Journal of Plastic, Reconstructive & Aesthetic Surgery (2014) 67, 1427e1435

Adipogenic differentiation potential of rat

adipose tissue-derived subpopulations of
stromal cells*
M. Gierloff a,*, L. Petersen a, H.-H. Oberg b, E.S. Quabius b,c,
il a
J. Wiltfang a, Y. Ac
a

Department of Oral & Maxillofacial Surgery, Christian-Albrechts-University, Kiel, Germany

Department of Immunology, Christian-Albrechts-University, Kiel, Germany
c
Department of Othorhinolaryngology, Head and Neck Surgery, Christian-Albrechts-University, Kiel,
Germany
b

Received 21 January 2014; accepted 20 May 2014

KEYWORDS
Adipose-derived
stromal cells;
ASCs;
Subpopulations;
Differentiation
potential;
Fat regeneration

Summary Adipose-derived stromal cells (ASCs) are mostly isolated by enzymatic digestion,
centrifugation and adherent growth resulting in a very heterogeneous cell population. Therefore, other cell types in the cell culture can comprise the differentiation and proliferation potential of the ASC population. Recent studies indicated that an antibody-aided isolation of
distinct ASC subpopulations provides advantages over the conventional method of ASC isolation. The aim of this study was to investigate the adipogenic differentiation potential of
CD29-, CD71-, CD73- and CD90-selected ASCs in vitro.
The stromal vascular fraction (SVF) was obtained from rat adipose tissue by enzymatic digestion and centrifugation. Subsequently, CD29-, CD71-, CD73- and CD90 cells were isolated
by magnetic activated cell sorting (MACS), seeded into culture plates and differentiated into
the adipogenic lineage. ASCs isolated by adherent growth only served as controls. Adipogenic
differentiation was assessed by Oil Red O staining and quantification of the adiponectin and
leptin concentrations in the cell culture supernatants. Statistical analysis was carried out using
one-way analysis of variance (ANOVA) followed by the Scheffes post hoc procedure.
The results showed that different subpopulations with different adipogenic differentiation
potentials can be isolated by the MACS procedure. The highest adipogenic differentiation potential was determined in the CD29-selected ASC population followed by the unsorted ASC

*
The work should be attributed to: Department of Oral & Maxillofacial Surgery, Christian-Albrechts, University Kiel, Arnold-Heller-Strasse
16, 24105 Kiel, Germany.
* Corresponding author. Department of Oral & Maxillofacial Surgery, Christian-Albrechts-University Kiel, Arnold-Heller-Strasse 16, 24105
Kiel, Germany. Tel.: 49 15142319383; fax: 49 4315972107.
E-mail address: matthias_gierloff@icloud.com (M. Gierloff).

http://dx.doi.org/10.1016/j.bjps.2014.05.042
1748-6815/ 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

1428

M. Gierloff et al.
population. The CD71-, CD73- and CD90-selected cells exhibited significantly the lowest adipogenic differentiation potential.
In conclusion, the CD29-selected ASCs and the unsorted ASCs exhibited a similar adipogenic
differentiation potential. Therefore, we do not see a clear advantage in the application of an
anti-CD29-based isolation of ASCs over the conventional technique using adherent growth.
However, the research on isolation/purification methods of adipogenic ASCs should continue
in order to make this stem cell source even more attractive for future adipose tissue engineering applications.
2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by
Elsevier Ltd. All rights reserved.

Introduction

Materials and methods

Adipose-derived stromal cells (ASCs) are multipotential

mesenchymal progenitor cells1,2 and represent promising
candidates for tissue engineering applications.3e6 ASCs
reside in the so-called stromal vascular fraction (SVF) of
adipose tissue. The SVF is obtained by enzymatic digestion
and centrifugation1,7,8 and consists of a heterogeneous cell
population containing haematopoietic cells, adipocytes,
endothelial cells, vascular smooth muscle cells, fibroblasts,
resident monocytes, macrophages, lymphocytes and their
particular precursors.5,9e13 The frequency of ASCs in the
SVF accounts for approximately 1e5%.14 Currently, the
major drawback in the clinical and experimental use of
ASCs is to clearly separate ASCs from other cells and to
isolate a pure ASC population which can be expanded and
differentiated in culture. To date, most experimental
research groups isolate ASCs by tissue digestion, centrifugation and the capacity of ASCs to adhere to cell culture
plastic surfaces.1,2,4 This technique allows to exclude all
non-adherent growing cells of the SVF and results in a
relatively homogeneous cell population9 that contains a
high number of differentiationable ASCs. However, the
adherent cell population also contains other cell types
which are not multipotential and comprise the proliferation
and differentiation potential of the population. In order to
overcome the problem of a contamination, Rada et al.
used an antibody-aided technique to purify ASCs.15 The
technique is based on immunomagnetic beads coated with
specific antibodies against surface molecules of mesenchymal precursors. The authors could show that CD105and CD29-ASCs exhibit a higher chondrogenic differentiation potential16 compared to a cell population obtained
solely by adherent growth. In a different study, they
demonstrated that Stro-1-ASCs represent ideal mesenchymal precursors for bone tissue engineering applications.17 To date, not many studies investigated the
antibody-aided selection of ASCs with regard to their adipogenic differentiation potential. There is slight evidence
that CD105-ASCs are more prone to differentiate into
adipocytes compared to CD105-ASCs.18 Yet, further niches
of ASC subpopulations and their adipogenic differentiation
potential are not known. The aim of the current study was
to investigate the adipogenic differentiation potential of
CD29-, CD71-, CD73- and CD90-selected cells of the SVF in a
rat model.

Animals
Use and care of the animals were approved by the Minister
of Nature, Environment, and Forestry of Schleswig-Holstein
and were in accordance with the local ethics committee
(application no. V312-72241.121-14 (53-5/10)). The experiments were conducted with cells of two isogenic adult
male rats of the Lewis strain. The animals were kept under
sterile housing conditions with a fixed day/night cycle at
the central animal facility of the University of Kiel. The
animals had free access to food and water.

Adipose tissue digestion, cell harvest and cell

culture
Two male rats of the Lewis strain were euthanized by CO2
asphyxiation and adipose tissue was explanted from the
intra-abdominal cavity and the inguinal area. ASCs were
isolated according to a modified protocol that has been
previously described.1 Briefly, the adipose tissue was
washed 3 with phosphate-buffered saline (Gibco,
Paisly, Scotland, UK). For digestion, the tissue was transferred into a sterile 50-ml centrifuge tube containing
0.075% collagenase Type II (Sigma Aldrich Co. LLC, Steinheim, Germany) and was incubated for 30 min at 5% CO2 at
37  C under constant shaking. The enzyme activity was
neutralized by adding equal amounts of Dulbeccos Modified Eagles Medium (DMEM, Biochrom AG, Berlin, Germany) and 10% foetal calf serum (FCS; Biochrom AG,
Berlin, Germany). The cell suspension was centrifuged at
1200 rpm for 5 min and the supernatant was discarded.
The cell pellet was resuspended carefully with 10 ml
phosphate-buffered saline (PBS) and centrifuged a second
time. After removing the supernatant, the pellet was
resuspended in 10 ml erythrocyte lysis buffer (Qiagen,
Hilden, Germany) and incubated for 10 min at room
temperature. Following the lysis of red blood cells, the
cell suspension was filtered through a 100-mm nylon filter
(VWR International GmbH, Damstadt, Germany) to remove
cellular debris. After another centrifugation, the cell
pellet was resuspended in cell culture medium consisting
of DMEM (Biochrom AG, Berlin, Germany) supplemented
with FCS 10% (Biochrom AG), 100 IU/ml penicillin

Adipogenic differentiation potential of rat adipose-derived stromal cells

1429

Figure 1 Flow cytometric dot plots showing the particular single colour fluorescence vs. forward scatter intensity (FSC-H) before
(right) and after (left) MACS separation. The percentage values indicate the percentages of positive and negative cells for the
particular surface marker.

(Biochrom AG, Berlin, Germany), 100 mg/ml streptomycin

(Biochrom AG), 25 mg/ml amphotericin (Biochrom AG),
2 mM L-glutamine (Biochrom AG, Berlin, Germany) and
1 mM ascorbat-2-phosphate (Sigma Aldrich Co. LLC,
Steinheim, Germany).

Magnetic cell separation

The immunomagnetic bead separation was performed using
the MACS Column Technology according to the manufacturers instructions. Briefly, 1  106 freshly isolated cells of

1430

M. Gierloff et al.

Figure 2 Photomicrographs (20) showing the Oil Red O staining of the ASC populations stimulated with adipogenic culture
medium (right) and control medium (left) for 14 days. Significant higher percentages of Oil Red O-positive cells were determined in
the CD29-enriched and the unsorted ASC populations compared to the CD90-enriched, the CD73-enriched or the CD71 enriched ASC
populations (p < 0.001).

the SVF were labelled with a primary antibody against

CD90, CD73, CD71 or CD29, respectively. Subsequently, the
primary antibodies were coupled with a magnetic secondary antibody (MicroBeads; Miltenyi Biotec GmbH, Bergisch

Gladbach, Germany). The following primary antibodies

were used: IgG1-Anti-Rat-CD90-PE (clone OX-7) (BD Biosciences, Heidelberg, Germany), IgG1-Anti-Rat-CD73 (clone
5F/B9) (BD Biosciences), IgG2-Anti-Rat-CD71-FITC (clone

Adipogenic differentiation potential of rat adipose-derived stromal cells

OX-26) (BD Biosciences) and IgM-Anti-Rat-CD29-PE (clone
Ha2/5) (BD Biosciences). All primary antibodies were used
at a concentration of 0.5 mg/ml. A total of 1  106 cells was
stained with 5.0 mg of each particular antibody. After
washing the cells in buffer (PBS containing 0.5% bovine
serum albumin (BSA) and 2 mM EDTA), the cells were
centrifuged at 300 g for 10 min. The supernatant was
discarded and the cell pellet was resuspended in 80 ml
buffer either 20 ml Anti-IgG1 MicroBeads or 20 ml Anti-PE
MicroBeads according to the manufacturers protocol. For
staining of the CD71-positive cells, 90 ml buffer 10 ml AntiFITC MicroBeads were used. After incubation at 7  C for
15 min the cells were, again, washed in buffer and centrifuged at 300 g for 10 min. The supernatant was discarded
and the cells were resuspended in 500 ml buffer for the
magnetic separation process. For this purpose, the MACS
MS Columns and MACS Separators (Miltenyi Biotec GmbH,
Germany) have been used according to the manufacturers
instructions. After the magnetic separation, the number of
viable cells of each cell lineage was determined in a haemocytometer after staining with trypan blue. Samples of
each cell population were measured prior to and after the
magnetic separation process on a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). The data
were analysed using the CellQuest Pro software (Becton
Dickinson).
Cell seeding and cell culture
The CD90-enriched, CD73-enriched, CD71-enriched, CD29enriched cell populations and the unsorted ASC population
were seeded at a density of 1250 cells/cm2 into 6-wellplates containing adipogenic induction medium consisting
of DMEM (Biochrom AG) supplemented with FCS 10% (Biochrom AG), 100 IU/ml penicillin (Biochrom AG), 100 mg/ml
streptomycin (Biochrom AG), 25 mg/ml amphotericine
(Biochrom AG), 2 mM L-glutamine (Biochrom AG), 1 mM
ascorbat-2-phosphate (Sigma Aldrich Co. LLC), 0.5 mM
dexamethasone, 0.5 mM isobutylmethylxanthine, 50 mM
indomethacin and 10 mg/ml insulin (Biochrom AG). ASC
populations cultured in normal culture medium without
adipogenic supplements served as control. The cells were
cultured under standard conditions in a humidified atmosphere with 5% CO2 at 37  C for 14 days. Medium change was
performed every 3 days.
Oil Red O staining
At day 14 of the culture period, the Oil Red O staining was
performed as previously described.19 Briefly, the Oil Red O
working solution was prepared using three parts of stock
solution in 0.5% isopropanol (all from Sigma Aldrich Co.
LLC, Steinheim, Germany) and two parts of water and
subsequently filtered through a filter funnel. After
washing the cell culture, 60% isopropanol was added for
4 min prior to staining with the Oil Red O working solution
for 5 min. Undifferentiated cells, cultured without adipogenic supplements, served as controls. In four different
wells of the 6-well-plate, two fields of view
(0.72  1.08 mm in size) were randomly picked and the
total number of cells and the number of Oil Red O-positive
cells was assessed using a haemocytometer. Numbers of
Oil Red O-positive cells were calculated as a percentage
of total cells.

1431

Enzyme-linked immunosorbent assay

Leptin
Leptin is a secreted proteohormone which is mainly produced by adipocytes. At day 14 of the culture period, the
cell culture supernatants were collected for determination
of the leptin concentration by an enzyme-linked immunosorbent assay (ELISA) using the Leptin Rat ELISA Kit (Abcam
plc, Cambridge, UK). The test is based on a polyclonal
antibody against rat leptin that is coated on a 96-well plate.
The test was performed according to the manufacturers
instructions. Briefly, standards and samples were prepared
and pipetted into the wells. After washing the wells, the
biotinylated Anti-Rat Leptin antibody was added. Unbound
biotinylated antibody was removed by another washing
step and horseradish peroxidase (HRP)-conjugated streptavidin was pipetted into the wells. The wells were washed
again, and in order to initiate the colour development, the
3,30 ,5,50 -tetramethylbenzidine (TMB) substrate solution
was applied to the wells. At last, the stop solution was
pipetted into the wells and the extinction was measured at
450 nm using the Roche Hitachi Modular P (Roche Diagnostic
GmbH, Mannheim, Germany). Three independent tests
were performed.
Adiponectin
Adiponectin is a secreted protein expressed exclusively by
differentiated adipocytes. At day 14 of the culture period,
the cell culture supernatants were collected for determination of the adiponectin concentration by an ELISA using
the Adiponectin Rat ELISA Kit (Abcam plc, Cambridge, UK).
The test is based on a polyclonal antibody, specific for rat
adiponectin, that has been pre-coated onto a 96-well
microplate. Adiponectin in standards and samples is sandwiched by an immobilized polyclonal antibody and a biotinylated polyclonal antibody against rat adiponectin,
which is recognized by a streptavidineperoxidase conjugate. The test was performed as described above (see
Leptin) according to the manufacturers instructions. Three
independent tests were performed.

Statistics
Normal distribution was proved by using the KolmogoroveSmirnoff test and the ShapiroeWilk test. Statistical
analysis was carried out using one-way analysis of variance
(ANOVA) followed by the Scheffes post hoc procedure. The
level of statistical significance was set at p  0.05.

Results
MACS
The sedimented cell pellet of the SVF contained 7.8% CD29positive cells, 11.9% CD71-positive cells, 14.2% CD73positive cells and 4.5% CD90-positive cells (Figure 1).
After the magnetic separation a 12.3-fold enrichment of
the CD29-positive cells (95.7%), an 8.0-fold enrichment of
the CD71-positive cells (94.9%), a 6.8-fold enrichment of

1432

M. Gierloff et al.

the CD73-positive cells (96.7%) and a 21.2-fold enrichment

of CD90-positive cells (95.3%) were achieved (Figure 1).

Adipogenic differentiation
Oil Red O staining
At day 14 of the culture period, Oil Red O-positive cells
were found in all five ASC populations that were treated
with adipogenic culture medium (Figure 2). By contrast, no
Oil Red O-positive cells could be detected in the populations that were cultured with standard culture medium
(Figure 2). Descriptive statistics data are summarized in
Table 1. Significant higher percentages of Oil Red-positive
cells were determined in the CD29-enriched and the unsorted ASC population compared to the other populations
(p < 0.001) (Figures 2 and 3). No significant differences
were determined between the CD29-selected and the unsorted ASC population (p Z 1.000), between the CD90selected and the CD73-selected ASC population
(p Z 0.997), between the CD90-selected and the CD71selected ASC population (p Z 0.048) and between the
CD73-selected and the CD71-selected ASC population
(p Z 0.145).

Leptin
The highest leptin concentration was determined in the
CD29-selected culture (184.33  4.86 pg/nl) (Table 2,
Figure 4). The differences compared to the CD90-selected,
CD73-selected and CD71-selected ASC population were
highly significant (p < 0.001). The second highest Leptin
concentration was determined in the unsorted ASC population (146.81  5.66 pg/nl) which was significantly higher
compared to the CD90-selected, the CD73-selected and
CD71-selected ASC culture (p < 0.05).
Interestingly, no significant difference was determined
between the adipogenically treated CD90-selected, CD73selected and CD71-selected populations and their particular controls. By contrast, significantly lower Leptin concentrations (p < 0.01) were determined in the control
cultures of the CD29-selected (92.11  14.59 pg/nl) and
unsorted ASC populations (24.32  4.17 pg/nl).

Adiponectin
The results of the adiponectin ELISA are presented in Table
3 and Figure 5. The highest adiponectin concentrations
were determined in the CD29-selected (33.61  2.30 ng/

Table 1

Figure 3 Graphical representation of the percentage values

(mean  SD) of Oil Red O-positive cells. Significantly higher
percentages were determined in the CD29-enriched and the
unsorted ASC population compared to the other ASC populations (n Z 8, one-way ANOVA: p < 0.001).

ml) and the unsorted ASC population (31.99  6.00 ng/ml).

The adiponectin levels of both populations were significantly higher compared to the CD90-selected, to the CD73selected and the CD71-selected ASC populations (p < 0.05).
No significant differences were determined between the
CD90-selected, the CD73-selected and the CD71-selected
ASC populations. No adiponectin was detected in any of
the control cultures (culture medium).

Discussion
The aim of this study was to investigate the adipogenic
differentiation potential of distinct ASC subpopulations
sorted according to their expression of typical ASC surface
markers. Recently, it could be demonstrated that specific
ASC subpopulations exhibit an improved osteogenic16,17
and/or chondrogenic20 differentiation potential compared
to ASC populations isolated by enzymatic digestion and
gradient solution only. A method to isolate a pure ASC
subpopulation with a high adipogenic potential could help
overcome clinical problems of fat grafts such as volume
loss21 or fibrosis.22 The major drawback of the antibodyaided isolation of ASCs is that surface markers, selectively
expressed by ASCs, are not known. To date, it is not
possible to define the exact ASC phenotype and to
discriminate clearly between these cells and, for example,
fibroblasts.23 A variety of studies showed that cultured ASCs

Descriptive statistics data of the percentage values of Oil Red O-positive cells.

Cell
n Mean Median Standard
Standard error Minimum Maximum Confidence interval [95%] of the mean (%)
population
(%)
(%)
deviation (%) of mean (%)
value (%) value (%) Lower value
Upper value
CD90
CD73
CD71
CD29
Unsorted
ASCs

8
8
8
8
8

37.31
35.16
23.75
80.74
80.22

37.49
35.24
23.04
80.37
79.93

7.72
11.73
7.61
4.15
5.25

2.73
4.15
2.69
1.47
1.86

25.00
19.55
14.61
76.56
72.56

45.74
53.97
40.20
89.61
90.05

30.86
25.36
17.39
77.27
75.82

43.77
44.97
30.11
84.20
84.61

Adipogenic differentiation potential of rat adipose-derived stromal cells

1433

Table 2 Descriptive statistics data of the leptin concentrations in the cell culture supernatants of the adipogenically induced
ASCs determined by ELISA.
Cell
population

Mean
(pg/ml)

Median

Standard
deviation

Standard error
of mean

Minimum
value

Maximum
value

Confidence interval [95%] of the mean

Lower value

Upper value

CD90
CD73
CD71
CD29
Unsorted
ASCs

3
3
3
3
3

43.38
59.27
92.47
184.33
146.81

39.22
59.67
94.97
184.04
147.56

9.51
11.27
21.48
4.86
5.66

5.49
6.51
12.40
2.80
3.27

36.66
47.80
69.85
179.63
140.81

54.26
70.33
112.58
189.33
152.05

19.76
31.27
39.12
172.27
132.75

67.00
87.26
145.81
196.40
160.86

Figure 4 Graphical representation of the leptin concentrations (mean  SD) in the cell culture supernatants of the adipogenically induced ASCs (red) and the ASCs cultured in
normal culture medium (white) on day 14 of the culture
period (n Z 3, one-way ANOVA).

are positive for CD166, CD151 CD105, CD90, CD73, CD63

CD71, CD49d, CD44, CD34, CD29, CD13 and negative for
CD146, CD45, CD31.4,5,9,10,24,25 However, none of these
markers allow the exact discrimination of ASCs from other
cell types. Another drawback is presented by the fact that
the expression of known ASC surface markers changes
dramatically with plating and successive passaging. For
example, freshly isolated cells of the SVF barely express the
stromal cell-associated markers CD13, CD29, CD44, CD73 or
CD90. Yet, during culture the expression of these markers
increases.9 The results obtained by the Oil Red O staining

and the quantification of adiponectin and leptin in the cell

culture supernatants demonstrated the highest adipogenic
differentiation potential for the CD29-selected ASC population. The differences compared to the CD71-selected, the
CD73-selected and the CD90-selected cell populations were
significant for all tests (p < 0.05). Interestingly, the unsorted ASCs showed a similar or only slightly lower adipogenic differentiation potential compared to the CD29selected ASCs. The reasons why the CD90-, CD73- and
CD71-selected ASCs exhibited a significantly lower adipogenic differentiation potential compared to the other two
populations remain unclear. One reason could be the
presence of fibroblasts which tend to overgrow other cells
in a culture. The transferrin receptor 1 (CD71) is highly
expressed not only on erythroid precursors26 and ASCs2,27
but also to a large amount on fibroblasts.28 It is possible
that the growth of CD71-selected ASCs was limited by CD71selected fibroblasts. Likewise, the surface molecules CD73
and CD90, which are known to be mesenchymal stromal cell
markers,29 are also expressed on specific fractions of
fibroblasts.30e32 A second explanation could be that the
selected CD71, CD73 and CD90 cells of the stromal
vascular fraction were in an earlier developmental stage of
adipocyte differentiation compared to CD29 cells. In
contrast to CD29, the other markers are not constitutively
expressed on all adipocyte precursors during the differentiation towards a mature adipocyte.27 Furthermore, it is
possible that specific cells, which are required for efficient
adipocyte differentiation, were not included in the CD90-,
CD73- and CD71-selected populations. It is known that the
preadipocyte growth and differentiation is controlled
notably by multiple cellecell interactions33 as, for
example, between preadipocytes and adipocytes.34 Yet,
adipocytes do not express regularly CD71, CD73 or CD90

Table 3 Descriptive statistics data of the adiponectin concentrations in the cell culture supernatants of the adipogenically
induced ASCs determined by ELISA.
Cell
population

CD90
CD73
CD71
CD29
Unsorted
ASCs

3
3
3
3
3

Mean
(ng/ml)

Median

12.17
17.75
20.82
33.61
31.99

11.41
17.60
19.25
34.84
29.46

Standard
deviation

Standard error
of mean

Minimum
value

Maximum
value

Confidence interval [95%] of the mean

Lower value

Upper value

2.19
0.72
3.52
2.30
6.00

1.27
0.42
2.03
1.33
3.46

10.37
17.12
18.35
30.96
27.68

14.57
18.54
24.85
35.03
38.84

6.68
15.96
12.07
27.90
17.10

17.55
19.55
29.57
39.32
46.89

1434

M. Gierloff et al.
financial support of this study. They also thank Mrs. Refrath
and Mrs. Neenius for their kind support and technical
assistance. The authors declare that they did not make
demand on any writing assistance.

References

Figure 5 Graphical representation of the adiponectin concentrations (mean  SD) in the cell culture supernatants of the
adipogenically induced ASCs on day 14 of the culture period.
No adiponectin was detected in any of the control cultures
(culture medium). The error bars indicate the standard error of
mean (n Z 3, one-way ANOVA).

and, therefore, were mainly excluded by the MACS procedure. In this case, the selection of a distinct ASC subpopulation would constitute a clear disadvantage compared to
choosing the heterogeneous SVF for adipogenic cultures.
In conclusion, the highest adipogenic potential was
determined in the CD29-selected population. The surface
molecule CD29 (integrin b1) is expressed by numerous
precursor cells during differentiation towards mature adipocytes35 including ASCs,4 early-stage adipocyte precursors
and committed adipocytes.24 However, all these different
cell types are also included in the SVF, which is reflected by
the comparably high adipogenic potential of the unsorted
population. Therefore, according to the results of the
current study, we do not see a clear advantage in the
application of the antibody-aided isolation of ASCs by using
the chosen surface molecules. However, as long as no
specific ASC surface markers are available, the search for
ideal surface molecules for a positive selection of ASCs or,
otherwise, for a negative selection in order to exclude nondifferentiationable cells such as fibroblasts from a pure ASC
culture, should continue.

Conflict of interest
None.

Funding
None.

Acknowledgements
The authors thank the medical faculty of the ChristianAlbrechts University Kiel (grant number: F342972) for the

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