably, induced pluripotent stem cells (iPSCs) provide a priori unlimited number of individual-specific stem cells that can be used for in vitro disease modeling, drug screening, and potential cell-based therapies [29,30]. ...
    Full-text available
    The generation of induced pluripotent stem cells through somatic cell reprogramming requires a global reorganization of cellular functions. This reorganization occurs in a multi-phased manner and involves a gradual revision of both the epigenome and transcriptome. Recent studies have shown that the large-scale transcriptional changes observed during reprogramming also apply to long non-coding RNAs (lncRNAs), a type of traditionally neglected RNA species that are increasingly viewed as critical regulators of cellular function. Deeper understanding of lncRNA function and regulation in reprogramming may not only help to improve this process but also have implications for studying cell plasticity in other contexts, such as development, aging, and cancer. In this review, we summarize the current progress made in profiling and analyzing the role of lncRNAs in various phases of somatic cell reprogramming, with emphasis on the re-establishment of the pluripotency gene network and X chromosome reactivation.
  • ... Despite the improved expansion of iKCs and successful construction of a human skin equivalent in vitro, clinicians have continually questioned their value versus the risks of retrovirus-mediated gene transfer, which can potential contribute to undesired genotoxicity, insertional mutagenesis, and tumorigenesis [59]. Various integration-free approaches have been developed to convert cells into iPSCs and adult stem/progenitor cells, including viral (adenoviruses, Sendai viruses, and Creexcisable viruses) [60][61][62][63] and non-viral (DNA expression vectors, minicircle vectors, and episomal vectors) [64][65][66][67] vectors and non-DNA-based systems (proteins, mRNAs, and chemicals) [68][69][70][71][72][73]. Thus, further studies of genetic delivery that avoids potential issues associated with viral integration are required to increase the feasibility of clinical trials. ...
    Full-text available
    Background: Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. Methods: Induced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition. Results: BN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro. Conclusions: This study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.
  • ... However, other study has found that XEN-like cells and iPSCs were induced concomitantly, but independently during reprogramming [5]. More recently, the generation of chemical-induced pluripotent stem cells (ciPSCs) from mouse [6][7][8] and goat [9] solely through the use of chemicals has been successful. And previous studies have confirmed that ciXEN cells represent an indispensable intermediate cell state during chemical-based multipotential reprogramming [10]. ...
    Full-text available
    Background: The development of somatic reprogramming, especially purely chemical reprogramming, has significantly advanced biological research. And chemical-induced extraembryonic endoderm-like (ciXEN) cells have been confirmed to be an indispensable intermediate stage of chemical reprogramming. They resemble extraembryonic endoderm (XEN) cells in terms of transcriptome, reprogramming potential, and developmental ability in vivo. However, the other characteristics of ciXEN cells and the effects of chemicals and bFGF on the in vitro culture of ciXEN cells have not been systematically reported. Methods: Chemicals and bFGF in combination with Matrigel were used to induce the generation of ciXEN cells derived from mouse embryonic fibroblasts (MEFs). RNA sequencing was utilised to examine the transcriptome of ciXEN cells, and PCR/qPCR assays were performed to evaluate the mRNA levels of the genes involved in this study. Hepatic functions were investigated by periodic acid-Schiff staining and indocyanine green assay. Lactate production, ATP detection, and extracellular metabolic flux analysis were used to analyse the energy metabolism of ciXEN cells. Results: ciXEN cells expressed XEN-related genes, exhibited high proliferative capacity, had the ability to differentiate into visceral endoderm in vitro, and possessed the plasticity allowing for their differentiation into induced hepatocytes (iHeps). Additionally, the upregulated biological processes of ciXEN cells compared to those in MEFs focused on metabolism, but their energy production was independent of glycolysis. Furthermore, without the cocktail of chemicals and bFGF, which are indispensable for the generation of ciXEN cells, induced XEN (iXEN) cells remained the expression of XEN markers, the high proliferative capacity, and the plasticity to differentiate into iHeps in vitro. Conclusions: ciXEN cells had high plasticity, and energy metabolism was reconstructed during chemical reprogramming, but it did not change from aerobic oxidation to glycolysis. And the cocktail of chemicals and bFGF were non-essential for the in vitro culture of ciXEN cells.
  • ... In diesen initialen Experimenten wurde die Reprogrammierung von Fibroblasten durch Überexpression der 4 Transkriptionsfaktoren Oct3/4, Sox2, cMyc und Klf4 durchgeführt. Mittlerweile ist es gelungen, auch durch Überexpression anderer Kombinationen von Transkriptionsfaktoren (Han et al. 2010, Feng et al. 2009) oder allein durch chemische Zellkulturzusätze induzierte pluripotente Stammzellen (iPS) zu reprogrammieren (Hou et al. 2013). ...
    Durch direkte Reprogrammierung können differenzierte Fibroblasten in unterschiedlichste andere differenzierte Zelltypen wie Kardiomyozyten, Neurone und Hepatozyten konvertiert werden, ohne dass zuvor Pluripotenz induziert wird. Für renale Zellen ist dies erstmals 2016 gelungen: basierend auf den Arbeiten der Gruppen Lienkamp und Arnold wurden durch Überexpression der vier Transkriptionsfaktoren Pax8, Hnf1b, Emx2 und Hnf4a aus murinen embryonalen Fibroblasten induzierte renale tubuläre Epithelzellen (iRECs) generiert (Kaminski et al. 2016). In der vorliegenden Arbeit wurde untersucht, inwiefern die Generierung von iRECs methodisch in murinen und humanen Zellen optimiert werden kann. Zudem wurden die reprogrammierten Zellen auf Expressionsebene näher charakterisiert. Im ersten Teil der Arbeit wurden Methoden für die Steigerung der Effizienz der Reprogrammierung zu iRECs untersucht. Der Einsatz verschiedener niedermolekularer Wirksubstanzen als Zusatz im Zellkulturmedium und auch die Transduktion aller 4 Transkriptionsfaktoren auf einem polycistronischen Vektor führten zu keiner Steigerung der Effizienz. Dahingegen führte die Erhöhung der verwendeten Virustiter bei der Transduktion zu einer mehr als 3-fach höheren Ausbeute an reprogrammierten Zellen. Auch die Immortalisierung der Zellen mit dem Simian Virus 40 steigerte die Effizienz und Ausbeute um ein vielfaches. Zudem konnte gezeigt werde, dass auch postnatale murine Fibroblasten erfolgreich und mit vergleichbarer Effizienz wie fetale Fibroblasten reprogrammiert werden können. Im zweiten Teil der Arbeit wird die Identifikation reprogrammierter humaner Fibroblasten mittels eines CDH16 / GFP-Reporterplasmids beschrieben. Reprogrammierte humane iRECs, die anhand des Reporters identifiziert wurden, waren in ihrem globalen RNA-Expressionsprofil einer biologischen humanen Niere ähnlicher als humanen Fibroblasten. Nierentypische Gene waren in den humanen iRECs im Vergleich zu Fibroblasten hochreguliert. Diese Methoden zur Steigerung der Effizienz der Reprogrammierung und Identifikation reprogrammierter humaner Zellen sind die Grundlage für die Gewinnung qualitativ guter und ausreichender Mengen an Zellmaterial für in vitro Modelle renaler Krankheitsbilder. Diese könnten sowohl als Basis für mechanistische Untersuchungen von Krankheitsverläufen, der Identifizierung von Biomarkern als auch als Plattform für Wirksamkeits- und Toxizitätstests potentieller Therapeutika genutzt werden.
  • ... To identify the optimal conditions for pancreatic islet expansion in vitro, we reviewed the chemicals, proliferation and regeneration factors used in the culture of other organoids, such as those generated by pancreatic duct cells, cholangiocytes and hepatocytes 14,16,20,21 , and pancreatic islet organoids derived from stem cells or fibroblasts ( Fig. 1) [1][2][3][4] . According to their reported functions, these chemicals and factors are classified into the following groups: islet identity, cell division and proliferation, and cell renewal and regeneration (Fig. 1). ...
    Full-text available
    Tissue regeneration, such as pancreatic islet tissue propagation in vitro, could serve as a promising strategy for diabetes therapy and personalised drug testing. However, such a strategy has not been realised yet. Propagation could be divided into two steps, in vitro expansion and repeated passaging. Even the first step of the in vitro islet expansion has not been achieved to date. Here, we describe a method that enables the expansion of islet clusters isolated from pregnant mice or wild-type rats by employing a combination of specific regeneration factors and chemical compounds in vitro. The expanded islet clusters expressed insulin, glucagon and somatostatin, which are markers corresponding to pancreatic β cells, α cells and δ cells, respectively. These different types of cells grouped together, were spatially organised and functioned similarly to primary islets. Further mechanistic analysis revealed that forskolin in our recipe contributed to renewal and regeneration, whereas exendin-4 was essential for preserving islet cell identity. Our results provide a novel method for the in vitro expansion of islet clusters, which is an important step forward in developing future protocols and media used for islet tissue propagation in vitro. Such method is important for future regenerative diabetes therapies and personalised medicines using large amounts of pancreatic islets derived from the same person.
  • ... Recently, several methods relying fully or partially on drug treatment to enhance cell conversion have emerged 12 . Many of these use fibroblasts as the starting cell type, reprogrammed towards pluripotency 13,14 or trans-differentiated to specialized cell types including neurons 15 , endothelial cells 16 , pancreatic like cells 17 , cardiomyocytes 18 , hepatocytes 19 or other cell types 20,21,22 . Such studies provide a proof of principle for drug-based reprogramming, the exact mechanisms of which, however, are often poorly understood, making extensive trial-and-error unavoidable. ...
    Full-text available
    Controlling cell fate has great potential for regenerative medicine, drug discovery, and basic research. Although numerous transcription factors have been discovered that are able to promote cell reprogramming and trans-differentiation, methods based on their up-regulation tend to show low efficiency. The identification of small molecules that can facilitate conversion between cell types can ameliorate this problem working through safe, rapid, and reversible mechanisms. Here we present DECCODE, an unbiased computational method for the identification of such molecules solely based on transcriptional data. DECCODE matches the largest available collection of drug-induced profiles (the LINCS database) for drug treatments against the largest publicly available dataset of primary cell transcriptional profiles (FANTOM5), to identify drugs that either alone or in combination enhance cell reprogramming and cell conversion. Extensive in silico and in vitro validation of DECCODE in the context of human induced pluripotent stem cells (hIPSCs) generation shows that the method is able to prioritize drugs enhancing cell reprogramming. We also generated predictions for cell conversion with single drugs and drug combinations for 145 different cell types and made them available for further studies.
  • ... Furthermore, they are relatively cost effective, stable, easy to synthesize, and readily standardized. The small molecule controlled activation or inhibition of target proteins can be reversible and adapted through adjustments in concentration [28]. It is important, however, not to underestimate serious issues related to low differentiation efficiency, poor quality, and the maturity of the differentiated cells. ...
    Full-text available
    Diabetes is a metabolic disease which affects not only glucose metabolism but also lipid and protein metabolism. It encompasses two major types: type 1 and 2 diabetes. Despite the different etiologies of type 1 and 2 diabetes mellitus (T1DM and T2DM, respectively), the defining features of the two forms are insulin deficiency and resistance, respectively. Stem cell therapy is an efficient method for the treatment of diabetes, which can be achieved by differentiating pancreatic β-like cells. The consistent generation of glucose-responsive insulin releasing cells remains challenging. In this review article, we present basic concepts of pancreatic organogenesis, which intermittently provides a basis for engineering differentiation procedures, mainly based on the use of small molecules. Small molecules are more auspicious than any other growth factors, as they have unique, valuable properties like cell-permeability, as well as a nonimmunogenic nature; furthermore, they offer immense benefits in terms of generating efficient functional beta-like cells. We also summarize advances in the generation of stem cell-derived pancreatic cell lineages, especially endocrine β-like cells or islet organoids. The successful induction of stem cells depends on the quantity and quality of available stem cells and the efficient use of small molecules.
  • ... Mouse skeletal myofibroblast and human dermal fibroblasts were successfully reprogrammed into adipocytes by different chemicals [83,84]. Chemical reprogramming can also convert somatic cells into pluripotent stem cells [85]. These established direct reprogramming methods therefore allow us to study the potential role of nuclear β-actin in controlling the state of chromatin and, consequently, expression of gene programs during cell fate changes using fibroblasts from β-actin knockout embryos as models. ...
    Full-text available
    In the eukaryotic cell nucleus, cytoskeletal proteins are emerging as essential players in nuclear function. In particular, actin regulates chromatin as part of ATP-dependent chromatin remodeling complexes, it modulates transcription and it is incorporated into nascent ribonucleoprotein complexes, accompanying them from the site of transcription to polyribosomes. The nuclear actin pool is undistinguishable from the cytoplasmic one in terms of its ability to undergo polymerization and it has also been implicated in the dynamics of chromatin, regulating heterochromatin segregation at the nuclear lamina and maintaining heterochromatin levels in the nuclear interiors. One of the next frontiers is, therefore, to determine a possible involvement of nuclear actin in the functional architecture of the cell nucleus by regulating the hierarchical organization of chromatin and, thus, genome organization. Here, we discuss the repertoire of these potential actin functions and how they are likely to play a role in the context of cellular differentiation.
  • ... Furthermore, we are rapidly appreciating that nuclear reprogramming-like phenomena inducing the acquisition of epigenetic plasticity and phenotype malleability should be viewed as a fundamental element of a tissue's capacity to undergo successful repair, aging degeneration or malignant transformation [73][74][75][76][77]. Thus, chronic or unrestrained cell plasticity would drive aging phenotypes by impairing the repair or the replacement of damaged cells and such uncontrolled phenomena of in vivo reprogramming might also generate CSC-like cellular states [73][74][75][76][77]. Pharmacological tools selectively targeting the LSD1-SOX2 axis might be appropriate to experimentally uncouple the apparently counterintuitive capacity of LSD1 blockade to promote reprogramming phenomena by regulating the balance between pluripotency and differentiation [78][79][80][81][82][83] while preventing SOX2-driven cancer stemness. This would raise the possibility of pharmacologically managing, in the appropriate direction and intensity, the physiological versus pathological processes of SOX2-related reparative cellular reprogramming in aging and cancer. ...
    SOX2 is a core pluripotency-associated transcription factor causally related to cancer initiation, aggressiveness, and drug resistance by driving the self-renewal and seeding capacity of cancer stem cells (CSC). Here, we tested the ability of the clinically proven inhibitor of the lysine-specific demethylase 1 (LSD1/KDM1A) iadademstat (ORY-100) to target SOX2-driven CSC in breast cancer. Iadademstat blocked CSC-driven mammosphere formation in breast cancer cell lines that are dependent on SOX2 expression to maintain their CSC phenotype. Iadademstat prevented the activation of an LSD1-targeted stemness-specific SOX2 enhancer in CSC-enriched 3-dimensional spheroids. Using high-throughput transcriptional data available from the METABRIC dataset, high expression of SOX2 was significantly more common in luminal-B and HER2-enriched subtypes according to PAM50 classifier and in IntClust1 (high proliferating luminal-B) and IntClust 5 (luminal-B and HER2-amplified) according to integrative clustering. Iadademstat significantly reduced mammospheres formation by CSC-like cells from a multidrug-resistant luminal-B breast cancer patient-derived xenograft but not of those from a treatment-naïve luminal-A patient. Iadademstat reduced the expression of SOX2 in luminal-B but not in luminal-A mammospheres, likely indicating a selective targeting of SOX2-driven CSC. The therapeutic relevance of targeting SOX2-driven breast CSC suggests the potential clinical use of iadademstat as an epigenetic therapy in luminal-B and HER2-positive subtypes.
  • Chapter
    The development of reprogramming technology to generate human induced pluripotent stem cells (iPSCs) has tremendously influenced the field of regenerative medicine and clinical therapeutics where curative cell replacement therapies can be used in the treatment of devastating diseases such as Parkinson’s disease (PD) and diabetes. In order to commercialize these therapies to treat a large number of individuals, it is important to demonstrate the safety and efficacy of these therapies and ensure that the manufacturing process for iPSC-derived functional cells can be industrialized at an affordable cost. However, there are a number of manufacturing obstacles that need to be addressed in order to meet this vision. It is important to note that the manufacturing process for generation of iPSC-derived specialized cells is relatively long and fairly complex and requires differentiation of high-quality iPSCs into specialized cells in a controlled manner. In this chapter, we have summarized our efforts to address the main challenges present in the industrialization of iPSC-derived cell therapy products with focus on the development of a current Good Manufacturing Practice (cGMP)-compliant iPSC manufacturing process, a comprehensive iPSC characterization platform, long-term stability of cGMP compliant iPSCs, and innovative technologies to address some of the scale-up challenges in establishment of iPSC processing in 3D computer-controlled bioreactors.
  • Article
    Eight adenosine analogs, 3-deaza-adenosine (DZA), 3-deaza-(±)aristeromycin (DZAri), 2′,3′-dideoxy-adenosine (ddAdo), 2′,3′-dideoxy-3-deaza-adenosine (ddDZA), 2′,3′-dideoxy-3-deaza-(±)aristeromycin (ddDZAri), 3-deaza-5′-(±)noraristeromycin (DZNAri), 3-deaza-neplanocin A (DZNep), and neplanocin A (NepA), were tested as inhibitors of human placenta S-adenosylhomocysteine (AdoHcy) hydrolase. The order of potency for the inhibition of human placental AdoHcy hydrolase was: DZNep ≈ NepA >> DZAri ≈ DZNAri > DZA >> ddAdo ≈ ddDZA ≈ ddDZAri. These same analogs were examined for their anti-HIV-1 activities measured by the reduction in p24 antigen produced by 3′-azido-3′-deoxythymidine (AZT)-sensitive HIV-1 isolates, A012 and A018, in phytohemagglutinin-stimulated peripheral blood mononuclear (PBMCs) cells. Interestingly, DZNAri and the 2′,3′-dideoxy 3-deaza-nucleosides (ddAdo, ddDZAri, and ddDZA) were only marginal inhibitors of p24 antigen production in HIV-1 infected PBMC. DZNAri is unique because it is the only DZA analog with a deleted methylene group that precludes anabolic phosphorylation. In contrast, the other analogs were potent inhibitors of p24 antigen production by both HIV-1 isolates. Thus it was postulated that these nucleoside analogs could exert their antiviral effect via a combination of anabolically generated nucleotides (with the exception of DZNAri), which could inhibit reverse transcriptase or other viral enzymes, and the inhibition of viral or cellular methylation reactions. Additionally, QSAR-like models based on the molecular mechanics (MM) were developed to predict the order of potency of eight adenosine analogs for the inhibition of human AdoHcy hydrolase. In view of the potent antiviral activities of the DZA analogs, this approach provides a promising tool for designing and screening of more potent AdoHcy hydrolase inhibitors and antiviral agents.
  • Article
    The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here, we report that, during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes, whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a "seesaw model" in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming.
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    One of the hurdles for practical application of induced pluripotent stem cells (iPSC) is the low efficiency and slow process of reprogramming. Octamer-binding transcription factor 4 (Oct4) has been shown to be an essential regulator of embryonic stem cell (ESC) pluripotency and key to the reprogramming process. To identify small molecules that enhance reprogramming efficiency, we performed a cell-based high-throughput screening of chemical libraries. One of the compounds, termed Oct4-activating compound 1 (OAC1), was found to activate both Oct4 and Nanog promoter-driven luciferase reporter genes. Furthermore, when added to the reprogramming mixture along with the quartet reprogramming factors (Oct4, Sox2, c-Myc, and Klf4), OAC1 enhanced the iPSC reprogramming efficiency and accelerated the reprogramming process. Two structural analogs of OAC1 also activated Oct4 and Nanog promoters and enhanced iPSC formation. The iPSC colonies derived using the Oct4-activating compounds along with the quartet factors exhibited typical ESC morphology, gene-expression pattern, and developmental potential. OAC1 seems to enhance reprogramming efficiency in a unique manner, independent of either inhibition of the p53-p21 pathway or activation of the Wnt-β-catenin signaling. OAC1 increases transcription of the Oct4-Nanog-Sox2 triad and Tet1, a gene known to be involved in DNA demethylation.
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    The induction of pluripotent stem cells (iPSCs) by defined factors is poorly understood stepwise. Here, we show that histone H3 lysine 9 (H3K9) methylation is the primary epigenetic determinant for the intermediate pre-iPSC state, and its removal leads to fully reprogrammed iPSCs. We generated a panel of stable pre-iPSCs that exhibit pluripotent properties but do not activate the core pluripotency network, although they remain sensitive to vitamin C for conversion into iPSCs. Bone morphogenetic proteins (BMPs) were subsequently identified in serum as critical signaling molecules in arresting reprogramming at the pre-iPSC state. Mechanistically, we identified H3K9 methyltransferases as downstream targets of BMPs and showed that they function with their corresponding demethylases as the on/off switch for the pre-iPSC fate by regulating H3K9 methylation status at the core pluripotency loci. Our results not only establish pre-iPSCs as an epigenetically stable signpost along the reprogramming road map, but they also provide mechanistic insights into the epigenetic reprogramming of cell fate.
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    The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.
  • Article
    Stem cell technology holds great promises for the cures of devastating diseases, injuries, aging, and even cancers as it is applied in regenerative medicine. Recent breakthroughs in the development of induced pluripotent stem cell techniques and efficient differentiation strategies have generated tremendous enthusiasm and efforts to explore the therapeutic potential of stem cells. Small molecules, which target specific signaling pathways and/or proteins, have been demonstrated to be particularly valuable for manipulating cell fate, state, and function. Such small molecules not only are useful in generating desired cell types in vitro for various applications but also could be further developed as conventional therapeutics to stimulate patients' endogenous cells to repair and regenerate in vivo. Here, we focus on recent progress in the use of small molecules in stem cell biology and regenerative medicine.
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    A central feature of epimorphic regeneration during amphibian limb regeneration is cellular dedifferentiation. Two questions are discussed. First, what is the origin and nature of the soluble factors involved in triggering local cellular and tissue dedifferentiation? Secondly, what role does the key stem cell transcription factor Sall4 play in reprogramming gene expression during dedifferentiation? The pattern of Sall4 expression during Xenopus hindlimb regeneration is consistent with the hypothesis that Sall4 plays a role in dedifferentiation (reprogramming) and in maintaining limb blastema cells in an undifferentiated state. Sall4 is involved in maintenance of ESC pluripotency, is a major repressor of differentiation, plays a major role in reprogramming differentiated cells into iPSCs, and is a component of the stemness regulatory circuit of pluripotent ESCs and iPSCs. These functions suggest Sall4 as an excellent candidate to regulate reprogramming events that produce and maintain dedifferentiated blastema cells required for epimorphic regeneration.
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    Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.
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    The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.