Mir-302 reprograms human skin cancer cells
into a pluripotent ES-cell-like state
SHI-LUNG LIN,1 DONALD C. CHANG,1 SAMANTHA CHANG-LIN,1 CHUN-HUNG LIN,2 DAVID T.S. WU,3
DAVID T. CHEN,3 and SHAO-YAO YING1
1Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA
2Dental Department, Taiwan Adventist Hospital, Taipei City 10556, Taiwan, Republic of China
3Department of Ear, Nose and Throat, Tzu Chi General Hospital, Hualien 970, Taiwan, Republic of China
ABSTRACT
Renewal of stem cells differs from cancer cell growth in self-controlled cell division. The mir-302 microRNA (miRNA) family
(mir-302s) is expressed most abundantly in slow-growing human embryonic stem (ES) cells, and quickly decreases after cell
differentiation and proliferation. Therefore, mir-302s was investigated as one of the key factors essential for maintenance of ES
cell renewal and pluripotency in this study. The Pol-II-based intronic miRNA expression system was used to transgenically
transfect the mir-302s into several human cancer cell lines. The mir-302 – transfected cells, namely, miRNA-induced pluripotent
stem (mirPS) cells, not only expressed many key ES cell markers, such as Oct3/4, SSEA-3, SSEA-4 ,Sox2, and Nanog, but also had
a highly demethylated genome similar to a reprogrammed zygotic genome. Microarray analyses further revealed that genome-
wide gene expression patterns between the mirPS and human ES H1 and H9 cells shared over 86% similarity. Using molecular
guidance in vitro, these mirPS cells could differentiate into distinct tissue cell types, such as neuron-, chondrocyte-, fibroblast-,
and spermatogonia-like primordial cells. Based on these findings, we conclude that mir-302s not only function to reprogram
cancer cells into an ES-like pluripotent state but also to maintain this state under a feeder-free cultural condition, which may
offer a great opportunity for therapeutic intervention.
Keywords: miRNA; mir-302; embryonic stem cell; induced pluripotent stem cell; stem cell generation; renewal; pluripotency;
embryoid body; feeder-free cell culture; epigenetic reprogramming; cell differentiation
INTRODUCTION
The concept of cancer stem cells indicates that transformed
stem cells within a tumor are able to self-renew and
differentiate into a heterogeneous tumor population (Reya
et al. 2001). However, there is no clear mechanism under-
lying such stem cell–cancer cell transformation or vice
versa. In the clinic, it is very frequently observed that cancer
progression is generally associated with poor differentiation
(high grade) of human tumor cells. Recent findings have
also shown that poorly differentiated tumors preferentially
overexpress genes normally enriched in human embryonic
stem (ES) cells, such as targets of Oct3/4, Sox2, and Nanog
transcription factors. Nevertheless, the concurrent expres-
sion of these transcription factors themselves is not often
detected in the poorly differentiated tumors (Ben-Porath
et al. 2008). It is conceivable that a different set of transcrip-
tional regulators in place of Oct3/4, Sox2, and Nanog may
function in poorly differentiated tumor cells to promote
their ‘‘stemness’’ signatures. Therefore, finding the way by
which stem cells substitute for these cancer-related tran-
scriptional regulators may lead to breakthroughs in both can-
cer therapy and stem cell generation. To this end, we report
here that the present study of mir-302 function provides the
first insight into the mechanism underlying reverse trans-
formation of human cancer cells into ES-like pluripotent cells.
The mir-302 family (mir-302s) consists of four highly
homologous microRNA (miRNA) members, which are
rna11627 Lin et al. ARTICLE RA
Reprint requests to: Shi-Lung Lin, Department of Cell and Neurobi-
ology, Keck School of Medicine, BMT-403, University of Southern
California, 1333 San Pablo Street, Los Angeles, CA 90033; e-mail: lins@
usc.edu; fax: (323) 442-3466; or Shao-Yao Ying, Department of Cell and
Neurobiology, Keck School of Medicine, BMT-403, University of South-
ern California, 1333 San Pablo Street, Los Angeles, CA 90033; e-mail:
sying@usc.edu; fax: (323) 442-3466.
Abbreviations: miRNA, microRNA; mir-302s, the mir-302 family; ES,
embryonic stem; iPS, induced pluripotent stem; mirPS, mir-302-induced
pluripotent stem; EB, embryoid body; CSC, cancerous stem cell; Pol-II,
RNA polymerase type-II; NMD, nonsense-mediated decay.
Article published online ahead of print. Article and publication date are
at http://www.rnajournal.org/cgi/doi/10.1261/rna.1162708.
RNA (2008), 14:2115–2124. Published by Cold Spring Harbor Laboratory Press. Copyright  2008 RNA Society. 2115
JOBNAME: RNA 14#10 2008 PAGE: 1 OUTPUT: Tuesday September 9 16:23:01 2008
csh/RNA/170255/rna11627

transcribed together as a noncoding RNA cluster contain-
ing mir-302b, mir-302c, mir-302a, mir-302d, and mir-367
in a 59-to-39 direction (Suh et al. 2004). They are expressed
most abundantly in slow-growing human ES cells and
quickly decrease after cell differentiation and proliferation
(Suh et al. 2004). Given that miRNAs are characterized as
small inhibitory RNAs capable of suppressing the trans-
lation of target genes with high complementarity (Bartel
2004), mir-302s is a likely candidate zygotic inhibitor of
premature cell differentiation during early embryonic
development. As shown in the miRBaseTSequences pro-
gram (http://microrna.sanger.ac.uk/sequences/), mir-302s
can target over 445 human genes, and most of these targets
are developmental signals involving the initiation and/or
facilitation of lineage-specific cell differentiation during early
human embryogenesis. These target genes are listed in the
target prediction sites linked to the miRBaseTSequences
program at the Sanger website, including TARGETSCAN
(http://www.targetscan.org/vert_42/) and PICTAR-VERT
(http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi?). Thus,
we hypothesize that mir-302s are key factors essential for
ES cell maintenance, possibly able to reprogram cancer cells
into a more ES-cell-like state.
To test the function of mir-302s, we developed a
retroviral Pol-II-based intronic miRNA expression system,
namely, pLNCX2-rT-SpRNAi (Fig. 1), and successfully
used it to generate several transgenic miRNA-expressing
cell lines and animals (Lin and Ying 2006; Lin et al. 2006).
The same transgenic approach has also been used to
generate gene-knockout mice for human disease research
(Xia et al. 2006). Intronic miRNA expression is a prevalent
event in mammals because z50% of mammalian miRNAs
are encoded within the introns of protein-coding genes
(Rodriguez et al. 2004). These miRNAs are transcribed by
type-II RNA polymerases (Pol-II) and excised by spliceo-
somes and other RNase III endonucleases to form mature
miRNAs (Danin-Kreiselman et al. 2003; Lin et al. 2003).
However, Drosha may not be required for this process
(Ruby et al. 2007). The composition of this mir-302-
expressing pLNCX2-rT-SpRNAi vector is shown in Figure
2A. Using this vector-based transfection strategy, we have
generated two mir-302-expressing mirPS cell lines, namely,
mirPS-Colo and mirPS-PC3, derived from human mela-
noma Colo and prostate cancer PC3 cells, respectively, and
confirmed that mirPS-Colo cells highly resembled human
ES H1 and H9 cells in stem cell renewal and pluripotency.
RESULTS
Generation of human ES-like mir-302-induced
pluripotent stem (mirPS) cell lines
and embryoid bodies
After the pLNCX2-rT-SpRNAi retroviral transfection with a
predesigned mir-302 pre-miRNA cluster transgene (Fig.
2B), z95%–98% of the transfected cells underwent apo-
ptosis with the remaining 2%–5% of the cells transformed
into ES-like mirPS cells. The transfection rates of mir-302s
into Colo and PC3 cells were 99.8% and 99.4%, respec-
tively, as determined by FACS flow cytometry sorting with
mir-302 maker RGFP and ES marker Oct3/4 antibodies
(Fig. 2C). These mirPS cells could grow in either DMEM/
F12 or RPMI 1640/B27 medium supplemented with 10%
charcoal-stripped FBS, 4 mM L-glutamine, 1 mM sodium
pyruvate, 5 ng/mL activin, 5 ng/mL noggin, 3 ng/mL bFGF,
and an equal mixture of 0.5 mM Y-27632 and 0.5 mM GSK-
3 inhibitor XV, at 37°C under 5% CO2. Under this feeder-
free cultural condition, the average cell cycle of the mirPS
cells was z20–24 h, indicating a very slow cell renewal rate
compared with their cancerous counterparts (z4–6 h per
cell cycle).
Flow cytometry analysis comparing DNA content to cell
cycle stages showed a greater than 67% reduction in the
mirPS mitotic cell population (Fig. 2D). The mitotic cell
population (M phase) was decreased from 36.5% to 11.5%
in mirPS-Colo and from 38.4% to 12.6% in mirPS-PC3
cells, whereas no change was found in the control cells
transfected with either an empty pLNCX2-rT-SpRNAi vec-
tor (cell+vector) or a vector encoding an off-target mir-gfp
pre-miRNA construct (cell+mir-gfp). However, transfec-
tion of a vector encoding mutated mir-302s abolished the
effects of mir-302s on cell cycle suppression and Oct3/4
gene activation (Supplemental Fig. 1). The mutated mir-
302s were formed by replacing the first eight nucleotides of
the mir-302 seed sequence (UAAGUGCU) with AUUAA
UUA in all four isoforms. Accordingly, the mirPS cell
morphology (lower panels) was changed from a spindle- or
asterisk-like form to a more rounded shape, indicating that
the mirPS cells may have lost their ability to migrate. As
shown in Figure 2E, metastatic PC3 cells quickly migrated
over time, whereas mirPS-PC3 cells remained stationary.
No morphological change was observed in all other con-
trols. Thus, such transgenic mir-302s expression is suffi-
cient to transform human cancer cells into a more ES-like
cell morphology and rate of cell division, suggesting a very
beneficial use in cancer therapy.
MirPS cells were able to form compact colonies remi-
niscent of embryoid bodies (EBs) derived from human ES
cells (Fig. 2F; Supplemental Fig. 2). When dissociated with
collagenase IV and then cultivated in RPMI 1640 medium
supplemented with 10% FBS, but without GSK and ROCK/
Ras inhibitors, many of these EB-like cells differentiated
into neuronal cells based on the presence of positive
neuronal markers Tuj1 and ABCA2. We further noted that
mirPS-PC3 EB cells could only differentiate into neuronal
cell types, while mirPS-Colo EB cells formed teratoma-like
primordial tissue cysts in immunocompromised SCID-
beige mice (Fig. 3), suggesting that different cancerous
stem cells may have different pluripotent potentials. In view
of the broad pluripotency in mirPS-Colo cells, we therefore
Lin et al.
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