close

Вход

Забыли?

вход по аккаунту

?

Oligopotent Mesenchymal Stem Cell-Like Clone Becomes Multinucleated Following Phorbol Ester TPA Stimulation.

код для вставкиСкачать
THE ANATOMICAL RECORD 120:1256–1267 (2007)
Oligopotent Mesenchymal Stem
Cell-Like Clone Becomes Multinucleated
Following Phorbol Ester,
TPA Stimulation
KEIICHIRO YOSHIDA,* MICHIO ONO, TATSUO MAEJIMA,
MICHIYO ESAKI, AND HAJIME SAWADA
Department of Histology and Cell Biology, Yokohama City University School of Medicine,
Kanazawa-ku, Yokohama, Japan
ABSTRACT
We established a mesenchymal stem cell clone, 5F9A, from rat bone
marrow substrate adherent cells by repeated limiting dilutions. The cells
have a fibroblastic shape and form intimate contacts with adjacent cells
with interdigitations and junctions similar to adherence and tight junctions in a semi-confluent culture. Analysis of the phenotypes of these cells
by RT-PCR and FACS demonstrated that they resembled mesenchymal
stem cells, and the cells could differentiate into adiopocytes and osteoblasts under appropriate conditions in vitro showing their oligopotency.
Furthermore, the cells were induced to become multinuclear cells by TPA
(12-o-tetradecanoylphorbol 13-acetate) stimulation. Anat Rec, 290:1256–
1267, 2007. Ó 2007 Wiley-Liss, Inc.
Key words: mesenchymal stem cell; junction; phorbol ester;
TPA; multinucleation
Mesenchymal stem cells (MSCs) reside in the bone
marrow, like hematopoietic stem cells, and can differentiate into various types of cells in appropriate conditions,
both in vitro and in vivo (Pittenger et al., 1999; Jiang
et al., 2002). MSCs can differentiate into tendon, endothelial, and nerve cells, as well as osteoblasts, adipocytes, and chondrocytes (Pittenger et al., 1999; Dezawa
et al., 2001, 2004; Jiang et al., 2002). MSCs can also
become stromal cells, supporting the differentiation of
hematopoietic stem cells into mature blood cells (Dennis
and Charbord, 2002).
MSCs are fibroblastic in shape, adhere to culture substrates, and do not phagocytose particles (Castro-Malaspina et al., 1980). By fluorescent-activated cell sorting
(FACS) analysis, MSCs were classified into small and
agranular cells (Colter et al., 2000). As for the cell surface markers, MSCs express CD105 (SH-2; Barry et al.,
1999), CD73 (SH-3 and SH-4; Barry et al., 2001), CD90,
STRO-1, and CD106, but not the hematopoietic markers
CD45 and CD34 (Tocci and Forte, 2003). However, it
was also reported that MSCs freshly prepared from bone
marrow aspirates are phenotypically different from
those cultured in vitro for some time (Gronthos et al.,
2003). Jiang et al. (2002) suggested that a single MSC
Ó 2007 WILEY-LISS, INC.
could differentiate into almost all types of cells in the
body.
Many cells develop junctional structures when they
make contact with each other. These structures include
tight junctions, adherence junctions, gap junctions, desmosomes, and interdigitations, and cells communicate
through these junctions. Although these junctional
structures are not prominent for cells of mesenchymal
origin, human mesenchymal stem cells are reported to
build gap junctions among themselves or with cardiomyocytes in some in vitro culture systems (Valiunas et al.,
Grant sponsor: The Ministry of Education, Culture, Sports,
Science and Technology, Japan Grant-in-Aid for Scientific
Research; Grant numbers: 14370008 and 16659048.
*Correspondence to: Keiichiro Yoshida, Department of Histology and Cell Biology, Yokohama City University School of Medicine, Fukuura 3–9, Kanazawa-ku, Yokohama, Japan 236-0004.
Fax: 81-45-787-2568. E-mail: k6552@yokohama-cu.ac.jp
Received 29 November 2006; Accepted 12 July 2007
DOI 10.1002/ar.20590
Published online in Wiley InterScience (www.interscience.wiley.
com).
MULTINUCLEATION OF MESENCHYMAL STEM CELL
2004; Beeres et al., 2005). These cells interact electrically and can repair experimentally induced conduction
blocks.
TPA is a potent tumor promoter and known to be a
protein kinase C (PKC) activator. TPA has been reported
to induce formation of multinuclear cells, either by cell–
cell fusion (syncytium; Hassan et al., 1989; David et al.,
1990) or by nuclear division without cell division (plasmodium; Menaya and Clemens, 1991).
Syncytia occur in striated muscle cells and macrophage-related cells, and their formation results in the
generation of multinuclear cells (Anderson, 2000; Taylor,
2003). Plasmodia were reported in some tumors stimulated with TPA (Menaya and Clemens, 1991), and it was
reported that this effect was dependent on PKCa and
PKCd activation (Watanabe et al., 1992; Yamaguchi
et al., 1995).
TPA has also been reported to induce endomitosis
(Murate et al., 1991; Bermejo et al., 2002). An endomitotic cell has multiple sets of its genome, for example,
4n, 8n, 16n, or more in one nucleus (Vitrat et al., 1998).
Endomitosis occurs in leukemic cells (e.g., K562, HEL
MEG-01) stimulated by TPA, and these cells change into
megakaryocytes (Murate et al., 1991; Bermejo et al.,
2002).
In this report, we established an oligopotent (Smith,
2006) mesenchymal stem cell clone that could differentiate into adipocytes and osteoblasts, and we investigated
the fine structure of these cells. Upon stimulation with
TPA, these cells converted into multinucleated cells.
MATERIALS AND METHODS
Cell Culture
Normal rat bone marrow (BM) was obtained from the
femurs of DA/Slc rats (Japan SLC. Inc., Hamamatsu, Japan). Eagle’s minimum essential medium with alpha
modification (a-MEM; Sigma, St. Louis, MO) including
10% fetal calf serum (Lot.S04301S1820, BioWest, Miami,
FL), 300 mg/ml L-glutamine (WAKO, Osaka, Japan), and
60 mg/ml kanamycin sulfate (WAKO) was used for culturing cells. Culture was performed at 378C in a humidified atmosphere containing 5% CO2 and 95% air. The
medium was changed every third day. A few MSC clones
were obtained through successive limiting dilutions, and
clone 5F9A was used in this study.
Transmission Electron Microscopy
Cells were fixed with 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 48C, post-fixed with 1% OsO4 for 1 hr on ice,
and further stained with 0.5% uranyl acetate for 30 min
at room temperature. Fixed and stained cells were dehydrated with ascending concentrations of ethanol, and
embedded in Epon 812. Ultrathin sections were obtained
and stained with 2% uranyl acetate in 70% ethanol and
0.4% lead citrate, and observed using a H-7500 transmission electron microscope (Hitachi, Tokyo, Japan)
operated at 80 kV.
Immunohistochemistry
Cells on coverslips were fixed with PLP solution
(0.01 M NaIO4, 0.075 M lysine, 0.0375 M phosphate
1257
buffer, and 2% paraformaldehyde, pH 6.2), blocked with
0.4% bovine serum albumin in phosphate buffered saline
(BSA-PBS), and permeabilized with 0.1% Triton X-100
in BSA-PBS. Thereafter, the cells were labeled with the
primary antibodies and secondary antibodies (Alexa
Fluor1488 goat anti-mouse IgG and anti-rabbit IgG;
Invitrogen, Carlsbad, CA). The stained samples were observed by confocal laser scanning microscopy (LSM510,
Carl Zeiss Ltd. Oberkochen, Germany).
For the primary antibodies, rabbit anti–ZO-1 antibody
(Invitrogen), mouse anti–b-catenin monoclonal antibody
(BD Transduction Lab., Lexington, KY), mouse anti-desmoglein antibody (BD Transduction Lab.), and mouse
anti–E-cadherin monoclonal antibody (BD Transduction
Lab.) were used in this study.
Reverse-Transcription Polymerase
Chain Reaction
Total RNA was extracted from 5F9A cells using TRI
REAGENTTM RNA, DNA, and protein isolation solution
(Sigma) according to the manufacturer’s instructions.
cDNA was synthesized by reverse transcription with
M-MLV reverse transcriptase (Invitrogen). Polymerase
chain reaction (PCR) was performed in a PCR thermal
cycler (Takara, Tokyo, Japan), and the products were
subjected to electrophoresis on 2% agarose gels after 35
cycles of amplification and stained with 100 ng/ml ethidium bromide. The sizes of the PCR products were estimated by comparison with Marker 4 (WAKO). Oligonucleotide primers used in PCR are shown in Table 1.
FACS
For FACS analysis, we used biotin–anti-CD29 antibody, fluorescein isothiocyanate (FITC) –anti-CD45 antibody (BD Biosciences Pharmingen, San Diego, CA), antiCD11b antibody, FITC–anti-CD44 antibody (Chemicon,
Tamecula, CA), and FITC–anti-CD90 antibody (eBioscience, San Diego, CA). As a secondary antibody, we
used FITC-Streptavidin (Vector, Burlingame, CA) and
FITC goat anti-mouse IgG (Fab0 ) antibody (PROTOS
IMMUNORESEARCH, San Francisco, CA). The expression of the antigens was analyzed using a MoFlo flow cytometer (Dako Cytomation, Fort Collins, CO).
Phagocytosis of Latex Beads
5F9A cells were cultured in medium containing latex
beads (1.07-mm diameter, Sigma) for 3 hr. These cells
were washed gently with PBS, fixed with methanol, and
stained with Giemsa solution (MERCK, Dermstadt, Germany). The cells were observed using a light microscope.
Induction of Differentiation
For 5F9A cells to differentiate into osteoblasts, they
were cultured in a-MEM containing 50 mM ascorbate-2phosphate (Asc-2-P, Sigma), 10 mM b-glycerophosphate
(Sigma), and 0.1 mM dexamethasone (WAKO; Zuk et al.,
2001). The medium was changed every third day. After
5 weeks, total RNA was extracted from the cells and
applied to reverse transcription (RT) PCR, and the cells
were stained by von Kossa staining for the detection of
calcium. Concerning the differentiation into adipocytes,
the cells were cultured in a-MEM containing 4 mg/ml
1258
YOSHIDA ET AL.
TABLE 1. Primers used for reverse-transcription polymerase chain reaction
Oligonucleotide sequence
Rat CD14
Rat CD29
Rat CD90
Rat CD104
Rat CD106
Rat CD126
Rat c-kit
Rat CD45
Rat CD44
Rat CD34
Mouse CD73
Rat CD105
Rat E-Cadherin
Rat N-Cadherin
Rat Runx2
Rat PTHR
Rat BSP
Rat Osteonectin
Rat GPIIb
Rat GPV
Rat MyoD
Rat Myf-5
Mouse Myogenin
Rat albumin
Rat a-Fetoprotein
Rat Integrin aV
Rat Integrin b3
Rat Cathepsin K
Rat c-fms
Rat TRAP
Rat RANK
Rat CTR
Rat b-actin
0
0
5 -AGTCGGAGGCGTATAACTCTG-3
50 -GTAGAGCGCTTGCCTAGTGA-30
50 -TAAGTGCACAGATCCCAAGTT-30
50 -TGATGTCGGGACCAGTAG-30
50 -CCGTCATGAGAATAACACCA-30
50 -CCCAACCAGTCACAGAGAAAT-30
50 -TCCCTATTGCGACTACGAAAT-30
50 -TGGCCAGGTTGATGATAGC-30
50 -ACGTTGCTCCGAAAGAAC-30
50 -AGATGGGAAGACTGTAAGCTG-30
50 -GTTCAGTTCGCACAGGTGTAT-30
50 -GTTTCCTTTCTCCCAAGACA-30
50 -ACAACGGCACGGTGGAGT-30
50 -GGGCACTTGGTTTGAGCATCT-30
50 -GATCCACCCAGTGACCCGTCT-30
50 -ACCCTGGGCAGCAATCATCAT-30
50 -GGGACTACTTTGCCTCTTACA-30
50 -ATGCGAGGAGGATATACACTC-30
50 -GCGTGAGAATGCCGGTCCA-30
50 -TCAGCAAACACTCGGGCCTAA-30
50 -CTGTGCTGCCCTTTGGAG-30
50 -CGGCTGGGTAAACTACTTTCA-30
50 -AGACAGCTGCCATCTGGATTT-30
50 -GGGCACGTGTGTGAGAATAGA-30
50 -GTGTGGGTCTGGAGATCGCATCTTAC-30
50 -AGAGATTCTTGTTCTGCGTCATTGTACTCA-30
50 -AGTGCAGAGAACTCGGAACAT-30
50 -TGCTAGTGCTGCCAGTACCA-30
50 -CCAGATGGGACTGTGGTTACC-30
50 -ACTTGGTGCAGAGTTCAGGG-30
50 -CCGGGTGCTTGCCACTAA-30
50 -GCTTTGCGCTTGAAGTCCAAC-30
50 -ATCGTAAATCAGGGCTGTTGA-30
50 -GGCGTCTCACACCGTGTAT-30
50 -CGCATGCGTGACTGGC-30
50 -CAAAGAAGCGAGTGGTGCAAT-30
50 -CCTGGAAGAAGCTGGAGATT-30
50 -CGAGTACGGTAGGGAGGAGA-30
50 -GCTCTTCCGTAACCTCAGCAG-30
50 -TTTCCCATTGCCTCAAACAAG-30
50 -TGGCGCCGCTGCCTTCTACG-30
50 -ACACGGCCGCACTCTTCCCTG-30
50 -CAGCCAAGAGTAGCAGCCTTCG-30
50 -GTTCTTTCGGGACCAGACAGGG-30
50 -TGGAGCTGTATGAGACATCCC-30
50 -TGGACAATGCTCAGGGGTCCC-30
50 -CAGGCCACTATCTCCAGCAAA-30
50 -GTGCCCACTCTTCCCAGGTT-30
50 -TGGCCGACATTTACATTG-30
50 -AAGAGGCCAGAGAAATCAGCA-30
50 -TGGGCCGATGAACTGTACTGC-30
50 -GGCGCTCTTCCCTCTATCCAG-30
50 -AAAATCCGTTCTAAAGTAGA-30
50 -ATCTCCCCTTTGTAGCGGA-30
50 -ATGTATAACGCCACGGCAAAG-30
50 -CAGATGGGCTGGCTGGCTTGA-30
50 -GGCGTAGATACCTATGTGGAG-30
50 -GGAGGATGCCGTAGGAC-30
50 -CAAGAACTTGAGGCCATTGTT-30
50 -CAGAGTACCAGGGCAGAGAAA-30
50 -TTAAGCCAGTGCTTCACGGG-30
50 -ACGTAGACCACGATGATGTCGC-30
50 -ACACCCTGACAGCAACCGAACCT-30
50 -GAACCCCCAGCCAAGTAAATAGTA-30
50 -AACTGGGACGATATGGAGAA-30
50 -GTAACCCTCATAGATGGGCA-30
Product (base pair)
Annealing (8C)
390bp
558C
335bp
568C
382bp
568C
392bp
568C
308bp
568C
443bp
558C
435bp
608C
306bp
608C
308bp
558C
744bp
608C
387bp
558C
374bp
558C
451bp
558C
353bp
558C
380bp
558C
315bp
588C
560bp
558C
316bp
588C
682bp
548C
549bp
548C
230bp
608C
440bp
608C
184bp
608C
503bp
588C
310bp
568C
220bp
558C
556bp
558C
382bp
558C
379bp
558C
438bp
618C
497bp
558C
447bp
558C
276bp
608C
MULTINUCLEATION OF MESENCHYMAL STEM CELL
1259
Fig. 2. Junctional structures between 5F9A cells. A–C: An adherence junction-like structure (A), a tight junction-like structure (B), and
an interdigitation-like structure (C) are indicated by arrows. D: Fluorescence micrograph of anti–ZO-1 antibody staining of 5F9A cells. The
borders of the cells were stained by the antibody (arrow). Scale bars
5 600 nm in A, 100 nm in B, 200 nm in C, 100 mm in D.
insulin (Sigma), 500 mM 3-isobutyl-1-methylxanthine
(IBM, WAKO), 0.4 mM dexamethasone (WAKO), and
60 mM indomethacin (WAKO) for 3 weeks, changing the
medium twice a week, and stained with Sudan III for
the detection of lipids. In both cases, the culture dishes
were coated with Growth Factor Reduced MATRIGEL1Matrix (40 mg/ml; BD Biosciences, Discovery Labware,
Bedford, MA), and the initial cell density was 2 3 104
cells per 2 ml of medium in a 35 mm dish.
Induction of Multinuclear Cells
For the induction of multinuclear cells (MNCs), cells
were cultured in a 35-mm dish in the presence of 20 ng/
ml TPA (Sigma) for 3 days. For light microscopy, the
cells were fixed with absolute methanol for 5 min and
stained with Giemsa solution (MERCK).
Additionally, TPA-stimulated cells were fixed with
10% formalin, permeabilized with 0.5% Triton X-100 in
PBS, and stained with SYBR Green I (SYBR-I; Molecular Probes, Inc., Eugene, OR). Twenty optical sections of
multinuclei along the z-axis (0.9 mm in thickness and
0.45 mm in focus step) were analyzed using confocal
laser scanning microscopy (LSM510, Carl Zeiss Ltd.).
Fig. 1. The 5F9A cells. A: Photograph of 5F9A cells. The 5F9A was
established from adherent cells in DA/Slc rat bone marrow by
repeated limiting dilutions. The cells were fixed with absolute methanol
and stained by Giemsa solution. B: Electron micrographs of 5F9A
cells. The arrow indicates heterochromatin, the arrowhead indicates
Golgi apparatus, the asterisk indicates polysomes, and the white
arrow indicates rough endoplasmic reticulum. C: Actin bundle is indicated beneath the plasma membrane at the basal surface (arrow).
Scale bars 5 25 mm in A, 1 mm in B,C.
1260
YOSHIDA ET AL.
Fig. 3. Phenotype of 5F9A cells. A: Reverse transcription-polymerase chain reaction was performed using total RNA extracted from
nontreated and TPA-treated 5F9A cells with specific primers, which
are shown in Table 1. The left column shows the results with total
RNA from 5F9A cells without TPA stimulation. The column in the cen-
ter shows the results from the TPA-stimulated cells. Total RNA prepared from normal rat spleen cells was used as a control (right column). B: Phenotype analyses using fluorescent-activated cell sorting.
5F9A cells stained with fluorescence were analyzed using a MoFlo
flow cytometer.
MULTINUCLEATION OF MESENCHYMAL STEM CELL
1261
RESULTS
5F9A cells
The 5F9A cell clone was obtained through repeated
limiting dilutions of bone marrow adherent cells. After
establishment, this clone was maintained continually by
dilution with fresh medium because it needed neither
feeder cells nor special supplements. The cells spread on
plastic surfaces and exhibited a fibroblast-like shape
(Fig. 1A). The population doubling time was approximately 20 hr.
Disperse heterochromatin could be observed in the
nuclei by electron microscopy. Golgi apparati, polysomes,
and rough endoplasmic reticulum developed in the cytoplasm, which suggested the presence of active protein
synthesis in 5F9A cells (Fig. 1B). Thick actin bundles
could be observed on the substratum at the basal surface
beneath the plasma membrane (Fig. 1C).
Junctional Structures Between 5F9A Cells
Conspicuously, the cells formed numerous junctional
structures among themselves. This observation is unique
considering their probable mesenchymal origin. Some
junctions resembled adherence junctions (Fig. 2A), some
resembled tight junctions (Fig. 2B), and some seemed to
be interdigitations formed by protrusions of one cell
ensheathed by the plasma membrane of the adjacent cell
(Fig. 2C). Actin bundles were also seen in the vicinity of
the adherence junction-like structures. The protrusions
were longer than 1 mm and the longest one we observed
was approximately 3 mm long. The width appeared to be
100–200 nm. The interdigitations contained actin filaments extending along their long axis. However, no
thickening or undercoating of plasma membranes reminiscent of junctional specialization could be observed
between these protrusions and their sheaths (Fig. 2C).
Next, we stained 5F9A cells with antibodies that recognize the molecules forming the junctions. This cell
clone did not react with anti–E-cadherin antibody (data
not shown). Neither could we detect any signal of b-catenin in the cells (data not shown). These findings suggest
that typical adherence junctions are not constructed
between 5F9A cells. However, TEM analysis suggested
the presence of adherence junction-like structures (Fig.
2A), which may indicate the presence of an atypical
member of the adherence junction family.
An antibody against the ZO-1 molecule, which is one
of the components of tight junctions, stained borders
between 5F9A cells (Fig. 2D), and, furthermore, a junction-like structure similar to a tight junction was
observed during TEM analysis. Fused outer leaflets of
the plasma membrane could also be detected (Fig. 2B).
In addition, we could not stain 5F9A cells with an
anti-desmogrein antibody that reacts with desmosomes
(data not shown), which suggests that desmosomes did
not exist between the cells. Therefore, the junctional
structures between 5F9A cells likely consist of tight
junction- and adherence junction-like structures, and
protrusions with no communication with the sheath.
Phenotype of 5F9A Cells
To examine the phenotype of the 5F9A clone, we performed RT-PCR using specific primers for various
markers (Table 1; Fig. 3A). The cells showed positive
Fig. 4. The 5F9A cells did not ingest latex beads. The 5F9A cells
were cultured with latex beads (1.07 mm diameter). Rat peritoneal adherent cells were used as a positive control, and they could internalize
beads (arrows). Scale bar 5 25 mm.
reactions for CD90 (Thy-1), CD29 (b1-integrin), CD73
(Ecto-50 -nucleotidase), CD105 (Endoglin), CD44, CD14,
and N-Cadherin, but did not express CD126 (IL-6 receptor), CD104 (b4-integrin), CD106 (VCAM-1), E-Cadherin,
CD45, CD34, or c-kit. The results of FACS were consistent with the results by RT-PCR. The expression of
CD29, CD44, and CD90 but not CD11b and CD45 could
be observed both in the presence and absence of TPA
(Fig. 3B). Additionally, each histogram shows a single
peak of staining except CD44 with TPA, which suggests
that the population of cells was homogenous. However,
TPA seemed to induce some changes of CD44 expression
in the cells. Because the 5F9A clone did not express
CD45, CD34, or c-kit (Fig. 3A,B), this clone did not
likely originate from hematopoietic lineage cells. Although this clone was positive for CD14 (Fig. 3A), which
is a macrophage marker, it did not ingest latex beads,
indicating that it was not a phagocyte (Fig. 4). The negative expression of CD11b (Fig. 3B) confirms these
results.
5F9A Cells Are Oligopotent
5F9A cells were able to differentiate to contain lipid
droplets positive for Sudan III staining in the cytoplasm
when cultured in a-MEM medium containing insulin,
IBM, dexamethasone, and indomethacin for 3 weeks
(Fig. 5A,B). This finding indicates that 5F9A cells can
differentiate into adipocytes, as previously reported for
mesenchymal stem cells. After reaching confluence, the
cell sheets often rolled up to form aggregates of cells. In
these aggregates, adipocytes were very often observed
(Fig. 5C).
When the cells were cultured for 5 weeks in the presence of Asc-2-P, b-glycerophosphate, and dexamethasone
(Zuk et al., 2001), brown deposits were detected on the
cells by von Kossa staining (Fig. 6A,B). These deposits
were regarded to be calcium, and the cells, therefore,
seemed to differentiate into osteoblasts. To confirm
osteoblastic differentiation, analysis by RT-PCR was performed. Cells cultured for 5 weeks in osteoblast differentiation medium were shown to express Runx-2 and bone
sialoprotein, which are markers of osteoblasts (Laino
et al., 2006; Friedman et al., 2006; Fig. 6C), whereas
control cells did not express these markers. These
results indicated that the 5F9A clone maintained the
capacity to differentiate into both adipocytes and osteoblasts under suitable culture conditions, suggesting its
oligopotent nature.
1262
YOSHIDA ET AL.
Fig. 5. Differentiation into adipocytes. The 5F9A cells were cultured
in adipogenic differentiation medium, as described in the Materials
and Methods section. A: The cells were fixed and stained by Sudan III
staining for adipocytes. Lipid droplets were stained (arrows). B: Sudan
III staining of cells cultured without supplements. C: Photograph of
transmission electron microscopy of an aggregate of cultured 5F9A
cells. There are many lipid droplets in the cells (arrows). Scale bars 5
25 mm in A,B, 5 mm in C.
MULTINUCLEATION OF MESENCHYMAL STEM CELL
1263
Fig. 6. Differentiation into osteoblasts. The 5F9A cells were cultured in osteogenic differentiation medium, as described in Materials
and Methods. A: The cells were fixed and stained by von Kossa staining for osteoblasts. Calcium deposits could be observed (arrows). B:
Shown is the von Kossa staining of the cells cultured without supplements. C: Reverse transcription-polymerase chain reaction analysis of
the expression of the osteoblast specific genes. Total RNA was
extracted from cells cultured for 5 weeks in the osteogenic differentiation medium (1) and the control medium (without supplements) (2),
and used as templates with the primers listed in Table 1. Asterisks
indicate specific bands of Runx-2 and bone sialoprotein. Scale bars 5
25 mm in A,B.
5F9A Cells Can Be Induced to Develop Into
Multinuclear Cells
optimal conditions, 1020% of the cells were multinucleated. Although some 5F9A cells spontaneously
became multinuclear, the frequency was less than 3%,
and the difference between nontreated and TPA-treated
5F9A cells was easily recognizable by microscopic examination. When 5F9A cells were seeded in a 35-mm culture vessel at cell densities of 1 3 104/ml (2 3 104/dish),
2.0 3 1044.0 3 104 cells/dish were multinuclear by day
3 when the cells reached confluence (approximately
When 5F9A cells were cultured in the presence of
TPA, they developed multiple nuclei, some of which
were large and irregular in shape (Fig. 7). This phenomenon was confirmed by confocal laser scanning microscopy, which showed that more than one distinct nucleus
existed in one cell following TPA stimulation (Fig. 8). In
1264
YOSHIDA ET AL.
Fig. 7. TPA-induced multinucleation. The 5F9A cells were cultured
in the presence or absence of 20 ng/ml of TPA for 3 days. Cells were
stained with Giemsa solution. Multinucleated cells were induced
(arrows). Scale bar 5 25 mm.
2.0 3 105/dish). Upon TPA stimulation, multinuclear cells
spread (Fig. 7), and highly distinct stress fibers could be
observed. It also appears that cells grew very slowly after
becoming multinucleated (data not shown). TPA stimulation did not change the phenotype of 5F9A cells as
assessed by RT-PCR (Fig. 3A), and this may reflect that
only approximately 1020% cells became multinucleated.
There are some naturally occurring multinuclear cells,
including megakaryocytes (although strictly speaking,
they are not multinucleated), striated muscle cells, hepatocytes, and osteoclasts. In RT-PCR analyses, neither
nonstimulated nor TPA-stimulated cells expressed GPIIb
or GPV (megakaryocyte markers; Lepage et al., 2000),
MyoD, Myf-5, or myogenin (striated muscle cell markers;
Berkes and Tapscott, 2005), or albumin or a-fetoprotein
(hepatocyte markers; Hay et al., 2007; Fig. 9). However,
some osteoclastic markers were expressed in 5F9A cells.
These cells were positive for integrin aV, integrin b3
(Ptaff and Jurdic, 2001), cathepsin K, M-CSF (macrophage-colony stimulating factor) receptor (c-fms), and
TRAP (tartrate-resistant acid phosphatase; Fujisaki
et al., 2007), but negative for RANK (receptor activator
of NF-jB) and CTR (calcitonin receptor; Myers et al.,
1999) both in the presence and absence of TPA stimulation
(Fig. 9). Because 5F9A cells are oligopotent, they would
likely be stem cells that retain some osteoclast markers.
We, therefore, could not acquire any evidence that
5F9A cells differentiated into megakaryocytes, striated
muscle, hepatocytes, or osteoclasts after TPA stimulation.
Although we tried to induce multinuclear cells with
various substances other than TPA during short-term
culturing (3 days), a significant number of multinuclear
cells could not be induced. The substances included dibutyryl cyclic-AMP (250 mg/ml), staurosporine (0.2 mM),
activin (10 ng/ml), vitamin A (50 pg/ml), retinoic acid
(50 nM), insulin (5 mg/ml), concanavalin A (2 mg/ml), lipopolysaccharide (10 mg/ml), and lysophosphatidic acid
(300 ng/ml; data of all materials listed above are not
shown). Additionally, rat normal skin fibroblasts did not
become multinucleated following TPA stimulation (data
not shown), which may suggest that multinucleation is a
specific phenomenon characteristic of 5F9A cells.
DISCUSSION
5F9A Cells Are Phenotypically Similar to
Mesenchymal Stem Cells
RT-PCR and FACS (Fig. 3) revealed that 5F9A cells
express CD14, CD29, CD44, CD90, CD73, CD105, and
N-Cadherin. They do not express E-Cadherin, CD104,
CD106, CD126, CD34, c-kit, CD45, or CD11b (Fig. 3).
Deans and Moseley (2000) reported that the phenotypes
of MSC were CD90, CD29, and CD106 positive, and
CD14, CD34, and CD45 negative. On the contrary, Reyes
et al. (2001) reported they were CD44 and CD90 positive, and CD34, CD106, c-Kit, and CD45 negative. The
phenotypes of 5F9A cells in the present study were consistent with the results of Reyes et al. and those of Deans
and Moseley except CD14 and CD106. 5F9A cells were
negative for CD45, CD34, and c-Kit, which suggests that
5F9A cells are not hematopoietic. In human mesenchymal stem cells, CD73 is recognized by SH-3 and SH-4
antibodies (Barry et.al., 2001) and CD105 is recognized
by SH-2 antibodies (Barry et al., 1999); Therefore, these
antibodies may be applied to the separation and characterization of rat mesenchymal stem cells by FACS or
other methods.
5F9A cells are CD44 positive. Deans and Moseley
(2000) also reported that CD34-positive hematopoietic
stem cells require CD44-mediated signals expressed by
mesenchymal stem cells for hematopoiesis. 5F9A cells
may, therefore, have the ability to maintain hematopoiesis.
5F9A cells are also positive for CD14, which is one of
the components of the LPS (lipopolysaccharide) receptor
(Miller et al., 2005) and is regarded as a specific marker
for macrophages and granulocytes (Fig. 3A). This finding
may suggest that 5F9A cells are of macrophage/granulocyte lineage. However, 5F9A cells were CD11b negative
and could not internalize latex beads (Figs. 3B, 4). Together with their lack of many hematopoietic markers,
we consider that 5F9A cells are not of macrophage/granulocyte lineage.
In RT-PCR analyses, 5F9A cells expressed many but
not all osteoclast markers. They were positive for integrin aV, integrin b3, cathepsin K, c-fms, and TRAP, but
negative for RANK and CTR (Fig. 9). Although osteoclasts were reported to be positive for CD45 and negative for CD14 (Athanasou and Quinn, 1990), 5F9A cells
showed the opposite pattern of results (Fig. 3). Although
these results suggest that 5F9A cells might be related to
osteoclasts, we could not definitively define 5F9A cells
as osteoclasts because of some phenotypic differences
between 5F9A cells and osteoclasts. When taken into
consideration that 5F9A cells had oligopotency (Figs. 5,
6), it is possible that they may instead be stem cells
expressing some osteoclast markers.
5F9A Cells Are Oligopotent
5F9A cells could differentiate into adipocytes and
osteoblasts in the presence of adipogenic and osteogenic
differentiation cocktails, as determined by Sudan III
staining and electron microscopy for adipocytes, and von
Kossa staining and RT-PCR analysis for osteoblasts
(Figs. 5, 6). Although osteonectin and PTHR (parathy-
Fig. 8. Three-dimensional analysis of multinuclei in the cell using
confocal microscopy. A,B: SYBR-I stained nuclei in one TPA-treated
cell were observed using confocal microscopy (0.9 mm in thickness
and 0.45 mm in focus step along the z-axis from the upper left to the
lower right). Twenty optical sections were analyzed in each cell, and
more than one distinct nucleus could be observed in one cell. Scale
bar 5 10 mm.
MULTINUCLEATION OF MESENCHYMAL STEM CELL
Figure 8.
1265
1266
YOSHIDA ET AL.
We detected N-cadherin on the surface of 5F9A cells
(Fig. 3), which was previously reported to be associated
with the osteoblastic niche for hematopoietic stem cells
(Zhang et al., 2003). Although at present, we do not
know if these molecules affect the function of 5F9A cells,
a more intensive study of the junctional structures of
5F9A cells would reveal their important roles in the
interaction of the cells during events such as cell–cell
fusion.
Multinucleation by TPA
Fig. 9. Analyses of the expression of differentiation markers by
TPA-induced multinucleated cells. Total RNA of 5F9A cells with or
without TPA treatment was analyzed by the reverse transcription-polymerase chain reaction. Total RNA of rat fetal limb was used as a positive control for MyoD, Myf-5, and myogenin (striated muscle markers).
Total RNA of the rat liver was used as a positive control for albumin
and a-fetoprotein (hepatocytes markers). Positive controls of GPIIb
and GPV (megakaryocyte markers) and other groups (osteoclast
markers) were also assessed.
roid hormone receptor) were expressed not only in osteogenically differentiated (Runx-2 and bone sialoprotein
positive), but also in undifferentiated cells (Fig. 6C), previous findings showed that osteonectin was expressed in
bone marrow-derived (undifferentiated) MSCs (Silva
et al., 2003), and MSCs in fetal circulating blood spontaneously became PTHR-positive cells (Naruse et al.,
2004). Therefore, our findings do not necessarily deny
the ability of 5F9A cells to undergo osteoblastic differentiation. Rather, our data suggest that 5F9A cells have
oligopotency. In brief, 5F9A cells would give us a valuable tool for studying rat MSCs because of their species
importance.
Cell Communication Between Mesenchymal
Stem Cells
Although adherence junctions, tight junctions, or desmosomes, that could be observed mainly in epithelial
cells have not been reported in MSCs, 5F9A cells were
shown to form junction-like structures by electron microscopy (Fig. 2). One type of junction (an interdigitation-like structure) had a bundle of microfilaments in
the interdigitations along their long axis. Actin cables in
the cells were connected to adherence junction-like
structures.
However, we could not detect E-cadherin or b-catenin
in 5F9A cells either by immunofluorescence techniques
or RT-PCR analysis (Fig. 3). This finding may suggest
that typical epithelial-type adherence junctions are not
constructed between these cells. We could not detect desmogrein either, indicating that few, if any, desmosomes
were formed (data not shown).
The tumor promoter TPA could cause 5F9A cells to
become cells with more than one nucleus (multinuclear;
TPA-response; Figs. 7, 8) and it induced 1020% of the
total cell population into multinucleated cells, whereas
nontreated control cells contained less than 3% multinucleated cells.
In general, it has been thought that genome amplification in a cell is carried out by one of the mechanisms of
syncytium, plasmodium, and endomitosis. All these processes could be induced by TPA stimulation, possibly
depending on the cell types (Hassan et al., 1989; David
et al., 1990; Murate et al., 1991; Menaya and Clemens,
1991; Bermejo et al., 2002). This is the first report indicating that MSCs could be multinucleated by TPA stimulation. Further studies should be done to clarify the
relationship between stem cells and multinucleation.
ACKNOWLEDGMENTS
We thank Dr. Y. Ikeda, Dr. K. Ohbo, and Dr. H.
Tanaka for helpful discussions and encouragement during the course of this work.
LITERATURE CITED
Anderson JM. 2000. Multinucleated giant cells. Curr Opin Hematol
7:40–47.
Athanasou NA, Quinn J. 1990. Immunophenotypic differences
between osteoclasts and macrophage polykaryons: immunohistological distinction and implications for osteoclast ontogeny and
function. J Clin Pathol 43:997–1003.
Barry FP, Boyton RE, Haynesworth S, Murphy M, Zaia J. 1999.
The monoclonal antibody SH-2 raised against human mesenchymal stem cells recognizes an epitope on Endoglin (CD105). Biochem Biophys Res Commun 265:134–139.
Barry F, Boyton R, Murphy M, Zaia J. 2001. The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Commun 289:519–524.
Beeres SLMA, Atsma DE, van der Laarse A, Pijnappels DA, van
Tuyn J, Fibbe WE, de Vries AAF, Ypey DL, van der Wal EE, Schalij MJ. 2005. Human adult bone marrow mesenchymal stem cells
repair experimental conduction block in rat cardiomyocyte cultures. J Am Coll Cardiol 46:1943–1952.
Berkes CA, Tapscott SJ. 2005. MyoD and transcriptional control of
myogenesis. Semin Cell Dev Biol 16:585–595.
Bermejo R, Vilaboa N, Cales C. 2002. Regulation of CDC6, Geminin,
and CDT1 in human cells that undergo polyploidization. Mol Biol
Cell 13:3989–4000.
Castro-Malaspina H, Gay RE, Resnick G, Kapoor N, Meyers P,
Chiarieri D, McKenzie S, Broxmeyer HE, Moore MAS. 1980.
Characterization of human bone marrow fibroblast colony-forming
cells (CFU-F) and their progeny. Blood 56:289–301.
Colter DC, Class R, DiGirolamo CM, Prockop DJ. 2000. Rapid
expansion of recycling stem cells in cultures of plastic-adherent
cells from bone marrow. Proc Natl Acad Sci U S A 97:3213–3218.
David JD, Faser CR, Perrot GP. 1990. Role of protein kinase C in
chick embryo skeletal myoblast fusion. Dev Biol 139:89–99.
MULTINUCLEATION OF MESENCHYMAL STEM CELL
Deans RJ, Moseley AB. 2000. Mesenchymal stem cells: biology and
potential clinical uses. Exp. Hematol. 28:875–884.
Dennis JE, Charbord P. 2002. Origin and differentiation of human
and murine stroma. Stem Cells 20:205–214.
Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H. 2001. Sciatic nerve regeneration in rats induced by transplantation of in
vitro differentiated bone-marrow stromal cells. Eur J Neurosci
14:1771–1776.
Dezawa M, Kanno H, Hoshino M, Cho H, Matsumoto N, Itokazu Y,
Tajima N, Yamada H, Sawada H, Ishikawa H, Mimura T, Kitada
M, Suzuki Y, Ide C. 2004. Specific induction of neuronal cells
from bone marrow stromal cells and application for autologous
transplantation. J Clin Invest 113:1701–1710.
Friedman MS, Long MW, Hankenson KD. 2006. Osteogenic differentiation of human mesenchymal stem cells is regulated by bone
morphogenetic protein-6. J Cell Biochem 98:538–554.
Fujisaki K, Tanabe N, Suzuki N, Kawato T, Takeichi O, Tsuzukibashi O, Makimura M, Ito K, Maeno M. 2007. Receptor activator of
NF-jB ligand induces the expression of carbonic anhydrase II, cathepsin K and matrix metalloproteinase-9 in osteoclast precursor
RAW264.7 cells. Life Sci 80:1311–1318.
Gronthos S, Zannettino ACW, Hay SJ, Shi S, Graves SE, Kortesidis
A, Simmons PJ. 2003. Molecular and cellular characterization of
highly purified stromal stem cells derived from human bone marrow. J Cell Sci 116:1827–1835.
Hassan NF, Kamani N, Meszaros MM, Douglas SD. 1989. Induction
of multinucleated giant cell formation from human blood-derived
monocytes by phorbol myristate acetate in in vitro culture. J
Immunol 143:2179–2184.
Hay DC, Zhao D, Ross A, Mandalam R, Lebkowski J, Cui W. 2007.
Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells
9:51–62.
Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD,
Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du
J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM.
2002. Pluripotency of mesenchymal stem cells derived from adult
marrow. Nature 418:41–49.
Laino G, Carinci F, Graziano A, d’Aquino R, Lanza V, De Rosa A,
Gombos F, Caruso F, Guida L, Rullo R, Menditti D, Papaccio G.
2006. In vitro bone production using stem cells derived from
human dental pulp. J Graniofac Surg 17:511–515.
Lepage A, Leboeuf M, Cazenave J, Salle C, Lanza F, Uzan G. 2000.
The aIIbb3 integrin and GPIb-V-IX complex identify distinct
stages in the maturation of CD341 cord blood cells to megakaryocytes. Blood 96:4169–4177.
Menaya J, Clemens MJ. 1991. Phorbol ester-induced inhibition of
proliferation of Daudi Burkitt’s Lymphoma cells by impairment of
cytokinesis. Exp Cell Res 194:260–266.
Miller SI, Ernst RK, Bader MW. 2005. LPS, TLR4 and infectious
disease diversity. Nat Rev Microbiol 3:36–46.
Murate T, Hotta T, Tsushita K, Suzuki M, Yoshida T, Saga S, Saito
H, Yoshida S. 1991. Aphidicolin, an inhibitor of DNA replication,
1267
blocks the TPA-induced differentiation of a human megakaryoblastic cell line, MEG-O1. Blood 78:3168–3177.
Myers DE, Collier FM, Minkin C, Wang H, Holloway WR, Malakellis M, Nicholson GC. 1999. Expression of functional RANK
on mature rat and human osteoclasts. FEBS Lett 463:295–
300.
Naruse K, Urabe K, Mukaida T, Ueno T, Migishima F, Oikawa A,
Mikumi-Takagaki Y, Itoman M. 2004. Spontaneous differentiation
of mesenchymal stem cells obtained from fetal rat circulation.
Bone 35:850–858.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca
JD, Moorman MA, Simonetti DW, Craig S. Marshak DR. 1999.
Multilineage potential of adult human mesenchymal stem cells.
Science 284:143–147.
Ptaff M, Jurdic P. 2001. Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin aVb3. J Cell Sci 114:2775–
2786.
Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM.
2001. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98:2615–2625.
Silva WA Jr, Covas DT, Panepucci RA, Proto-Siqueira R, Siufi JLC,
Zanette DL, Santos ARD, Zago MA. 2003. The profile of gene
expression of human marrow mesenchymal stem cells. Stem Cells
21:661–669.
Smith A. 2006. A glossary for stem-cell biology. Nature 441:1060.
Taylor MV. 2003. Muscle differentiation: signaling cell fusion. Curr
Biol 13:R964–R966.
Tocci AL, Forte L. 2003. Mesenchymal stem cell: use and perspectives. Hematol J 4:92–96.
Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. 2004.
Human mesenchymal stem cells make cardiac connexins and
form functional gap junction. J Physiol 555:617–626.
Vitrat N, Cohen-Solal K, Pique C, Pierre Le Couedic J, Norol F,
Larsen AK, Katz A, Vainchenker W, Debili N. 1998. Endomitosis
of human megakaryocytes are due to abortive mitosis. Blood
91:3711–3723.
Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K,
Kikkawa U, Nishizuka Y. 1992. Cell division arrest induced by
phorbol ester in CHO cells overexpressing protein kinase C-d subspecies. Proc Natl Acad Sci U S A 89:10159–10163.
Yamaguchi K, Ogita K, Nakamura S, Nishizuka Y. 1995. The protein kinase C isoforms leading to MAP-kinase activation in CHO
cells. Biochem Biophys Res Commun 210:639–647.
Zhang J, Niu C, Huang H, He X, Tong W, Ross J, Haug J, Johnson
T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L. 2003.
Identification of the haematopoietic stem cell niche and control of
the niche size. Nature 425:836–841.
Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim
P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human
adipose tissue: implications for cell-based therapies. Tissue Eng
7:211–228.
Документ
Категория
Без категории
Просмотров
1
Размер файла
774 Кб
Теги
stem, following, phorbol, stimulating, cells, like, multinucleate, become, mesenchymal, oligopotent, esters, tpa, clones
1/--страниц
Пожаловаться на содержимое документа