Cell Cycle–Dependent Variation of a CD133 Epitope in Human
Embryonic Stem Cell, Colon Cancer, and Melanoma Cell Lines
Marie Jaksch, Jorge Mu ´nera, Ruchi Bajpai, Alexey Terskikh, and Robert G. Oshima
Tumor Development Program, Cancer Research Center, Burnham Institute for Medical Research, La Jolla, California
CD133 (Prominin1) is a pentaspan transmembrane glycopro-
tein expressed in several stem cell populations and cancers.
Reactivity with an antibody (AC133) to a glycoslyated form of
CD133 has been widely used for the enrichment of cells with
tumor-initiating activity in xenograph transplantation assays.
We have found by fluorescence-activated cell sorting that
increased AC133 reactivity in human embryonic stem cells,
colon cancer, and melanoma cells is correlated with increased
sorted on the basis of high and low AC133 reactivity results in a
with low AC133 reactivity can generate highly reactive cells as
they resume proliferation. The association of AC133 with
for tumor-initiating activity. [Cancer Res 2008;68(19):7882–6]
Tumors may be composed of a hierarchy of cells in which only a
subset is responsible for self renewal, while the remainder may not
be tumorgenic. Putative cancer stem cells (CSC) have been
identified in multiple types of human cancers by their ability to
initiate tumors in immune compromised mice (1). However, some
tumor cells that do not express CD133 are capable of self-renewal
and are tumorigenic (2–4), and not all human tumor cell lines that
are capable of generating tumors, at low cell numbers, are AC133
positive. Nevertheless, markers that allow enrichment for CSCs
from whole tumor tissues are essential for the purification,
characterization, and eventual targeting of CSCs. A very specific
antibody designated AC133 (5) against a glycosylated form of the
cell surface protein CD133 (Prominin1) has been widely used to
enrich for CSC (6). Reaction with the AC133 antibody (Miltenyi
Biotech) is not identical with CD133 protein detection but rather
seems to be due to a glycosylated form of membrane-associated
CD133 (6). The AC133 epitope is expressed on some human stem
and progenitor cells but is not present on mouse cells (6). Cells that
react with AC133 are reported to be more likely to form tumors in
transplantation tests than cells that are negative (7–10). AC133
reaction has been used to enrich for cells with tumor-initiating
activity from human brain tumors, colon cancers, and prostate
We have found that in culture, AC133 reactivity is correlated with
the cell cycle DNA profile of colon cancer, melanoma, and human
embryonic stem cells. In some cell types, differential AC133
expression may more accurately reflect cycling cells rather than a
differentially expressed stable stem cell lineage marker.
Materials and Methods
Cell culture. The human colon epithelial cancer cell line Caco2 was
obtained from American Type Culture Collection. Caco2 cells at passage 10
were infected with lentivirus reporter vectors that contain the mouse
maternal embryonic leucine zipper kinase (MELK) promoter driving
enhancer green fluorescent protein (MELK-GFP; ref. 11) or a control PGK
promoter-driven H2B-GFP vector. Individual clones were isolated, and two
of the clones were used for further experiments. Caco2 cells were cultured
as suggested by the supplier. Caco2 cells were cultured 2 d (subconfluent),
3 d (confluent), and 14 d (postconfluent) to generate differentiated cells.
The melanoma cell line WM115, provided by Boris Fichtman and Zeev
Ronai (Burnham Institute for Medical Research), was cultured in RPMI 1640
supplemented with 10% fetal bovine serum. The H9 hES cells were provided
by Brandon Nelson and Mark Mercola (Burnham Institute for Medical
Research). They were cultivated with mouse embryo fibroblast feeder cell
conditioned medium supplemented with basic fibroblast growth factor, as
described (12). The undifferentiated state of the hES cells was routinely
monitored by staining for Oct4 and other hES markers.
Immunocytochemistry. Single-cell suspensions were applied to glass
slides with a Shandon Cytospin 3 centrifuge at 500 rpm for 5 min. The cells
were fixed for 5 min in 2% paraformaldehyde (Sigma-Aldrich) at room
temperature, washed twice in PBS, and then blocked with 1.5% normal goat
serum for 1 h at room temperature. The cells were incubated with a 1:10
dilution of the primary antibody (anti–CD133-PE, AC133, Miltenyi Biotec) at
37jC for 1 h and subsequently washed twice with 0.1% Tween 20 (Sigma-
Aldrich) in PBS and twice with PBS. The primary antibody was detected
with Alexa 568–conjugated goat anti-mouse IgG 1:100 (Invitrogen), and
nuclei were stained with 4¶,6-diamidino-2-phenylindole. The stained cells
were mounted in Vectashield mounting medium (VECTOR).
Flow cytometry analysis. AC133 reaction was identified by direct
immunofluorescent staining using the AC133 mouse monoclonal antibody
directly conjugated with phycoerythrin. All cells were stained according to
manufacturer’s recommendations. In brief, 2 ? 105live cells were suspended
in 100 AL of buffer (0.5% FCS and 2 mmol/L EDTA) and stained for 10 min
at 4jC with 10 AL of the AC133 antibody (1:11). Cells were analyzed for
phycoerythrin and GFP expression by flow cytometry on a FACSort
cytometer (Becton Dickinson). Ten thousand events were acquired and
analyzed using FlowJo software.
Cell cycle analysis. DNA in MELK-GFP expressing Caco2 cells was
stained using Hoechst 33342 (Invitrogen), whereas WM115 and hES cells
were stained with Draq5 (Biostatus Ltd.). For the Hoechst 33342 staining,
2 ? 105to 3 ? 105cells, previously stained for AC133, were washed 1? in
wash buffer (0.5% FCS and 2 mmol/L EDTA in PBS). Cells were resuspended
in 250 AL of culture media (DMEM), and Hoechst 33342 was added to a final
concentration of 15 Ag/mL. Cells were incubated at 37jC for 90 min. For the
Draq5 staining, 1 ? 105cells, previously stained for AC133, were washed
1? in wash buffer. The cells were resuspended in DMEM and Draq5 at a
final concentration of 10 Amol/L. Cells stained with Hoechst 33342 were
analyzed on a FACSDiVa flow cytometer (Becton Dickinson), and cells
Note: Supplementary data for this article are available at Cancer Research Online
Current address: Ruchi Bajpai CCSR 3130, Chemical and Systems Biology, 269
Campus Drive, Stanford University, Stanford, CA 94305.
Requests for reprints: Robert G. Oshima, Burnham Institute for Medical Research,
10901 North Torrey Pines Road, La Jolla, CA 92035. Phone: 858-646-3147; Fax: 858-646-
3199; E-mail: email@example.com.
I2008 American Association for Cancer Research.
Cancer Res 2008; 68: (19). October 1, 2008
stained with Draq5 were analyzed on a FACSort cytometer (Becton
Dickinson). All fluorescence-activated cell sorting (FACS) data were
analyzed using FlowJo software.
FACSort. For sorting cells expressing AC133, the cells were removed
from the culture dish with 0.05% trypsin and 0.02% EDTA (Invitrogen),
washed in PBS containing 1% FCS, stained as described above, and
resuspended at 106cells/mL in the same buffer. The cells were filtered
through a 35-Am nylon filter before FACSort. Sorting was performed on a
FACSDiVa flow cytometer (Becton Dickinson). Side and forward scatter
profiles and propidium iodide staining were used to eliminate cell doublets
and dead cells. The top 10% of the AC133-reactive cells and the AC133-
negative cells were collected. An aliquot was removed at the end of the sort
and reanalyzed to evaluate purity.
Colony formation assay. Caco2 cells sorted on AC133 reactivity were
cultured in 24-well plates at concentrations of 100, 300, 1,000, and 5,000 cells
per well in triplicates. After 7 d, the cells were fixed in methanol, stained
with giemsa stain, and counted with a dissection microscope at 10?
Proliferation assay. AC133 high-sorted and negative-sorted cells were
plated at a concentration of 50, 500, and 5,000 cells per well in duplicate.
Cells were cultured for 1, 2, or 3 d. The cells were washed 2? in PBS before
they were frozen at ?70jC in the 96-well plate. The CyQUANT Cell
Proliferation Assay kit (Invitrogen) was used according to manufacturer’s
Microarray analysis. Total RNA from AC133 high-sorted and negative-
sorted Caco2 and WM115 cells was extracted using the TRI Reagent
(Molecular Research Center, Inc.) according to the manufacturer’s protocol.
Two samples each of two cell lines were analyzed as biological duplicates.
Labeled cRNA was prepared from 500 ng RNA using the Illumina RNA
Amplification kit from Ambion. The biotin-labeled cRNA (750 ng) was
hybridized 18 h at 58jC to the HumanRef-8 v2 Expression BeadChip
(>22,000 gene transcripts; Illumina) according to the manufacturer’s
instructions. BeadChips were scanned with an Illumina BeadArray Reader,
and hybridization efficiency was monitored using BeadStudio software
(Illumina). BeadStudio software was used for the normalization and quality
control of the data. To identify statistically significant changes, the data
were evaluated by GeneSpring software. A volcano plot was used to identify
genes with changed at least 2-fold and had reproducibility P values of 0.05
or less. The list of genes passing these thresholds was compared with
publicly available data using the Nextbio search engine. Complete primary
data are available through the Gene Expression Omnibus support by the
National Center for Biotechnology Information as GEO accession number
Results and Discussion
AC133 expression in Caco2 and hES cells. A screen of seven
human cell lines for AC133 expression revealed three that were
positive. We did not detect reaction with MDA-MB231, MCF7,
Du145, or U87 cells. FACS analysis and immunofluorescence
staining of Caco2 and H9 hES cells detected high levels of the
AC133 epitope (Fig. 1). Ninety-four percent and 70% of Caco2 and
hES cells, respectively, showed positive staining. These results
confirm previous reports of AC133 reactivity on Caco2 (13) and hES
cells (14). The human melanoma cell line WM115 was also positive
(15). Interestingly, immunoreactivity for the AC133 antigen, but not
CD133 mRNA level, is reported to be down-regulated upon
differentiation of Caco2 cells for 40 days (13). In our study we
did not see a significant difference in AC133 expression in
subconfluent, confluent, and 14-day postconfluent cells (Fig. 1C).
AC133 expression and DNA profile in subpopulations. FACS
analysis of the DNA contents of AC133-reactive cells revealed that
cells with greatest AC133 reaction were enriched for cells with DNA
content of 4N or even greater, in the case of hES cells (Fig. 2). When
equal subpopulations of AC133+cells (Fig. 2A) were analyzed for
DNA content, the fraction of cells with 4N DNA content or greater
was found to correlate with increasing AC133 reactivity, whereas
the fraction of cells with 2N DNA (G1and G0portion of the cell
cycle) was inversely correlated with AC133 reaction (Fig. 2B). In
cells with the least AC133 binding, almost 70% of the cells
contained 2N DNA content compared with only 20% of the cells
with highest levels of AC133 (Fig. 2C, left). Similar results were
found for subcloned populations of Caco2 cells. Eighty percent of
the cells that expressed the lowest levels of AC133 showed a DNA
profile similar to cells with 2N DNA content compared with only
14% of the cells expressing the highest levels of AC133. These
results were also confirmed by the AC133 expression in the
melanoma cell line WM115. Figure 2D summarizes the results from
all three cell lines. Cell cycle profiles on cells gated on the highest
10% and the lowest 10% of AC133 expression are shown.
Expression of MELK protein is cell cycle–dependent (16). We
compared the expression of a MELK-GFP reporter gene with its
corresponding DNA content profile. The expression of the GFP
protein from a MELK-GFP reporter gene in cloned Caco2 cells has
the same correlation with DNA content as the AC133 antigen
(Fig. 2C, right). These data are consistent with previous reports that
Figure 1. AC133 expression in Caco2 and hES cells. A, immunofluorescent
analysis of AC133 staining in Caco2 colon cancer cells and cytospin preparation
of H9 hES cells (40? magnification). Scale bar, 100 Am. B, flow cytometry
analysis of AC133 staining on live cells (gray, negative controls). C, AC133
expression on confluent, subconfluent, and postconfluent Caco2 cells.
Cell Cycle–Dependent Variation of CD133 Epitope
Cancer Res 2008; 68: (19). October 1, 2008
CD133+cells have a much higher expression of MELK mRNA
compared with autologous CD133-negative tumor cells from
glioblastoma patients. Grskovic and colleagues documented that
CD133+cells are more mitotically active than CD133?cells after
the first week of cultivation (17, 18).
Gene expression in AC133 high-sorted and negative-sorted
cells. The gene expression profiles for AC133 high versus negative-
sorted Caco2 and WM115 cells were compared. Figure 3A and B
shows scatter plots from the obtained microarray data with a fold
difference of 2 or more. The r2values (r2= 0.9859 and r2= 0.9842)
indicate high similarity in gene expression between cells sorted on
extremes of AC133. An unsupervised cluster analysis of individual-
gene expression data sets did not distinguish AC133-reactive cells
(Fig. 3C). Noteworthy, the most striking difference was the up-
regulated expression of Prominin1 in AC133 high expressing cells,
15? and 9? for Caco2 and WM115, respectively. This was the only
gene that was differentially expressed in both cell lines. This
indicates that AC133 reaction correlates well with Prominin1 RNA.
A total of only 39 Caco2 RNAs and 7 WM115 RNAs (Supplementary
Table S1) were significantly different (z2-fold and reproducibility;
P < 0.05) between AC133 high and negative cell fractions.
Cultivation of high-sorted and negative-sorted cells. To
determine the stability of AC133 antigen expression, high and
negative AC133 sorted Caco2 cells were cultivated. A colony
formation assay showed a significant difference in colony formation
frequency between the two sorted cell populations (P < 0.05;
Fig. 4A). However, although the AC133-negative cells were less likely
to grow from single cells, the proliferation rate for the two cell
populations were not significantly different (P > 0.05; Fig. 4B). After
a few passages, AC133-negative cells expressed similar levels of
AC133 as the starting population (Fig. 4C). Similarly, cells sorted on
high expression of AC133 generated cells with less expression
(Fig. 4C). Hence, continued culturing of cells with extremes of
AC133 reactivity leads to redistribution of the degree of reaction.
Also, the morphology of the colonies formed after cultivation is very
similar between the two different subpopulations (Fig. 4D). Chang
and colleagues (19) have recently showed similar results with
another stem cell marker, Sca-1. In clonal populations of mouse
hematopoietic progenitor cells, they found that spontaneous
‘‘outlier’’ cells with either extremely high or low expression levels
of Sca-1 reconstitute the parental distribution of Sca-1 after 1 week.
These extremes of Sca-1 expression were associated with differen-
tial gene expression consistent with increased probability of
differentiation along two different lineages. However, in our study,
the high and negative expressing sorted cells do not seem to belong
to two distinct cell populations based on gene expression profiles.
The AC133 epitope seems not to be a stable marker for a particular
population of cells but rather may reflect mitotic activity. Cell cycle
dependence might be one of the metastable variables contributing
to transcriptome variation (15).
The cell cycle–associated reactivity of AC133 is similar to
the cell cycle–dependent expression of CD34, a hematopoietic
Figure 2. Correlation of AC133 reaction and DNA content. A, the AC133-reactive populations of hES cells were divided into five groups (1–5), each representing
20% of the total population. B, DNA contents for the individual fractions of AC133-reactive cells. Graph 6 shows the cell cycle profile for all AC133-positive cells.
C, graphs show the percentage of cells with 2N DNA content and cells with 4N DNA content for each subpopulation of cells with AC133 reactivity and cells with
MELK-GFP expression. D, DNA content profiles for cells gated on the highest 10% (black line, no fill) and the lowest 10% (gray fill) of AC133 expression in three
different cell lines, hES cells, melanoma cells WM115, and colon cancer Caco2 cells.
Cancer Res 2008; 68: (19). October 1, 2008
progenitor marker that is also commonly detected by a
glycosylation-dependent epitope. CD34 expression has been a
valuable tool for identification and purification of human hemato-
poietic stem cells. However, CD34 expression in mice is not required
for survival, and murine hematopoiesis could be reconstituted by
CD34?cells (20). Recently, Dooley and colleagues (21) showed that
CD34 expression increased as CD34?cells shifted from quiescence
to proliferation. Cultured CD34?cells up-regulate CD34 antigen
expression in as little as 42 hours, and CD34+precursors lost
expression in culture if they remained in G0for >2 days.
Tumor-initiating cells with stem-like characteristics might have
differential resistance to chemotherapy or radiotherapy. CD133+
glioblastoma cells are significantly more resistant to conventional
chemotherapeutic agents, and resistance is correlated with higher
expression of survivin, an antiapoptotic acting protein in CD133+
cells (18, 22). However, survivin expression is cell cycle–dependent,
increasing in the G2-M phase of the cell cycle followed by a rapid
Figure 3. Gene expression in AC133 high versus negative cells. A and
B, scatter plots of AC133 high versus negative cells for Caco2 and WM115 cells,
respectively. Fold changes of z2 are indicated above and below the lines
parallel to the diagonal. Not all 2? changes are statistically significant. C, cluster
analysis of microarray data. Gene expression in AC133 high-sorted and
negative-sorted Caco2 (sample A-D) and WM115 (sample E-H). The Pearson
correlation using a 1 ? r distance measure was applied.
Figure 4. Colony formation, proliferation, and AC133 expression in AC133
high-sorted and negative-sorted cells. A, colony formation of AC133 high-sorted
and negative-sorted Caco2 cells [colony forming units (cfu)/seeded cells].
B, proliferation rate in AC133 high-sorted and negative-sorted Caco2 cells.
C, AC133 expression in sorted Caco2 cells before and after cultivation of
negative-sorted and high-sorted cells, respectively. Gray fill, AC133 expression
of the sorted cells; black lines, AC133 expression after three passages of
cultivation. D, morphology of AC133 high-sorted and negative-sorted Caco2
cells after 7 d of cultivation (20? magnification). Scale bar, 100 Am.
Cell Cycle–Dependent Variation of CD133 Epitope
Cancer Res 2008; 68: (19). October 1, 2008
decline in the G1phase. The selection of AC133-reactive cells in this Download full-text
system might also be expected to enrich for cells with high survivin
expression and, thus, increased resistance to apoptotic agents.
Primary human cancers are commonly heterogeneous with both
host and tumor-related components. The proportion of actively
proliferating cancer cells varies greatly, depending on the tumor
and its progression. The general strategy of targeting proliferative
cells of cancers is being challenged by the CSC model that may
include extrapolations from the behavior of slow cycling normal
stem cells. However, the essential defining characteristic of a CSC,
supported by the biology of teratocarcinoma and certain
leukemias, is the directional, moderating influence of differentia-
tion to a stable benign state. AC133 reactivity may be used for
enrichment for tumor-initiating activity without necessarily
supporting a CSC theory.
To summarize, our study indicates that within three different cell
types that express AC133, the antigen expression is highest in cells
with 4N DNA content and lowest in cells with 2N DNA. This is
consistent with higher expression in actively proliferating cells and
low expression in cells in G1or G0. We show that cultivation of cells
with the extremes of AC133 reactivity resulted in a redistribution
of the antigen expression, which suggests that high and low
expressing cells do not belong to stable distinct populations. AC133
reactivity may be valuable to identify cells with increased tumor-
genicity. However, the basis of this utility may be due to the
distinction between proliferative and quiescent cells and should be
used cautiously as a putative marker of a stable, distinct stem cell–
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Received 2/28/2008; revised 7/11/2008; accepted 7/29/2008.
Grant support: California Institute for Regenerative Medicine grant RS1-00283-1.
M. Jaksch and R. Bajpai were both supported by California Institute for Regenerative
Medicine postdoctoral training grant T2-00004. J. Mu ´nera was supported by a research
supplement to promote diversity in health-related research from National Cancer
Institute grant PO1 CA102583.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Yoav Altman of Burnham Cell Analysis Shared Resource for expert
technical help, Roy Williams of Burnham Bioinformatics Shared Resource for help
with the evaluation of the microarray data, Brandon Nelson and Mark Mercola for hES
cells, and Boris Fichtman and Zeev Ronai for WM115 cells.
Cancer Res 2008; 68: (19). October 1, 2008
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