Advance Publication Experimental Animals
Received: 2017.11.7 Accepted: 2018.2.14 J-STAGE Advance Published Date: 2018.3.8
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Original paper: Bioresource
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Subchondral Bone Derived Mesenchymal Stem Cells
3
Display Enhanced Osteo-Chondrogenic Differentiation,
4
Self-renewal and Proliferation Potentials
5
Hao Zhang1, 2, Zhong-Li Li1,*, Xiang-Zheng Su1, Li Ding3, Ji Li1, Heng Zhu2,*
6
1. Department of Orthopedics, Sports Medicine Center, People's Liberation
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Army General Hospital, Beijing 100853, China
8
2. Department of Cell Biology, Institute of Basic Medical Sciences, Beijing
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100850, P.R. China
10
3. Department of Hematology, General Hospital of Air Forces, PLA, Beijing,
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China
12 13
Corresponding author: Zhong-Li Li, Department of Orthopedics, Sports
14
Medicine Center, People's Liberation Army General Hospital, Beijing 100853,
15
China. Tel: 86-10-66938206. Fax: 86-10-88219862. Email:
[email protected]
16
Heng Zhu, Department of Cell Biology, Institute of Basic Medical Sciences,
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Taiping Road 27, Beijing 100850, P.R. China, and Tel: 861066930913, Fax:
18
861068213039 E-mail:
[email protected]
19 20 21
1
2
22
Abstract
23
Rabbit mesenchymal stem cells (MSCs) are important seed cells in regenerative
24
medicine research, particularly in translational research. In the current study, we
25
showed that rabbit subchondral bone is a reliable source of MSCs. First, we
26
harvested subchondral bone (SCB) from the rabbit knee-joint and initiated the
27
MSC culture by cultivating enzyme-treated SCB. Adherent fibroblast-like cells
28
that outgrew from SCB fulfill the common immuno-phenotypic criteria for
29
defining MSCs, but with low contamination of CD45+ hematopoietic cells.
30
Interestingly, differentiated SCB-MSCs expressed osteogenic and chondrogenic
31
markers at significantly higher levels than those in bone marrow cell
32
suspension-derived MSCs (BMS-MSCs) (P < 0.05). No differences in the
33
expression of adipogenic markers
34
0.05) were observed. Moreover, the results of the colony forming unit-fibroblast
35
assay and sphere formation assay demonstrated that the SCB-MSCs had
36
increased self-renewal potential. SCB-MSCs expressed higher levels of the
37
stemness markers Nanog, OCT4, and Sox-2 compared to in BMS-MSCs (P
2
3
44
properties of SCB-MSC are important for the potential treatment of tissue
45
damage resulting from disease and trauma.
46 47
Keywords: Bioresource; Experimental Animals; Mesenchymal Stem Cell;
48
Rabbit; Subchondral Bone
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4
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Introduction
51
Mesenchymal stem cells (MSCs), also known as multipotent stromal cells, were
52
first identified in the bone marrow[14]. In postnatal organisms, loosely woven
53
and highly vascularized bone marrow form a unique niche for stem cells[14,31].
54
Like hematopoietic stem cells, the multi-potency and self-renewal of MSCs are
55
tightly controlled by the bone marrow microenvironment[8,31,36]. According to
56
the requirements of the hosts, MSCs migrate out from connective tissue of bone
57
marrow and regenerate mesenchymal tissues[25]. Increasing data have shown
58
that MSCs play a role as promising seed cells for cellular replacement therapy
59
for diabetes, rheumatoid arthritis, and bone repair[8,17,18,31,38]. The rabbit is a
60
commonly used experimental animal for orthopedic application and tissue
61
engineering because of its easy accessibility and convenient maneuverability.
62
However, MSC-based therapies in rabbit models are limited because of
63
contamination by hematopoietic cells. Notably, the structure of the MSC niche
64
was typically destroyed, while rabbit MSCs were routinely cultured in bone
65
marrow cell suspension[2,16,36]. We previously isolated and characterized
66
MSCs and examined the potential application of these cells[24,45,48]. In our
67
previous studies, we found that collagenase digestion efficiently loosened the
68
tissue microstructure and facilitated MSC outgrowth from tissues without
69
reducing cell viability. In addition, enzymic treatment induced the release of
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hematopoietic cells and made it easier to deplete them[19,47].
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Because mesenchymal stem cells were first described in the 1970s, many types 4
5
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of biological tissue have been developed as stem cell resources[15,30].
73
Particularly,
74
nonimmunogenic, and easily available stem cells[1,22,26,35]. However, many
75
studies have shown that the sources of origin and microenvironment greatly
76
impact the differentiation ability of MSCs[26]. Researchers revealed that
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adipose tissue-derived MSCs show decreased osteogenic and chondrogenic
78
differentiation capacity compared to bone marrow-derived MSCs[9,42].
79
Therefore, attention should be given to subchondral bone (SCB), which is
80
accessible in orthopedics surgery and has a similar microenvironment, to
81
facilitate bone and cartilage injury regeneration.
82
Therefore, we hypothesized that culturing the SCB and allowing MSCs to
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migrate out from their stem cell niche may be an efficient strategy for obtaining
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viable and homogeneous rabbit MSC populations. We digested SCB and
85
conducted MSC (SCB-MSC) culture using these cells. Our results showed that
86
SCB-MSCs display enhanced osteo-chondrogenic differentiation, self-renewal,
87
and proliferation potential compared to bone marrow suspension-derived MSCs
88
(BMS-MSCs).
adipose
tissue
is
an
optimal
source
of
proliferating,
89 90
Materials and methods
91
Isolation and culture of SCB-MSCs
92
MSCs were isolated from male New Zealand White rabbits (3–4 weeks of age,
93
from the Laboratory Animal Center of the Academy of Military Medical 5
6
94
Sciences of China, Beijing, China). All experiments in this study were
95
performed in accordance with the Academy of Military Medical Sciences Guide
96
for Laboratory Animals. To isolate SCB-MSCs, the knee joints of the rabbit
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were carefully excised with scissors, and the subchondral bones were collected
98
using forceps. Subchondral bone fragments were cultured in α-MEM containing
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10% (vol/vol) fetal bovine sum (FBS) (Solarbio, Hyclone, Logan, UT, USA) in
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the presence of 1 mg mL−1 (wt/vol) of collagenase II (Gibco, Grand Island, NY,
101
USA) at 37°C for 20 min. The digestion medium and released cells were
102
discarded, and the enzyme-treated SCBs were seeded into a plastic culture dish
103
(250 mL) in the presence of α-MEM supplemented with 10% (vol/vol) FBS. The
104
culture medium was changed on the third day of culture, and the tissue debris
105
was maintained to allow more MSCs to outgrow. To isolate BMS-MSCs, the
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bone marrow was flushed out from the marrow cavity in the tibiae and femurs
107
and mononuclear cells were isolated from the bone marrow suspensions by
108
routine density gradient centrifugation. The cells were then seeded on a plastic
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dish (BD Biosciences, Franklin Lakes, NJ, USA, 100 × 15 mm), and the MSCs
110
were allowed to adhere for 72 h before the total volume of the culture medium
111
was changed.
112 113
Flow cytometry analysis
114
SCB-MSCs and BMS-MSCs were harvested at passages 3–6 by trypsin
115
digestion and stained individually with 6
phycoerythrin- or fluorescein
7
116
isothiocyanate-conjugated monoclonal antibodies against rabbit CD44, CD45,
117
CD14, CD79a, CD81, or CD90 (BD Biosciences and Abcam, Cambridge, UK)
118
for 30 min in the dark at 4°C. After two washes with PBS, the cells were
119
collected with a FACScan (BD Biosciences) and the data were analyzed using
120
WinMDI 2.9 software.
121 122
Multi-differentiation of MSCs
123
Multi-differentiation analysis of MSCs was performed as described previously
124
with minor modifications[19,47]. Briefly, for osteogenic differentiation, MSCs
125
at passage 3 were seeded into 24-well culture plates (1 mL/well) at a density of
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5 × 103 cells/cm2, grown in osteogenic induction medium for 14 days, and
127
subjected to alkaline phosphatase (ALP) staining. The osteogenic induction
128
medium consisted of culture medium, 0.1 µM dexamethasone, 10 mM
129
β-glycerophosphate, and 50 µM ascorbic acid (Sigma-Aldrich, St. Louis, MO,
130
USA). The osteogenic differentiation of MSCs was assayed by in situ ALP
131
staining with a commercial kit (Sigma-Aldrich).
132
For adipogenic differentiation, MSCs at passage 3 were seeded into 24-well
133
culture plates at a density of 1 × 104 cells/cm 2, incubated in adipogenic induction
134
medium for 14 days, and subjected to Oil-Red-O staining. The adipogenic
135
induction medium consisted of culture medium, 1 µM dexamethasone, 0.2 mM
136
indomethacin, 0.5 mM 3-butyl-L-methylxanthine (IBMX), and 0.01 mg/mL
137
insulin (Sigma-Aldrich). The accumulation of lipid vacuoles in MSCs was 7
8
138
evaluated by in situ Oil-Red-O staining.
139
For chondrogenic differentiation, 4 × 105 MSCs were centrifuged in
140
polypropylene tubes to form a pelleted micromass and maintained in
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chondrogenic induction medium consisting of α-MEM supplemented with 10−7
142
M dexamethasone, 1% (vol/vol) insulin-transferrin-sodium selenite, 50 µM
143
ascorbate-2 phosphate, 1 mM sodium pyruvate, 50 µg/mL (wt/vol) proline, and
144
20 ng/mL (wt/vol) TGF-β3. On day 21, the pellets were fixed and sectioned as
145
previously described[47]. The development of chondrocytes and accumulation
146
of the cartilage matrix were evaluated by toluidine blue staining.
147 148
Colony-forming unit-fibroblast (CFU-F) assay
149
The clonogenic potential of MSCs was tested in a colony-forming unit-fibroblast
150
(CFU-F) assay as described previously with minor revisions[11]. Briefly, MSCs
151
at passage 1 were seeded into a 6-well plate (Corning, Inc., Corning, NY, USA,
152
16.8 mL/well) at a density of 1 × 103/well and maintained in culture medium. To
153
detect the formation of CFU-F, the cultured cells in three replicates were stained
154
with 3% crystal violet in methanol for 10 min at days 5, 10, and 15. All visible
155
colonies larger than 5 mm in diameter were counted.
156 157
Sphere formation assay
158
The clonogenic potential of the MSCs was further tested in a sphere formation
159
assay[20,31]. MSCs at passage 1 were seeded at 2 × 105/cm 2 on an ultra-low 8
9
160
attachment dish (Corning) in α-MEM supplemented with 10% (vol/vol) FBS.
161
Primary cell spheres were counted after 3 days in culture, trypsinized, and
162
re-plated. Secondary spheres were counted on day 6.
163 164
CCK8 assay
165
MSC proliferation assays were performed using the Cell Counting Kit-8 (CCK-8;
166
Dojindo Laboratories, Kumamoto, Japan)[41]. Briefly, MSCs at passage 3 were
167
seeded into 96-well plates (Thermo Scientific, Waltham, MA, USA) at a density
168
of 1×103 cells/cm 2, cultured in α-MEM medium with 10% FBS (6 wells in each
169
group), added to CCK-8 solution at a ratio of 100 µL/µL, and incubated at 37°C
170
for 1 h. Absorbance was then measured at a wavelength of 450 nm using a
171
microplate reader (BMG LABTECH, Offenburg, Germany). In the current study,
172
CCK-8 experiments were performed on days 1, 5, 7, 10, and 13.
173 174
CFSE dilution assay
175
Moreover, the proliferation of SCB-MSCs and BMS-MSCs was also examined
176
in a CFSE dilution assay. Briefly, MSCs were suspended at a concentration of
177
107 cells/mL in PBS containing 2% FBS. MSCs were incubated in the presence
178
of 10 µM CFSE for 20 min in the dark, followed by blockage of CFSE
179
incorporation by FBS. The cells were then washed twice before they were
180
re-plated. MSCs were harvested on days 2 and 4. The dye dilution was assayed
181
with a FACSCalibur instrument and data were analyzed using WinMdi2.8 9
10
182
software.
183 184
Cell cycle assay
185
MSCs were seeded at 5 × 103 cells/cm 2 and cultured in α-MEM medium with 10%
186
FBS. At 80–90% confluence, the MSCs were collected for cell cycle analysis.
187
Briefly, the MSCs were washed and fixed overnight in 70% ethanol at -20°C in
188
1.5-mL microcentrifuge tubes (Biologix, Shandong, China). The fixed cells were
189
then washed and incubated in 100 µg/mL propidium iodide (Sigma-Aldrich) and
190
20 ng/mL RNase (Sigma-Aldrich) in PBS for 30 min. Cell cycle analysis was
191
then conducted by flow cytometry. Independent experiments were replicated at
192
least three times. The cell subpopulations in the G0/G1 and S phases were
193
calculated by gating analysis based on differences in DNA content.
194 195
Real-time polymerase chain reaction
196
Aliquots of MSCs (2 × 105) at passages 3–6 were seeded in 6-well culture plates
197
and maintained in osteogenic/adipogenic/chondrogenic induction medium for 7
198
days before they were harvested. Total RNA was extracted from MSCs with
199
TRIzol reagent (Invitrogen) and reverse-transcribed using the mRNA Selective
200
PCR Kit (TaKaRa, Shiga, Japan). Rabbit HPRT, Runx-2, osteopontin (OPN),
201
CEBP/α, PPARγ, Sox-9, collagen I, Nanog, OCT4, and Sox2 cDNA were
202
amplified by real-time PCR using the SYBR Green PCR kit (Sigma). The
203
primer sequences used for real-time PCR are shown in Table 1. 10
11
204 205
Western blotting
206
MSCs at passage 3 and 6 were plated in 6-well plates at a density of 1 × 105
207
cells/cm2 and starved in serum-free α-MEM medium for at least 6 h. Protein
208
lysis buffer (Bio-Rad, Hercules, CA, USA) was added, and thawed lysates were
209
vortexed and centrifuged. The proteins were separated by 10% sodium dodecyl
210
sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose
211
membranes. The membranes were blocked by incubation with 5% wt/vol nonfat
212
dry milk. Membranes were then incubated with anti-ERK, anti-phospho-ERK,
213
and β-actin (Sigma) Abs at the appropriate dilutions overnight at 4°C. After
214
incubation, the membranes were washed in Tris-buffered saline containing
215
Tween-20 (TBST). Secondary antibody conjugated to horseradish peroxidase
216
was added to the membranes in 5% nonfat dry milk in TBST. The negative
217
control was used as described previously. The western blotting assay was
218
performed at least 3 times independently, representative results are shown.
219 220
Statistical analysis
221
The data were expressed as the mean values with the standard deviation.
222
Statistical significance was analyzed by Student’s t test and two-tailed p-values
223
were calculated, and P < 0.05 was considered statistically significant. The error
224
bars in all figures represent the standard deviation.
225 11
12
226
Results
227
SCB-MSC exhibit morphological features and surface antigens similar to
228
those of BMS-MSCs
229
Forty-eight hours after the primary culture, fibroblast-like cells migrated out
230
from the digested SCB fragments and adhered to the dish (Figure 1A-a),
231
whereas a few elongated adhesion cells were observed in the dish in which the
232
bone marrow cell suspension cells were seeded (Figure 1A-b). An adherent layer
233
of vortex-shaped cells developed within 6 days (Figure 1A-c), whereas a culture
234
confluence of only 30–40% was achieved when the nuclear cells were cultivated
235
(Figure 1A-d). Further, the results of immuno-phenotyping showed that both
236
SCB-MSCs and BMS-MSCs were homogenously positive for the mesenchymal
237
markers CD44 and CD81 but negative for the hematopoietic markers CD14 and
238
CD45 and co-stimulating molecule CD79α (Figure 1B). Unlike human MSCs, it
239
remains controversial whether rabbit MSCs are positive for CD90[2,28,36]. Our
240
results showed that SCB-MSCs and BMS-MSCs were negative for CD90
241
(Figure 1B). In addition, the percentage of CD45+ cells in the SCB-MSCs (3.31
242
± 0.78%) was significantly lower than that in BMS-MSCs (13.93 ± 1.63%) (**P
243
＜ 0.01), demonstrating that a homogeneous cell population was expanded from
244
the digested subchondral bone (Figure 1C).
245 246
SCB-MSCs display enhanced osteogenic and chondrogenic differentiation
247
potential 12
13
248
Although the SCB-MSCs and BMS-MSCs shared similar morphologic and
249
immuno-phenotypic features, SCB-MSCs display enhanced differentiation
250
capacity compared to BMS-MSCs. Analysis of osteogenic differentiation
251
showed higher ALP activity in SCB-MSCs than in BMS-MSCs after 14 days of
252
induction (Figure 2A). Additionally, the analysis of chondrogenic differentiation
253
showed that more SCB-MSCs developed into toluidine blue-positive
254
chondrocytes, indicating that the cells secreted sulfated proteoglycan at a higher
255
level to form a cartilage extracellular matrix (Figure 2A). However, no
256
significant differences were observed in the accumulation of intracellular
257
Oil-Red-O-stained lipids, indicating that SCB-MSCs and BMS-MSCs shared a
258
similar adipogenic differentiation capacity (Figure 2A). Complementing the
259
results of histochemical analysis, SCB-MSCs after induction exhibited high
260
levels of mRNA expression of osteogenic markers (Runx-2 and OPN) and
261
chondrogenic markers (Sox-9 and Collage I) (*P < 0.05; **P < 0.01, Figure 2B).
262
The mRNA expression of adipogenic transcription factor CEBP/α and PPARγ in
263
SCB-MSCs was similar to that in BMS-MSCs (Figure 2B).
264 265
SCB-MSCs display higher self-renewal potential
266
Functional MSCs were initially identified by their capacity to form clonogenic
267
cell clusters in vitro, a common feature different to other stromal cell
268
populations. In the current study, self-renewal potential was measured in a
269
CFU-F assay and sphere formation assay. As indicated in Figure 3A and 3B, the 13
14
270
CFU-F frequency remained relatively higher in SCB-MSCs than in BMS-MSCs
271
(SCB-MSCs versus BMS-MSCs: 6.33 ± 0.94 versus 3 ± 0.82, 11 ± 1.63 versus
272
5.67 ± 0.94, 17 ± 0.82 versus 10.67 ± 1.25 for days 5, 10, and 15, respectively.
273
*P < 0.05; **P < 0.01).
274
Sphere formation assays have long been used to evaluate progenitor/multipotent
275
cell populations in epithelial systems. Recent studies suggested that MSCs can
276
also produce spheres[20,31]. Three days after culture on ultra-low adherent
277
tissue culture plates, sphere formation was evident in the SCB-MSC group and
278
BMS-MSC group (Figure 3C). These spheres were disassociated and re-plated
279
on non-adherent plates. Fewer spheres developed after another 3 days of culture.
280
Interestingly, there was a noticeable difference in primary and secondary sphere
281
number in SCB-MSC culture compared with to in BMS culture (SCB-MSCs
282
versus BMS-MSCs: 38 ± 9.53 versus 22 ± 0.82, 24.67 ± 3.21 versus 10 ± 1.63
283
for primary spheres and secondary spheres, respectively. Figure 3D, *P < 0.05;
284
**P < 0.01).
285
The results of the CUF-F and sphere formation assays strongly suggest that
286
SCB-MSCs have an increased stem cell population that can self-renew. To
287
further explore the cause of enhanced self-renewal, we next measured the
288
mRNA expression of several stemness markers (Nanog, OCT4, and Sox-2) in
289
SCB-MSCs[4,7,27,34].
290
significantly higher transcription levels of Nanog, OCT4, and Sox-2 than in
291
BMS-MSCs (Figure 3E, *P < 0.05; **P < 0.01).
The
data
indicated
14
that
SCB-MSCs
displayed
15
292 293
SCB-MSCs display enhanced proliferative capacity
294
To investigate the proliferation ability of SCB-MSCs, a CCK-8 assay and CFSE
295
dilution assay were performed. The results of the CCK-8-based cell proliferation
296
assay (Figure 4A) showed that SCB-MSCs exerted stronger proliferative effects
297
than BMS-MSCs (*P < 0.05). Consistently, the CFSE data showed that a higher
298
proportion of SCB-MSCs underwent cell division on days 2 and 4 (Figure 4B),
299
indicating that these cells had an enhanced proliferation capacity.
300
Enhanced cell proliferation is also reflected by an increased number of cells in
301
the S phase and decreased number of cells arrested in the G0/G1 phase. A higher
302
percentage of SCB-MSCs (50 ± 1.41%) were in S phase compared to
303
BMS-MSCs (36.5 ± 3.55%) (Figure 4C), indicating that an increased number of
304
cells proceeded into G2/S phase (*P < 0.05).
305
Because
306
proliferation[6,13], we further examined the phosphorylation of ERK-MAPK in
307
the cells. The data in Figure 4D shows enhanced Erk1/2 phosphorylation in
308
passages 3 and 6 SCB-MSCs. The results support that SCB-MSC harbors an
309
enhanced proliferation capacity.
ERK-MAPK
signaling
is
involved
in
controlling
cell
310 311
Discussion
312
Rabbit MSCs are important seed cells in regenerative medicine research,
313
particularly in translational research. A variety of healthy tissues have been 15
16
314
developed as stem cell resources, including bone marrow, blood, umbilical cord,
315
placenta, fat, heart, brain, skin, muscle, liver, gonads, and teeth[37]. Many
316
studies have shown that the differentiation ability of MSCs varies greatly from
317
different resources. In the orthopedics field, SCB has received attention in
318
regeneration research[10,21,40,44,46].
319
To identify MSCs, surface antigen markers were tested. It remains controversial
320
weather rabbit MSC express CD90 based on previous studies. Tan et al. (2013)
321
characterized rabbit MSCs and found that they expressed CD90[36]. Bakhtina
322
(2014) and Lee (2014) compared the surface markers between human and rabbit
323
MSCs and found rabbit MSCs did not express CD90[2,28]. The results of flow
324
cytometry analysis in the present study showed that rabbit MSCs were
325
CD90-negative, which is in accordance with the previous reports. The adult
326
bone marrow contains niches that control the multi-differentiation potential and
327
self-renewal capacity of stem cells[3]. Several studies demonstrated that
328
implanted bone marrow could support long-term repopulating cells in vivo[5,39].
329
Therefore, maintaining the bone marrow niche in primary culture may be
330
beneficial for MSC properties. In the present study, we initiated MSC culture
331
using digested rabbit SCBs, which are mainly composed of adipose tissue and
332
vessel networks.
333
Our results suggest that SCB-MSCs meet the generally accepted criteria,[12]
334
including the fibroblast-like morphology, typical cell surface profile, and
335
multi-lineage differentiation capacity. It had been widely accepted that MSCs 16
17
336
cultured from different tissues share many common features, but the
337
differentiation potential vary[9,26]. In this study, the results showed that
338
SCB-MSCs gain enhanced osteogenic and chodrogenic differentiation potential
339
that is comparable to that of BMS-MSCs, which is important for the potential
340
treatment of tissue damage resulting from disease and trauma.
341
Several factors have been reported to influence MSC self-renewal capacity,
342
including cell passages, differentiation, and other factors[23,33,43]. In the
343
present
344
differentiation into osteoblasts and chondrocytes, maintain a higher self-renewal
345
capacity. The results of the CFU-F and sphere forming assays suggest that
346
SCB-MSCs contain more potent cells. Nanog, OCT4, and Sox-2 are crucial
347
stemness transcription factors, and lower expression of these proteins leads to a
348
deficiency of self-renewal[4,7,27,34]. Based on the results of the colony
349
formation assay, SCB-MSCs expressed high levels of Nanog, OCT4, and Sox-2.
350
High proliferation is a fundamental property of MSCs and is important for the
351
potential treatment of tissue damage resulting from disease and trauma. The
352
CCK-8 assay and CFSE dilution assay are widely used to analyze the
353
proliferation of stem cells[29,32]. Because ERK-MAPK signaling is involved in
354
controlling cell proliferation, phosphorylation of ERK-MAPK in MSCs was also
355
detected in this present study. The results showed SCB-MSCs grew at a higher
356
rate than their marrow counterparts. These results demonstrate that the
357
proliferation of MSCs was improved in SCB culture.
study,
we
demonstrated
that
17
SCB-MSCs,
when
undergoing
18
358
There were also many limitations in our study. First, the most widely used MSC
359
resource in regenerative medicine domain is fat tissue, umbilical cord, and
360
placenta. We only compared bone marrow-derived MSCs and SCB-derived
361
MSCs in the present study. Second, all tests were performed in vitro in this study,
362
and an animal joint injury model would be useful in further studies to explore
363
the differences between different source origin-derived MSCs in vivo. Third, the
364
mechanism of differentiation and proliferation potential changes should be
365
evaluate in further studies.
366 367
Conclusion
368
In conclusion, our results support that maintaining the bone marrow niche in
369
MSC culture minimizes the negative impact on cell yield and purity while
370
retaining enhanced multi-potency, self-renewal, and proliferation potential of
371
MSCs. However, the precise mechanism regulating the fate of SCB-MSCs
372
requires further investigation. The results also suggest that SCB is a novel
373
resource for rabbit MSCs and may provide helpful information for
374
understanding MSC niches.
375 376
Acknowledgements
377
This study was supported by the National Natural Science Foundation
378
(81572159, and 81371945) and the Beijing Natural Sciences Grants (No.
379
7182123). 18
19
380
The authors declare no competing financial interests.
381 382
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Figure Legends
585
Figure 1. Morphologic and immuno-phenotypic features of SCB-MSCs and
586
BMS-MSCs
587
588 589
A: The morphologic characteristics of MSCs in two groups. B: The
590
immuno-phenotypic features of two groups. Both groups were homogenously
591
positive for mesenchymal markers but negative for hematopoietic markers. C:
592
The comparison of CD45+ cells between the SCB-MSC group and BMS-MSC
593
group (3.31 ± 0.78% vs 13.93 ± 1.63%) (**P < 0.01). The bar represents 200 µm
594
in Figure 1A. SCB-MSCs, subchondral bone-derived MSCs; BMS-MSCs, bone
595
marrow suspension-derived MSCs.
596 597
Figure 2. Results of multi-differentiation induction and RT-PCR assay
598 29
30
599 600
A: ALP and Oil-Red-O staining showed higher osteogenic and chondrogenic
601
potential in the SCB-MSC group after induction. There were no significant
602
differences in adipogenic potential between the two groups. B: Comparison of
603
mRNA expression levels of osteogenic (Runx-2 and OPN), chondrogenic (Sox-9
604
and collagen I) and adipogenic (CEBP/α and PPARγ) markers between the two
605
groups. The bar represents 200 µm in Figure 2A. SCB-MSCs, subchondral
606
bone-derived MSCs; BMS-MSCs, bone marrow suspension-derived MSCs.
607 608 609
Figure 3. CFU-F assay, sphere formation assay, and stemness markers
610 30
31
611 612
A, B: CFU-F frequency remained relatively higher in the SCB-MSC group than
613
in the BMS-MSC group. The bar represents 1 cm in Figure 3A. SCB-MSCs,
614
subchondral
615
suspension-derived MSCs. C, D: The results of primary and secondary sphere
616
culture revealed a significant difference between the two groups. The bars
bone-derived
MSCs;
31
BMS-MSCs,
bone
marrow
32
617
represent 100 µm in Figure 3C upper and 200 µm in Figure 3C low, respectively.
618
E: Comparison of mRNA expression of several stemness markers (Nanog,
619
OCT4, and Sox-2) between two groups.
620
Figure 4. SCB-MSCs display enhanced proliferative capacity
621
622 623
A: CCK-8-based cell proliferation assay indicated that the SCB-MSC group
624
harbors stronger proliferative potential than the BMS-MSC group (*P < 0.05). B:
625
CFSE data on days 2 and 4 showed that a greater proportion of SCB-MSCs
626
underwent cell division, indicating enhanced proliferation potential. C: The
627
results of cell cycle analysis showed a higher percentage of SCB-MSCs (50 ±
628
1.41%) were in the S phase compared to BMS-MSCs (36.5 ± 3.55%). D: The 32
33
629
data showed enhanced Erk1/2 phosphorylation in passages 3 and 6 SCB-MSCs.
630
SCB-MSCs, subchondral bone-derived MSCs; BMS-MSC, bone marrow
631
suspension-derived MSCs.
632 633
Table 1. Table 1: Primer sequences
634
genes HPRT Runx2 OPN
CEBP/α
PPARγ
primer sequences forward, reverse, forward, reverse, forward, reverse,
5′5′5′5′5′5′-
forward,
5′- GGGACGCTAGGTGACAGAAT -3′
reverse,
5′- GAAAGGACGCTGGCTGAAAA -3′
forward,
5′- TTGCTGTGGGGATGTCTCAT-3′
reverse,
5′- TTTCCTGTCAAGATCGCCCT-3′
Annealing temperature
GACCAGTCAACAGGGGACAT -3′ ACACTTCGAGGGGTCCTTTT -3′ ATTTCTCACCTCCTCAGCCC -3′ TCCCAAGTTTCCCTCATCCC -3′ TTTTGTCTCTTGGGCATGGC -3′ GCATTCTGCGGTGTTAGGAG-3′
60 °C Sox9
Collage I
Nanog
4-Oct
forward,
5′- ATGAAGATGACCGA CGAGCA -3′
reverse, forward,
5′- ACTTGTCCTCTTCGCTCTCC -3′ 5′- CCAAGGGAGAGCAAGGAGAA-3′
reverse,
5′- CCTTTGGGGCCTTCTTTTCC- 3′
forward,
5′-AAAACTCCCGACTCTGCAGA -3′
reverse,
5′-AGGCTGGAGAGTTCTTGCAT -3′
forward,
5′-CGGAAGAGAAAGCGAACGAG -3′ 33
34
Sox2
reverse,
5′-TGGCCTCAAAATCCTCTCGT -3′
forward,
5′-AAGGGAAATGGGGAGAGGTG - 3′
reverse,
5′-TGGATGGGATTGGTGGTCTC -3′
635
34
