IN VITRO BMSC DIFFERENTIATION IS PRIMARILY DRIVEN BY A NETWORK OF ENDOGENOUS BMP
EXPRESSION
Edgar C.E.; Chakravarthy V.; Barnes G.L.; Einhorn T. A.; Gerstenfeld L.C.
Boston University Medical Center, Boston, MA
INTRODUCTION
Bone morphogenetic proteins (BMPs) are multifunctional members of
the transforming growth factor- superfamily known to induce ectopic
bone formation. Bone Marrow Stromal Cells (MSCs) are a primary focus
of bone research as a primary stem cells population due to their ability to
differentiate into chondrogenic, adipogenic, or osteogenic lineages. Our
results demonstrate that BMPs are endogenously expressed by MSCs and
are critical to osteogenic differentiation. Furthermore, BMPs do not
appear to act independently but it appears that multiple BMPS act as part
of an autoregulatory network. Our results further show that the treatment
of MSCs with exogenous BMPs produce a variable autoregulatory effects
on the endogenous BMP expression.
METHODS
Marrow stromal cells were isolated from tibia and femora of 4-6 week
old Balb/c mice using the following protocol. Animals were euthanized
according to the IACUC approved protocol. Limbs were removed
aseptically, metaphyseal ends of the bones were cut off, and the marrow
cavity contents were flushed with alpha-modified MEM ( MEM)
supplemented with 10% FBS and 1% penn/strep. Cells were plated at 10
x 106 cells/well in 6 well plates. On day 4 of culture, half of the media
was removed and replaced with fresh MEM. On day 5 media was
removed and replaced with complete media [ MEM supplemented with
10% FBS, 1% Penn/Strep, Dexamethasone (10-8 M), Ascorbic acid
(50ug/ml) and beta-glycerol phosphate (8mmol)] with or without BMP-7
or BMP-2 at 250ng/ml. The media was replaced every 2 days. All
cultures were maintained for 21 days and subsequently either fixed for
Von Kossa staining or total RNA isolated using the Trizol reagent. Gene
expression levels were analyzed by RNase Protection Analysis (RPA)
using commercially available template sets per the manufacturer’s
recommended protocol (Pharmingen, Inc., San Jose, CA).
RESULTS:
A. Total Nodule count
C.
Days 10 14 21 10 14
21
140
OPN
Control
120
Noggin Treated
100
BSP
80
60
40
20
Col 10A1
0
14
16
21
B. Avg. Area per Nodule
Col 2A1
140.00
Col 1A1
120.00
100.00
80.00
60.00
OC
40.00
20.00
0.00
14
16
L32
21
Control
Noggin treatment
Figure 2. BMPs are critical to sustain osteogenic differentiation in MSC
primary cultures. A. Total Von Kossa positive nodules in normal media
or media supplemented with 300ng/ml Noggin. B. Average area per Von
Kossa positive nodule with or with out 300ng/ml Noggin
supplementation. C. RPA of predominantly expressed matrix genes
from primary MSC cultures with or with out 300ng/ml Noggin treatment.
Exogenously added BMP-2 and BMP-7 demonstrate variable autoregulatory effects on endogenous BMP expression.
A.
Control
BMP-7 Treated
Days 10 14 16 21 10 14 16 21
B.
Days
BMP-8a
BMP-7
BMP-8a
BMP-6
BMP-5
BMP-6
BMP-2 Treated
6 7
8 10 14 16 21
BMP-7
BMP-5
BMP-4
Endogenous BMP expression increases as MSC osteogenic
differentiation progresses.
BMP-4
BMP-3b
A.
BMP-3
C.
Days
140
10
14
BMP-2
21
BMP-8A
BMP-1
BMP-7
BMP-8b
BMP-6
BMP-5
L32
80
60
40
20
0
14
16
21
BMP-3
BMP-2
120
100
BMP-3b
BMP-1
BMP-8b
L32
Days
BMP-4
B.
BSP
OC
6
5
Figure 3. BMPs demonstrate variable auto-regulatory effects on
endogenous BMP expression. A. RPA analysis BMP expression in
Control compared to BMP-7 (250ng/ml) treated MSC over a 21 day time
course. B. RPA analysis of BMPs expressed over a 21 day time course
following BMP-2 treatment (250ng/ml) of MSC cultures.
BMP-3B
BMP-3
BMP-2
7
4
3
BMP-1
2
1
0
7
14
16
Days
21
L32
Figure 1. BMP expression increases as osteogenic differentiation occurs
in MSCs. A. Total nodule count of Von Kossa positive mineralized
nodules at days 14, 16, and 21 after plating. B. The relative expression
levels of bone sialoprotein (BSP) and osteocalcin (OC) as determined by
densitometry analysis form a RPA gel at days 7, 14, 16, and 21 after
plating. C. RPA analysis of BMPs expressed during osteogenic
differentiation of MSC at days 10, 14 and 21 after plating.
REFERENCES
1. Gerstenfeld, LC et al. (2002) JBMR 17:221-230.
2. Gerstenfeld, LC et al. (2001) Cell Tissue Ogans. 169(3) 285-294.
3. Cho, TJ; Gerstenfeld, LC; Einhorn, TA (2002) JBMR. 17(3) 513-520.
4. Cheifetz, S. et al (1996) Con. Tiss. Res., Vol 35(1-4) 71-78
Endogenous BMPs are critical to MSC osteogenic differentiation.
DISCUSSION
Our results show that MSCS produce multiple BMPs and that the
expression of these BMPs increase over the time course of MSCs
differentiation. The antagonism of endogenous BMP expression with
Noggin blocks MSC differentiation based both on the formation of
mineralized nodule formation and the expression of ECM mRNAs
indicative of osteogenic differentiation. The exogenous addition of
either BMP-2 or BMP-7 uniquely down regulates the endogenous MSCs
expression of specific BMPs. Specifically BMP-7 treatment down
regulated all normally expressed BMP examined except for BMP-2,
BMP-8a, and BMP-1. Exogenously added BMP-2 stimulates BMP-5,
and BMP-4 expression compared to the endogenous levels without BMP
treatment. In aggregate these data suggest that a BMP network exists
that auto-regulates their own expression and upon which skeletalgenic
cell differentiation is dependent.
50th Annual Meeting of the Orthopaedic Research Society
Poster No: 0769