Phenotypic and Functional Characterization of a Marrow

Research Article
Phenotypic and Functional Characterization of a
Marrow-derived Stromal Cell Line, M210B4 and its
Comparison with Primary Marrow Stromal Cells
Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale*
Stem Cell Laboratory, National Centre for Cell Science, NCCS Complex, University of Pune Campus, Ganeshkhind,
Pune, India
In vitro co-culture system consisting of bone marrow stromal cells (BMSCs) or mesenchymal stromal cell
lines of marrow origin has provided important clues about the regulation of hematopoietic stem cells (HSCs)
by their microenvironment or niche. In the current studies, we have compared phenotypic and functional
characters of a marrow-derived mesenchymal stem cell line, M210B4, with BMSCs. We demonstrate that
M210B4 resembles BMSCs in terms of phenotypic characters. Unlike the BMSCs, M210B4 differentiated
only towards adipogenic lineage, and was refractory towards osteogenic differentiation. However, M210B4
cells exhibited a higher HSC-supportive ability as assessed by flow cytometry analyses of the output cells
from co-cultures. We observed that M210B4 cells show a constitutively higher activation of p44/42 and p-38
MAPK pathways compared to BMSCs, contributing to their higher HSC-support in vitro. Overall, the results
show that M210B4 forms a suitable in vitro system to study HSC regulation in vitro.
INTRODUCTION
Hematopoiesis takes place in bone marrow, a
various cytokines and extra-cellular molecules
complex micro-environment comprised of
(ECMs).
various cells like osteoblast, endothelial cells,
emanating from these cells also contribute
CXCL12-abundant reticular (CAR) cells,
actively in the HSC fate decision (Blank et al.,
adipocytes,
cells
2008; Eckfeldt et al., 2005). Mesenchymal
(MSCs), pericytes, etc. (Calvi et al., 2003;
stromal cells (MSCs) form a very important
Ding et al., 2012; Kiel et al., 2005; Mendez-
part of the HSC microenvironment. Although
Ferrer et al., 2010; Omatsu et al., 2010; Park et
these cells have been used as feeder layers for
al., 2012; Sacchetti et al., 2007; Sugiyama et
several years (Jang et al., 2006), their precise
al., 2006; Zhang et al., 2003). Hematopoietic
participation
stem cells (HSCs) occur in close contact with
documented recently (Jing et al., 2010;
the cells of the microenvironment, which
Mehrasa et al., 2014; Walenda et al., 2010).
control the fate of the HSCs via secretion of
Subsequent studies have demonstrated that
mesenchymal
stromal
Various
in
signaling
the
HSC
mechanisms
niche
was
Key words: Mesenchymal stromal cells, M210B4, HSC.
*Corresponding Author: Vaijayanti Kale, National Centre for Cell Science, NCCS complex, University of Pune Campus,
Ganeshkhind, Pune, India.
Email: [email protected], [email protected]
Biomed Res J 2015;2(1):120-133
Singh et al.
121
MSCs actively participate in regulation of
whether they express MSC-like phenotype and
hematopoiesis and play an important role in
support hematopoiesis with efficiency at par
homing and engraftment of transplanted HSCs
with BMSCs. In the present study, we have
(Bensidhoum et al., 2004; Sohni et al., 2013).
compared M210B4 cell line with the primary
In vitro co-culture of HSCs with stromal
feeder layers forms an excellent model to
bone marrow-derived stromal cells (BMSCs)
using phenotypic and functional parameters.
study molecular mechanisms involved in the
regulation of hematopoiesis in general, and
MATERIALS AND METHODS
HSC fate in particular. Murine stromal cells
Cells
were isolated and characterized by Tropel et
The protocols used in animal experimentation
al. (2004) and constituted an important in vitro
were approved by the institutional animal
tool to study stromal cell biology. To have a
ethics committee (IAEC). The C57BL/6J
constant supply of feeder cells, several stromal
(CD45.2)
cell lines were generated. Some of the clonal
(Ptprc;
stromal cell lines that have been established
Laboratory, Bar Harbor, USA) were housed
are PA6 (Piacibel et al., 1996), M210B4
and bred in our experimental animal facility
(Sutherland et al., 1991), S17 (Winwman et
(EAF). BMSCs were isolated from bone
al., 1993), and MS5 (Tordjman et al., 1999).
marrow of 6–8 weeks C57BL6/J mice by
These cells have been successfully used as
flushing the femurs with complete medium,
feeder layers and possess hematopoiesis-
constituting Iscove's modified Dulbecco's
supportive ability in vitro.
medium (IMDM) (HiMedia, Mumbai, India)
and
B6.SJL-PtprcaPepcb/BoyJ
CD45.1)
mice
(The
Jackson
The M210B4 cell line is a clone derived
supplemented with 20% mesenchymal stem
from bone marrow stromal cells from
cell FBS (Mesen-FBS; Stem Cell Technology,
(C57BL/6J × C3H/HeJ) F1 mouse (Lemoine
Vancouver, British Columbia, Canada) and
et al., 1988), and supports hematopoiesis when
plated in a petridish. After 8–10 days of
used as feeder layer for long-term culture-
incubation, with intermittent removal of non-
initiating cell (LTC-IC) assay (Burroughs et al
adherent cells and addition of fresh medium,
1994). Our group has used this cell line to
the adherent cells were used for the
study various aspects of hematopoiesis (Bajaj
experiments (Anjos-Afonso et al., 2008).
et al., 2011; Hinge et al., 2010).
M210B4 cell line was purchased from ATCC
In spite of its extensive use in LTC-IC
and maintained in RPMI1640 (HiMedia,
assays, these cells have not been critically
Mumbai, India) supplemented with 10% FBS
evaluated
primary
(GIBCO, Invitrogen, Carlsbad, California,
marrow-derived stromal cells. It is not known
USA). Lineage negative (lin-) cells were
in
comparison
with
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
122
isolated from bone marrow mononuclear cells
irradiated (8000 rads of gamma ray, Co60)
(MNCs) of Ptprc mice by using biotin-labelled
feeders for 7 days in either IMDM
anti-mouse lineage antibody cocktail prepared
supplemented with 10% MSC-FBS (MSC
from Biotin Mouse Lineage panel (BD
qualified
Pharmingen, San Diego, California, USA) and
California, USA) or Myelocult medium (Stem
®
Dynabeads
biotin
binder
(Invitrogen,
Calrsbad, California, USA).
Cell
FBS,
Invitrogen,
technology,
Columbia,
Carlsbad,
Vancouver,
Canada). The
British
medium
was
supplemented with 25 ng/ml murine IL-6, 25
Differentiation towards adipocytes and
ng/ml murine SCF and 10 ng/ml murine IL-3
osteoblasts
(Peprotech, Rocky Hill, USA). We compared
For adipocytic differentiation, BMSCs or
the HSC-supportive ability of M210B4 and
M210B4 cells were treated with standard
BMSCs
adipogenic
conditions:
differentiation
medium
under
four
A)
different
Non-irradiated
culture
BMSCs/
comprising insulin (4 μg/ml), 3-isobutyl, 1-
M210B4 in (IMDM + 10% Mesen-FBS),
methyl
B) Irradiated BMSCs/M210B4 in (IMDM +
xanthine
(IBMX)
(500
µM),
dexamethasone (0.25 μM), indomethacin (200
10%
Mesen
FBS),
C)
Non-irradiated
µM) and 2 µg/ml insulin for 15-18 days. The
BMSCs/M210B4 in Myelocult medium and
adipogenic differentiation was confirmed by
D) Irradiated BMSCs/M210B4 in Myelocult
staining lipid droplets with Oil Red O dye
medium. After 7 days of co-culture, the cells
osteoblastic
were harvested and analyzed for LSK (lin-
differentiation, BMSCs or M210B4 cells were
Sca1+ c-Kit+) stem cell population using flow
treated with β glycerophosphate (10 nmol/L),
cytometry. The LSK population was further
dexamethasone (100 nmol/L) and ascorbic
analyzed as long-term HSCs (LT-HSCs) or
acid (0.05 nmol/L) for 15–18 days. To confirm
short-term HSCs (ST-HSCs) based on CD34
osteoblastic differentiation, cells were stained
expression. The absolute numbers were
with Alizarin Red S to detect calcium deposits
calculated based on % of cells obtained on
(Sila-Asna et al.,2007).
flow cytometer and total yield, while the fold
(Bajaj
et
al.,
2011).
For
increase
was
calculated
by
comparing
Co-culture assay
absolute numbers of input population to that of
BMSCs or M210B4 cells were seeded in
the output population.
collagen-coated (50 µg/ml) 24-well plate as
feeder layer. After 24 h, 1 × 105 lin- cells
Flow Cytometry
isolated from mouse bone marrow were co-
For the phenotypic characterization, BMSCs
cultured
and M210B4 cells were stained with APC-
with
either
non-irradiated
Biomed Res J 2015;2(1):120-133
or
Singh et al.
123
conjugated anti-mouse CD44, PE-conjugated
membranes (Biorad, California, USA). The
anti-mouse CD73, APC-conjugated anti-
blots were incubated with specific primary
mouse CD90.1, PE-conjugated anti-mouse
antibodies
CD105, APC-conjugated anti-mouse CD106
(Thr202/Tyr204), anti-p38, and anti-p-p38
(eBioscience, San Diego, California, USA),
(Thr180/Tyr182); and horseradish peroxidase-
PE-conjugated anti-mouse Sca-1, FITC-
conjugated
conjugated anti-mouse CD45 and FITC-
Signaling Technology, Danvers, Massachu-
conjugated
(BD
settes, USA). The signals were detected using
Pharmingen, San Diego, California, USA).
Lumiglo reagent (Cell Signaling Technology,
For LSK profiling, cells were stained with
Danvers, Massachusettes, USA) and the
APC-conjugated mouse lineage antibody
signals were captured on X-Ray films.
cocktail,
anti-mouse
PECy7-conjugated
CD117(c-Kit),
Sca-1
CD34
(BD
PE-conjugated
anti-p44/42,
secondary
anti-p-p44/42
antibodies
(Cell
anti-mouse
anti-mouse
Data were analyzed by Sigma Stat software
California, USA) and FITC-conjugated anti-
using one-way repeated measure analysis of
mouse CD34 (eBioscience, San Diego,
variance (One-Way RM ANOVA; Jandel
California, USA). The isotype-stained cells
Scientific Software, California, USA). The
were used as controls. The stained cells were
plots represent the values as mean ± standard
acquired
error of mean (SEM), and p value ≤ 0.05 was
FACS
Canto
San
Statistical analysis
Diego,
on
Pharmingen,
–
II
(Becton
Dickinson, New Jersey, USA) and analyzed
considered significant.
using BD FACS-DIVA SOFTWARE version
5.0.
RESULTS
M210B4 cell line is comparable to BMSCs
Western blots
at phenotypic level
Whole cell lysates were prepared using RIPA
BMSCs are phenotypically characterized by
lysis buffer supplemented with Phosphatase
surface markers as Sca1+CD44+ CD73+
Inhibitor-1(PI-1), Phosphatase Inhibitor-2 (PI-
CD90.1+ CD105+ CD106+ CD45- CD34-
2), Protease Inhibitor Cocktail (PIC), 1 mM
(Chamberlain et al., 2007; Boxall SL et al.,
Phenyl Methyl Sulphonyl Fluoride (PMSF), 1
2012; Mabuchi et al., 2013). When M210B4
mM Sodium-orthovanadate and 1 mM
cells
Sodium Fluoride (NaF) (Sigma-Aldrich, St.
phenotypic analyses using the standard
Louis, Missouri, USA). The samples having
markers, we observed that both BMSCs as
equal protein concentration were separated by
well as M210B4 are highly positive for CD44,
9% SDS-PAGE and transferred to PVDF
CD106 and Sca-1; and moderately positive for
and
BMSCs
were
subjected
to
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
124
Table 1: Percent and mean fluorescence intensity (MFI) of different surface markers in M210B4 and BMSCs.
S. No.
Surface Markers
1
CD44
2
CD73
3
CD90.1
4
5
CD105
CD106
6
Sca-1
7
CD34
8
CD45
M210B4
BMSCs
a)
% population
99.99
73.89
b)
MFI
35445
1680
a)
% population
8.92
25.84
b)
MFI
216.43
178.41
a)
% populati1on
16.17
28.73
b)
MFI
253.06
173.34
a)
% population
31.48
56.75
b)
MFI
230.91
595.31
a)
% population
99.96
93
b)
MFI
4872
2432
a)
% population
98.99
86.51
b)
MFI
24088
5751
a)
% population
Nil
Nil
b)
MFI
Nil
Nil
a)
% population
Nil
Nil
b)
MFI
Nil
Nil
CD73, CD90.1 and CD105 (Fig. 1A). In terms
differentiation potential. We observed that
of percentages, more number of BMSCs
M210B4 cells differentiated into adipocytes
expressed CD73, CD90.1 and CD105. Both
(Fig. 1B), and not towards the osteogenic
cells were negative for CD34 and CD45. The
lineage. The data showed that M210B4
data showed that M210B4 cells compare well
efficiently differentiates towards adipogenic
with BMSCs at phenotypic level, albeit with
lineage, but not towards the osteoblastic one.
minor numerical differences (Table 1).
M210B4 cells expand long-term HSCs in
M210B4 cell line differentiates towards
vitro
adipocytic lineage
The results showed that in type 'A' (Non-
The marrow-derived Mesenchymal stromal
irradiated feeders in IMDM supplemented
cells are expected to differentiate towards
with 10% MSC-FBS) and type 'B' (irradiated
osteoblastic, and adipocytic lineages under
feeders in IMDM supplemented with 10%
appropriate stimuli (Prockop DJ., 1997;
MSC-FBS) co-cultures, the total number of
Pittenger MF et al., 1999; Dominici et al.,
cells harvested was significantly low in
2006). However, when these cells are cultured
M210B4 set (Fig. 2A-a, 2B-a). The absolute
for long periods they lose their differentiation
numbers of LT-HSCs (LSK CD34-) were
capacity. So we investigated whether M210B4
significantly increased, whereas the absolute
cells possess both adipogenic and osteogenic
numbers of LSK HSC (Lin-Sca-1+ c-Kit+), and
Biomed Res J 2015;2(1):120-133
Singh et al.
125
Figure 1: Characterization of murine bone marrow stromal cells and M210B4 cell-line. A) Flow cytometric analysis
for the MSC surface markers expressed on BMSCs versus M210B4 cell-line; B) The panel illustrates images of
adipocytes formed from M210B4 cells and BMSCs stained with Oil Red O. The panel shows that BMSCs differentiate
towards the osteoblastic lineage as evident by Alizarin Red S staining; M210B4 cells do not show such differentiation.
(Original magnification 100X).
Biomed Res J 2015;2(1):120-133
126
Characterization of a marrow-derived stromal cell line, M210B4
Figure 2: Co-culture of murine hematopoietic stem cells with BMSCs versus M210B4 cell-line. Total numbers of
hematopoietic cells obtained in various co-cultures are depicted (a). Quantitative data showing absolute number of
HSCs (b) and fold change over input cells (c) with respect to total cells harvested, LSK, LT-HSC and ST-HSC, at 4
different conditions (2A) Non-irradiated BMSCs/M210B4 in (IMDM+10%MSC-FBS), (2B) Irradiated BMSCs/M210B4 in
(IMDM+10%MSC-FBS), (2C) Non-irradiated BMSCs/M210B4 in Myelocult medium and (2D)Irradiated BMSCs/M210B4
in Myelocult medium. The data of one representative experiment are depicted and are represented as mean ± SEM. N=3.
ST-HSC (LSK CD34+) were significantly
(Fig. 2A-c and 2B-c). There was no significant
decreased in the M210B4 set as compared to
difference in M210B4 set versus BMSCs set in
the BMSCs set (Fig. 2A-b and 2B-b; Table 2).
type 'C' (non-irradiated feeders in Myelocult
In terms of fold increase over input, the result
medium) co-cultures with respect to LSK-
was the same as that of the absolute numbers
HSC, LT-HSC and ST-HSC populations in
Biomed Res J 2015;2(1):120-133
Singh et al.
127
Table 2: The absolute numbers (mean ± SEM, n=3) and fold increase over input from one representative experiment
have been tabulated.
3
Absolute numbers × 10
Culture
condition
LSK
LT-HSC
ST-HSC
BMSCs
M210B4
BMSCs
M210B4
BMSCs
M210B4
A
30.53±0.85
13.81±0.93
3.22±0.25
10.59±1.26
26.84±1.02
2.11±0.57
B
19.15±1.12
12.67±0.61
1.10±0.07
4.40±0.52
17.94±1.06
7.95±0.10
C
9.69±1.06
13.38±3.86
7.57±1.03
11.63±3.52
1.31±0.07
0.90±0.23
D
3.27±0.15
10.42±0.44
1.88±0.16
7.99±0.34
1.17±0.06
1.75±0.30
Fold increase over input
Culture
condition
LSK
LT-HSC
ST-HSC
BMSCs
M210B4
BMSCs
M210B4
BMSCs
M210B4
A
10.60±0.29
4.80±0.32
1.62±0.12
5.33±0.63
26.62±1.01
2.10±0.56
B
6.65±0.39
4.40±0.21
0.55±0.03
2.22±0.26
17.80±1.05
7.88±0.09
C
3.36±0.37
4.65±1.34
3.81±0.52
5.85±1.77
1.29±0.07
0.89±0.23
D
1.14±0.05
3.62±0.15
0.95±0.08
4.02±0.17
1.16±0.06
1.74±0.29
terms of absolute numbers and fold increase
A, B and D culture conditions used in the
over input (Fig. 2C-b–c; Table 2), with the
experiments, with respect to the primitive LT-
exception of significantly lower total cells
HSC population (Table 2). Under the culture
harvested in M210B4 as compared to that of
condition C, however, both BMSCs and
BMSCs (Fig. 2C-a). Type 'D' co-cultures
M210B4 gave comparable output of various
(irradiated feeders in Myelocult medium) did
HSC populations (Fig. 2C).
not show any difference in the total cells
harvested in M210B4 and BMSCs (Fig. 2D-a),
Signaling pathways operative in M210B4
but the absolute numbers and fold increase
vs. BMSCs
over input with respect to LSK and LT-HSC
Earlier data showed that M210B4 exhibits a
were significantly higher in M210B4 (Fig. 2D-
better HSC-supportive ability compared to the
b–c; Table 2). The absolute number and fold
BMSCs. To examine whether this was related
increase over input of ST-HSC between
to the difference in their signaling gamut, we
M210B4 and BMSCs was non-significant.
subjected the lysates of these cells to western
Flow cytometry graphs for all the 4 sets of co-
blot analyses to detect phosphorylation of p38
culture conditions (A–D) are depicted in
and p44/42 MAPK. The four different culture
supplementary Fig. S1. The data showed that
conditions described above were used.
M210B4 cells have a better HSC-supportive
We observed that under most of the culture
potential in vitro as compared to BMSCs under
conditions the levels of phospho p-38
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
128
Figure 3: Western blot analyses of phosphorylated forms of p38 and p44/42 in BMSCs versus M210B4 cell-line.
Total cell lysate of 48 h cultured BMSCs and M210B4 at different conditions, as shown in the figure were subjected to
Western blot analyses. The blots were probed with (i) anti p-p38 and total p38 and (ii) anti p-p44/42 and total p44/42
antibodies. Equal input of proteins was ensured by probing the blots with antibody to α-tubulin. The intensity of the bands
was quantitated by densitometry using ImageJ software.
(Thr180/Tyr182) were higher in M210B4 as
regulation of HSC fate. The cell line models
compared to the BMSCs (Fig.3A). The levels
become especially useful, when one needs to
of phospho p44/42 (Thr202/Tyr204) were
use a genetic approach to over-express or
higher in M210B4 under culture conditions A,
silence any particular gene. Primary cells can
B and D as compared to BMSCs (Fig. 3B).
also be genetically modified, but need viral
Under the culture condition C, the level of p-
vectors to get sufficient numbers of modified
p38 was high in M210B4, whereas the level of
cells. Secondly, being primary cells, the
p-p44/42 was comparable to BMSCs.
modified cells wither off, necessitating their
Collectively, the data suggests that
reestablishment. Cell lines give a distinct
increased levels of phospho p38 and phospho
advantage of unlimited supply of cells, and
p-44/42 in M210B4 may have conferred upon
also allow use of simple plasmid-based system
M210B4 a better HSC-supportive ability
for gene manipulations. They also facilitate
under culture conditions A, B and D.
generation of independent clones showing
stable expression.
Several cell lines like PA6 (Piacibel et al.,
DISCUSSION
In vitro co-culture system using non-irradiated
1996), M210B4 (Sutherland et al., 1991), S17
or irradiated feeder layers formulated with
(Winwman et al., 1993), and MS-5 (Tordjman
primary
mesenchymal
et al., 1991) have been established and
stromal cells or cell lines, is a useful tool to
successfully used in co-culture studies. Our
understand
group has mainly used M210B4 cell line in our
marrow
the
derived
stromal
Biomed Res J 2015;2(1):120-133
cell-mediated
Singh et al.
129
previous work (Hinge et al., 2010; Bajaj et al.,
differentiate towards chondrocytic, and also
2011). Presently, our group is involved in
towards other lineages like neural, muscular,
genetically modifying these cells using
etc.
plasmid vectors expressing various molecules
We subjected both M210B4 and primary
involved in regulation of hematopoiesis.
stromal cells in co-culture studies. Since the
When we initiated these studies, we noticed
feeder cells can be used in non-irradiated or
that though this cell line has been frequently
irradiated form, we employed both the
used in experiments (Sutherland et al., 1991),
conditions and used two different kinds of
its phenotype in comparison with BMSCs has
media typically used in such experiments. The
not
its
data showed that though under most of the
hematopoiesis support in comparison with
culture conditions the total output of
BMSCs has not been examined.
hematopoietic cells in M210B4 set was low,
been
established.
Secondly,
Therefore we compared phenotype of
except in co-culture condition 'D', the fold
M210B4 with BMSCs. We showed that these
increase of LT-HSCs over the input was
cells are phenotypically comparable to
consistently high in M210B4 set, except under
primary BMSCs. On examination of the
condition 'C'. The data suggest that M210B4
differentiation
and
cells possess better ability to support the LT-
adipocytic lineages, we observed that the
HSCs. In culture condition 'C', the absolute
M210B4 cells differentiated into adipocytes
number and the fold increase over input for LT-
(Fig. 1B), but were refractive for osteoblastic
HSCs were higher than in the BMSCs set, but
differentiation. BMSCs are most commonly
the data did not reach significance. This could
known to differentiate into osteoblastic,
be related to milder activation of p44/42
adipocytic and chondrogenic lineages. We
MAPK activation under this culture condition
have shown such tri-lineage differentiation of
(Fig. 3B). Adipocytes are considered as
placental MSCs (Sharma et al 2012).
negative
Adipocytes and osteoblasts form important
(Naveiras et al., 2009), but M210B4 cells in
components of the HSC niche and thus the
spite of being able to differentiate to
differentiation of BMSCs towards these two
adipocytes supported HSC proliferation. This
lineages is relevant in the context of
shows that differentiated adipocytes, but not
hematopoiesis. Therefore, in the present study
pre-adipocytes, may exert negative effect on
we examined the ability of M210B4 to
the HSCs. The overall low output of
differentiate
and
hematopoietic cells thus appears to be
osteoblastic lineages. However, it will be
primarily due to low proliferation of
interesting to see whether these cells
committed progenitors.
towards
towards
osteoblastic
adipogenic
modulators
of
hematopoiesis
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
130
The signaling mechanisms operative in
the stromal cells are known to affect HSC fate.
that alteration of p44/42 and p38 pathways in
the stromal cells can affect HSC fate.
Our data showed that constitutively activated
In summary, our study shows that
p44/42 and p38 pathways in M210B4 cells
M210B4 cell line shows phenotypic similarity
under all culture conditions except for the
with the primary BMSCs and has higher HSC-
levels of p-p44/42 under the culture condition
supportive properties by the virtue of the
'C,' may be responsible for their better HSC
signaling gamut present in them. This cell line
support. Stromal cells regulate the HSC fate
thus forms a suitable model to examine the
via secretion of various cytokines and ECM
stromal cell-mediated regulation of stem cell
molecules (Scadden, 2006; Baraniak et al.,
fate. Similarly, this cell line is a suitable model
2010). Cell–cell interactions are also known to
to study adipogenesis (Bajaj et al., 2011).
play important role in this process. In our
future experiments we propose to examine
ACKNOWLEDGEMENTS
whether
show
The authors acknowledge Department of
differential ECM and adhesion molecule
Biotechnology (DBT), Government of India,
profile.
New Delhi (Grant
M210B4
and
BMSCs
BT/PR14036/MED/31/
Our group has shown that p44/42 and p38
101/2010), Director, NCCS, FACS core
pathways are coupled and inversely regulated
facility, and Council of Scientific and
in primitive stem cells (Kale 2004; Kale et al.,
Industrial Research (CSIR) for fellowship to
2004;
SS, SG and MRD).
Kale
autonomous
2005). Although
the
role
signaling
of
MAPK
cell
pathways in the regulation of hematopoiesis is
CONFLICT OF INTEREST
known (Geest et al., 2009), our study showed
The authors claim no conflict of interest.
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