First published online February 8, 2008
doi: 10.1242/10.1242/dev.017400
Development 135, 909-918 (2008)
Published by The Company of Biologists 2008
Identification and characterization of subpopulations in undifferentiated ES cell culture
Yayoi Toyooka1,2,*,
Daisuke Shimosato1,3,
Kazuhiro Murakami1,
Kadue Takahashi1 and
Hitoshi Niwa1,3,*
1 Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental
Biology (CDB), 2-2-3 Minatojima-minamimachi, Chu-o-ku, Kobe, Hyogo 650-0047,
Japan.
2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road,
Oxford OX1 3RE, UK.
3 Laboratory for Development and Regenerative Medicine, Kobe University
Graduate, School of Medicine, 7-5-1 Kusunokicho, Chu-o-ku, Kobe, Hyogo
650-0017, Japan.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1. Structure of knock-in vectors for Oct3/4 and Rex1.
Structure of knock-in vector for Oct3/4 constructed to replace the
coding region of the mouse Oct3/4 gene with Oct3/4- ECFP (or
YFP) fusion gene and IRES-puromycin resistance gene to express Oct3/4-ECFP (or
YFP) fusion protein from the recombinant allele, and structure of knock-in
vector for Rex1 for insertion of an EGFP (or herpes simplex virus 2
thymidine kinase gene) and IRES-blasticidin resistance gene into exon 4 of the
mouse Rex1 gene. Black boxes represent coding regions. Southern
blotting analyses of the Oct3/4 locus and the Rex1 locus are
shown at the bottom. Genomic DNA was digested with EcoRI for analyses
of the Oct3/4 locus; a 300 bp probe from intron 5 produced an 11.6 kb
band from the wild-type locus and a 10.3 kb band from the targeted locus. For
analyses of the Rex1 locus, genomic DNA was digested with
EcoRI, and hybridization with a 300 bp probe produced a 7.9 kb band
from the wild-type locus and a 5.7 kb (5.9 kb for tk2 vector) band from the
targeted locus. BSD, blasticidin S deaminase; E, EcoRI; P, positions
of probes; Puro, puromycin-N-acetyltransferase; S, SpeI; tk2, herpes
simplex virus 2 thymidine kinase (HSVtk2).
|
|
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2. Observation of Rex1-GFP/Oct3/4-CFP double knock-in ES
cells by microscopy and flow cytometry. (A-H) Morphology and
fluorescence of colonies of
Oct3/4+/Rex1+ and
Oct3/4+/Rex1- cells in culture of
Rex1-GFP/Oct3/4+-CFP double-knock-in ES cell line under
puromycin selection (selection for cells expressing Oct3/4). (A,C,F)
GFP fluorescence, (D,G) CFP fluorescence and (B,E,H) bright field. (A,B) Low
magnification of knock-in ES cells. A small number of GFP
(Rex1)-negative cells was observed. Arrows in B indicate colonies of
Rex1-/Oct3/4+ (GFP-) cells. (C,D,E)
High magnification of colonies in which Rex1+ cells were
dominant. (F,G,H) High magnification of colonies in which
Rex1- cells were dominant. (I,J) Result of analysis of
knock-in cells by flow cytometry. (I) Comparison of GFP fluorescence of
Rex1-knock in cells with the parental cell line (OLC2-1). (J)
GFP fluorescence of Rex1 knock-in cells disappeared within 3 days
when differentiated by withdrawal of LIF. (K) Rex1 knock-in
cells were stained with anti-PECAM-1 monoclonal antibody conjugated with PE
and analyzed by flow cytometry. Most GFP-positive cells reacted with PECAM-1
antibodies. Scale bars: 50 µm.
|
|
View larger version (63K):
[in this window]
[in a new window]
|
Fig. 3. Expression patterns of genes associated with early development in mouse
Rex1+/- populations. (A) Ratio of expression
levels of early genes in Rex1+ and
Rex1- populations were examined by Q-PCR. The averages of
values from RNA of Rex1+ and Rex1-
cells sorted three times independently are shown in the graph. (B)
Immunostaining of Rex1-EGFP/Oct3/4-ECFP double-knock-in cells with antibodies
for proteins expressing in the peri-implantation embryos. Fluorescence of
antibody reaction is shown as red signal (Alexa 594) for Nanog, Klf4 and Tbx3,
or purple signal (Alexa 647) for Esrrb and Foxd3.
|
|
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 4. Reversible phenotypes of mouse Rex1+ and
Rex1- populations. (A)
Rex1+/Oct3/4+ and
Rex1-/Oct3/4+ fractions were sorted,
cultured and analyzed by flow cytometry. Oct3/4-CFP/Rex1-GFP
double-knock-in ES cells were cultured under puromycin selection, then
Rex1+ and Rex1- populations were
purified at more than 98% purity. Rex1- cells emerged from
the purified Rex1+ population, and
Rex1+ cells also appeared from purified
Rex1- populations within 1-2 days. It was confirmed that
GFP fluorescence reflected Rex1 expression by RT-PCR. (B)
Clonal analysis of reversibility of Rex1+ and
Rex1- cells. Scale bar: 50 µm.
|
|
View larger version (71K):
[in this window]
[in a new window]
|
Fig. 5. Blastocyst injection was performed with mouse Rex1+
and Rex1- ES cell populations to compare their capacities
for contributing embryonic tissue. (A-D) Rex1/Oct3/4
double-knock-in ES cells were marked by forced expression of the DsRedT4 gene
driven by the CAG promoter, and EGFP+ and EGFP- cells
were purified after culture under puromycin selection. Some of the 10.5 dpc
embryos derived from blastocysts injected with the Rex1+
fraction showed DsRed fluorescence. The embryo on the left has cells showing
strong fluorescence. Embryos injected with the Rex1-
fraction did not show DsRed fluorescence.
|
|
View larger version (67K):
[in this window]
[in a new window]
|
Fig. 6. Examination of mouse ES cells for differentiation ability of
Rex1-/Oct3/4+ cells selected with
thymidine kinase gene/gancyclovir system into primitive endoderm lineage.
(A) Thymidine kinase gene knock-in ES cell lines under selection for
puromycin and gancyclovir (GANC). (Left) Cells selected with puromycin
(selection for Oct3/4+ cells); (right) cells selected with
both puromycin and GANC (selection for Oct3/4+ and
exclusion of Rex1+ cells). (B) Expression of marker
genes was examined by RT-PCR in puromycin and GANC/puromycin double-selected
fractions. (C) Expression of primitive endoderm marker genes in
differentiated Rex1+, Rex1- and `reverted'
Rex1+ cells by withdrawal of LIF. Before starting
induction of differentiation, Rex1+ cells were selected
with blasticidin S whereas Rex1- cells were selected with
GANC and puromycin for 1 week. Reverted Rex1+ cells were
derived from Rex1- cells re-seeded and cultured without
GANC for 3 days, and then cultured with blasticidin S for 4 days. Scale bar:
100 µm.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7. Differentiation ability of
Rex1-/Oct3/4+ cells into mesodermal
and neural lineages. (A) Dot-plots of analysis at day 0 and day 4
for mesodermal induction from unselected Rex1-tk2 knock-in ES cells.
Percentage of Flk1+/E-cadherin- cells (indicted by
rectangles) was measured by analyzer. (B) The results of induction of
mesoderm from Rex1+/Oct3/4+ and
Rex1-/Oct3/4+ populations selected
with GANC and puromycin. Note that
Rex1-/Oct3/4+ (PrE-like cells)
differentiated into mesodermal cells more efficiently and with faster kinetics
than non-fractionated ES cells. (C) Analysis of induction of neural
cells (dot-plot on day 4). Anti-NCAM antibody and Alexa-633-conjugated
anti-goat IgG antibody were used as first and second antibodies, respectively,
for measurement of the proportion of neural cells. Green line, control ES
cells stained with only secondary antibody; blue line, fraction of
Oct3/4+ cells on day 5; red line, enriched
Rex1-/Oct3/4+ fraction on day 5.
(D) Summary of experiment for induction of neural cells on days 2, 4
and 5.
|
|
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 8. Morphology and results of flow cytometric analysis of mouse
Rex1/Oct3/4 double-knock-in ES cells cultured in different serum-free
media under puromycin selection (selection for Oct3/4
expression). (A) Morphology and GFP fluorescence of cells cultured
in GMEM supplemented with KSR, ACTH and LIF (above) and those cultured in
N2B27 medium supplemented with Bmp4 and LIF (below). (B) Flow
cytometric analysis of cells cultured in GMEM+FCS, +LIF (left), in GMEM+KSR,
+ACTH, +LIF (center) and in N2B27 medium +Bmp4, +LIF (right). Scale bar: 100
µm.
|
|
View larger version (9K):
[in this window]
[in a new window]
|
Fig. 9. Illustration showing the expression patterns of mouse marker genes
expressed in the pluripotent cell population at each embryonic stage (upper),
and the subpopulation in ES cells (lower) that could be detected by expression
of these marker genes. ICM-like (Rex1+) and early
PrE-like (Rex1-) populations have different abilities for
in vitro differentiation and could convert into each other under normal
conditions for ES cell maintenance.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2008
