Stromal cells cultured from omentum express pluripotent markers, produce high amounts of VEGF, and engraft to injured sites.

REGULAR ARTICLE
Stromal cells cultured from omentum express pluripotent
markers, produce high amounts of VEGF, and engraft
to injured sites
Ashok K. Singh & Jilpa Patel & Natalia O. Litbarg &
Krishnamurthy P. Gudehithlu & Perianna Sethupathi &
Jose A. L. Arruda & George Dunea
Received: 20 July 2007 /Accepted: 20 November 2007 / Published online: 15 January 2008
# Springer-Verlag 2007
Abstract When rat omentum becomes activated by intra-
peritoneal injection of inert polydextran particles, these
particles are rapidly surrounded by cells that express
markers of adult stem cells (SDF–1α, CXCR4, WT–1)
and of embryonic pluripotent cells (Oct–4, Nanog, SSEA–
1). We have cultured such cells, because they may offer a
convenient source of adult stem cells, and have found that
they retain stem cell markers and produce high levels of
vascular endothelial growth factor for up to ten passages.
After systemic or local injection of these cultured cells into
rats with acute injury of various organs, the cells specifi-
cally engraft at the injured sites. Thus, our experiments
show that omental stromal cells can be cultured from
activated omentum, and that these cells exhibit stem cell
properties enabling them to be used for repair and possibly
for the regeneration of damaged tissues.
Keywords Omentum . Stem cells . Oct–4 . Nanog .WT-1 .
Rat (Sprague Dawley)
Introduction
In previous experiments, we have shown that, when inert
polydextran particles (size: ∼120 μM) are placed in the
abdominal cavity of rats, the omentum recognizes them as
foreign bodies and expands rapidly to surround and
encapsulate them. Once the omentum becomes activated,
it greatly increases in size and in mass (>20 fold increase)
by producing new tissue that consists mainly of stromal
cells, interstitial cells, and blood vessels. Fat cells in the
omentum, which in the native state amount to 95% of the
total tissue, decrease to less than 30%, so that stromal cells
now make up more than 70% of the total omental mass.
The non-fat stromal cells in the expanded omental tissue,
especially those immediately surrounding the polydextran
particles, express markers of stem cell activity, viz.,
stromal-cell-derived factor (SDF–1α), chemokine receptor
4 (CXCR4), and Wilms’ tumor antigen 1 (WT–1; Litbarg
et al. 2007; Singh et al. 2007a, b). If such omental cells
could be cultured, they would represent a readily available
source of adult stem cells that could be used to repair and
regenerate damaged tissue. Accordingly, we have cultured
cells from the activated omentum, characterized them for
stem cell markers during several passages, and tested their
Cell Tissue Res (2008) 332:81–88
DOI 10.1007/s00441-007-0560-x
DO00560; No of Pages
A. K. Singh : K. P. Gudehithlu : J. A. L. Arruda : G. Dunea
The Division of Nephrology,
John Stroger Jr. Hospital of Cook County,
Chicago, IL, USA
J. A. L. Arruda
Section of Nephrology,
University of Illinois at Chicago and the Chicago VAMC,
Chicago, IL, USA
A. K. Singh : J. Patel : P. Sethupathi : G. Dunea
Hektoen Institute of Medicine,
Chicago, IL, USA
N. O. Litbarg
Section of Nephrology,
Loyola-Hines Medical Center,
Maywood, IL, USA
A. K. Singh (*)
Stroger Hospital of Cook County,
637 South Wood St (Durand Bldg, 2nd Floor),
Chicago 60612 IL, USA
e-mail: singhashok@comcast.net

ability to home to experimentally injured organ sites
(kidney, subcutaneous tissue).
Materials and methods
Culture of omental cells from activated rat omentum
Animal experiments were conducted with the approval of
the Institutional Animal Care and Use Committee
(IACUC).
Sprague-Dawley rats (200–250 g) were injected intra-
peritoneally with 5 ml polydextran particle slurry (Biogel
P–60, 120 μM; Biorad Laboratories, Richmond, Calif.; 1:1
in normal saline). At 1 week after polydextran injection, the
expanded omentum was harvested aseptically and placed in
mesenchymal stem cell growth medium (MSCGM) supple-
mented with growth factors supplied by the manufacturer
(Lonza, Walkersville, Md., USA), glutamine, and anti-
biotics. The MSCGM contained 10% fetal bovine serum.
The tissue was gently chopped and scraped over a no. 60
stainless steel sieve (pore size: approximately 300 μM).
The minced tissue was forced through the sieve by ice-cold
MSCGM and then centrifuged, and the pellet was washed
several times with fresh MSCGM. The pellet was finally
suspended in fresh MSCGM and placed in culture dishes
that were incubated in a 5% CO2–95% air environment at
37°C for 7–10 days without changing the medium. Once
the cells had reached approximately 80% confluence, they
were removed from the dishes by trypsin treatment and re-
cultured on fresh dishes (passage 1; split ratio 1:3). As
controls, native omenta were likewise processed from age-
matched normal rats injected intraperitoneally with normal
saline.
For photo-documentation, the cells were fixed in 95%
ethanol (30 min) and stained with 1% safranin O dye solution.
Labeling cultured omental cells and fresh kidney cells
with a vital fluorescent dye
Cultured cells were detached from the dishes by trypsini-
zation, washed with MSCGM, and re-suspended in the
medium for labeling. The cell suspension was incubated
with 10 μg/ml dye (acetoxymethyl ester also known as
calcein AM; Molecular Probes, Eugene, Ore.) for 1 h at
37°C. Fresh kidney cells obtained by gently scraping
whole adult rat kidney tissue over a no. 200 stainless steel
sieve (pore size: <100 μM) were similarly labeled with the
fluorescent dye. The labeled cells were finally washed
with medium and resuspended in fresh medium for
injection into a cell counter. Cells were counted in a
hemocytometer.
Histological processing and immunostaining of omentum
and cultured omental cells
Pieces of native and activated omenta were fixed for
histology and immunochemistry by immersion in Histochoice
(Amersco, Solon, Ohio). Following dehydration and paraffin
embedment, tissues were sectioned (5 μM thick). The
sections were pressure-cooked for 10 min in a solution of
BorgDecloaker (Biocare Medical, Walnut Creek, Calif.,
USA) for antigen enhancement. The sections (or cultured
cells on LabTek chamber slides) were stained by first
incubating them with the primary antibody (rabbit anti-
Nanog, mouse anti-Oct–4 (octamer–4), mouse anti-SSEA-1
(stage-specific embryonic antigen 1), rabbit anti-CXCR4, or
rabbit anti-SDF–1α from Chemicon International, Temecula,
Calif., or mouse anti-WT–1 from DAKO, Glostrup Den-
mark) followed by washes and re-incubation of the sections
with the secondary antibody (biotin-conjugated anti-rabbit
IgG or biotin-conjugated anti-mouse IgG) followed by
avidin-horseradish peroxidase (Vector Laboratories, Burlin-
game, Calif.). The slides were washed and finally developed
with diaminobenzidine–H2O2 (brown color). The stained
slides were permanently mounted, viewed under a light
microscope, and digitally photographed (Nikon, New York).
For visualizing the injected cells in the subcutaneous and
kidney tissues, the tissues were embedded in OCT cryogenic
compound, frozen at −85°C, and sectioned in a cryostat (5μM
thick). Sections were rapidly washed in phosphate-buffered
saline (PBS; 10 mM phosphate, 125 mM NaCl pH 7.4), wet-
mounted in glycerol-PBS (1:1), and viewed as above.
Determination of rate of vascular endothelial growth factor
synthesis
For determination of the rate of synthesis of vascular
endothelial growth factor (VEGF), medium from cultured
omental cells (primary culture and passages 1–10) was
sampled hourly and from cultured rat glomerular epithelial
cells (passage 13) or from primary rat kidney mesangial
cells was sampled every 24 h. VEGF was measured by a
sandwich enzyme-linked immunosorbent assay by using a
commercial murine VEGF assay kit supplied by R & D
Systems (Minneapolis, Minn.). The glomerular epithelial
cell line was originally obtained from Dr. J. L. Kreisberg
(Kreisberg et al. 1978) and has been used in many of our
previous studies (e.g., Sam et al. 2006). Primary rat
mesangial cells were cultured from freshly isolated glomeruli
as previously described (Singh et al. 2004).
Induction of kidney and subcutaneous injuries
Rats were laparotomized under total anesthesia, and subcuta-
neous tissue injury was induced by the surgical insertion of a
82 Cell Tissue Res (2008) 332:81–88

polyvinyl tube on the back of the rats. This resulted in a rapid
growth of granulation tissue around the tube, simulating
wound-healing tissue as previously described (Gudehithlu
et al. 2005; Singh et al. 2007a, b).
Ischemia-reperfusion injury of the kidney was induced by
occluding the left renal artery for 40 min by using non-
traumatic clamps followed by de-occlusion and reperfusion
of the kidney. This is a well-studied model of unilateral renal
ischemia in which renal proximal tubules are extensively
damaged but regenerate within a few days after injury (Park
et al. 2001; Togel et al. 2005).
Tests for migration of cultured omental cells to sites
of injury
To test the migration of cultured omental cells to injured
kidney, 2–3 million fluorescein-labeled cells were injected
via the left ventricle of the heart, and rats were sacrificed
after 24 h. Controls tissues consisted of the uninjured
contralateral kidney.
Migration of cells to sites of subcutaneous injury was
examined after local injection of labeled cells. In this case,
controls consisted of locally injecting freshly isolated and
labeled adult rat kidney cells.
The injured tissues were harvested, surrounded with
generous amounts of OCT compound and snap-frozen in dry
ice. Cryostat sections (5 μM thick) of the tissues were briefly
washedwith PBS plus 1%Tween–20 (PBS-T), counterstained
with 10 μg/ml ethidium bromide for 10 min (kidney only),
washed again with PBS–T, mounted with PBS:glycerol (1:1),
and examined under an epifluorescent microscope (Nikon).
Results
Activated omental tissue has abundant stromal cells
expressing pluripotent markers
We have previously reported that, in the activated omen-
tum, those cells in close contact with polydextran particles
express adult stem cell markers, such as SDF–1α, CXCR4,
and WT–1 (Litbarg et al. 2007). We now show that the
activated omentum also expresses pluripotent embryonic
markers, such as Nanog, Oct–4, and SSEA–1 (stage-
specific embryonic antigen 1).
As seen previously for the expression of adult stem cell
markers, reactivity for all three embryonic markers was
confined to cells immediately surrounding the polydextran
particles in the activated omentum (Fig. 1). Native
omentum, on the other hand, showed activity for Nanog,
Oct–4, and SSEA–1 only in islands of non-fat stromal cells,
considered to be the rat equivalent of milky spots usually
seen in the human omentum (not shown). As expected,
normal adult rat tissues such as kidney and liver were
mostly negative for the three embryonic markers, except in
unremarkable focal areas (not shown).
Fig. 1 Rat omentum activated
with polydextran particles and
immunostained for embryonic
markers. Activated omentum
showed strong immunoreactivity
for Nanog (a) and lower reac-
tivity for Oct–4 (b) and SSEA–1
(c). Control sections immuno-
stained with secondary antibody
only were negative (d). Reac-
tivity for all three markers was
limited to the cells immediately
surrounding the polydextran
particles in the activated omen-
tum (arrows spaces occupied by
polydextran particles). Normal
adult rat tissues (kidney and
liver) were negative for the three
markers (not shown). Bar
100 μm
Cell Tissue Res (2008) 332:81–88 83

Culture, propagation, and characterization of omental
stromal cells
When dispersed 7-day activated omental tissue was placed
in culture, cells that had originally clustered around the
polydextran particles started to attach to the dish and
multiply by days 4–5. The morphology and phenotype of
these cells resembled that of smooth muscle cells (Fig. 2a)
in that they were positive for α–smooth muscle actin but
negative for cytokeratin–17 (epithelial marker) and von
Willebrand factor (endothelial marker; not shown). Com-
pared with the growth rate of the passage 0 cells (primary
culture), the proliferation rate of the passage 1–4 cells was
faster (Fig. 2b), after which the rate declined. The cells
could be maintained successfully for 10 passages. Similar
cultures were obtained from cells harvested from omentum
activated for 1 or 4 days (not shown). Activated omentum
older than 2 weeks (after polydextran injection) was
difficult to culture because the cells did not attach or
multiply as readily as those from younger activated omenta.
Because the proliferating cells in culture appeared to be
derived from the cells surrounding the polydextran par-
ticles, the cultures were immunostained for both adult stem
cell markers and embryonic markers to determine whether
these cultured cells retained the stem cell characteristics of
the intact tissue. The omental cells in culture retained the
adult stem cell markers WT–1, CXCR4, and SDF–1α
(Fig. 3a–c). Among the embryonic markers, whereas Nanog
and Oct–4 (Fig. 3d,e) activities were high in omental
cultures, SSEA–1 activity became negative (not shown),
suggesting a partial loss of pluripotency on culture. The
cells retained these stem cell markers for up to 10 passages.
VEGF synthesis rate in cultured omental cells is higher
than in other cultured cells
Cultured omental cells synthesized VEGF at a rate that
was 10–20 times higher than that observed in primary
mesangial cells and glomerular epithelial cells (Table 1).
This high rate of VEGF secretion was maintained in
omental cells for up to 10 passages (data not shown).
Cultured omental stem cells migrate to injured sites
Before injection of cultured omental cells to the rats,
they were uniformly labeled with a vital fluorescent dye
to track them in the injured tissue (Fig. 4a). When
labeled omental cells were injected in the vicinity of
subcutaneous granulation tissue, they appeared to migrate
and integrate into the growing granulation tissue by 24 h.
In contrast, labeled control adult kidney cells injected
near the granulation tissue remained at the injection site
and did not appear to migrate into the granulation tissue
(Fig. 4b,c).
When labeled omental cells were injected systemically
into rats with unilateral ischemic injured kidney (3 days
after injury), the cells migrated into the injured tubules
and appeared to attach to these tubules. Moreover, the
cells seemed to have altered from their original round
shape to a more elongated forms suggesting their
participation in the healing process (Fig. 5a). The non-
ischemic contralateral kidney, on the other hand, did not
contain green fluorescent cells suggesting that omental
cells specifically migrated to only injured sites in the body
(Fig. 5b).
Fig. 2 Omental stromal cells cultured from omentum tissue activated
by polydextran particles for 7 days. Staining: Safranin O. a Primary
culture. After 4–5 days in culture, cells that originally clustered around
the polydextran particles started to attach to the dish and multiply (one
such particle surrounded by attached cells is shown in the middle of
the field). Cell morphology and phenotype resembled that of smooth
muscle cells and bone-marrow-derived human mesenchymal stromal
cells. b Passage 3 cells showing robust growth. The growth rate of the
cells started to gradually decline from passage 4 onward and became
extremely slow at passage 10. Bar 50 μm
84 Cell Tissue Res (2008) 332:81–88

Discussion
The omentum has long been known to have the power to
heal injured organs once it has adhered to the damaged site,
either naturally or deliberately by surgery (Cannaday 1948;
Liebermann-Meffert 2000; Goldsmith 1994, 1997, 2004;
Vernik and Singh 2007). We have previously shown that
the omentum, especially after its activation by injury,
becomes a reservoir of stromal cells that express stem cell
markers. We have further expanded the population of these
cells in the omentum by injecting polydextran gel particles
intraperitoneally, a stimulus that mimics injury (Litbarg et al.
2007; Singh et al. 2007a, b), and found that the cells in
immediate contact with the polydextran particles express
these stem cell markers (Litbarg et al. 2007; Singh et al.
2007a, b). We have therefore carried out additional studies to
determine whether cells cultured from the omentum can
continue to exhibit stem cell-like properties. Such cultured
cells could then be targeted to remote injured organs and
could repair them without surgically bringing the omentum
in contact with the injured site.
After culturing these cells, we have found that they
continue to express adult stem cells markers (SDF–1α,
CXCR4, WT–1) and pluripotent embryonic cell markers
(Oct–4, Nanog, SSEA–1; Litbarg et al. 2007; Hatch et al.
2002; Kayali et al. 2003; Pritchard-Jones et al. 1990;
Kreidberg et al. 1993; Gerrard et al. 2005; Chambers et al.
2003; Hatano et al. 2005; Solter and Knowles 1978). They
also display a high secretion rate of VEGF, a powerful
growth factor that induces new blood vessel formation and
that is highly expressed in bone marrow stem cells (Togel
et al. 2005). We have also substantiated the stem cell
potency of these cultured cells by showing that, after
systemic or local injection, they specifically migrate to
injured kidney and subcutaneous sites.
At present, proposed therapies involving the use of
embryonic cells pose ethical questions and carry the risk of
Fig. 3 Primary cultures of omental stromal cells stained for adult and
embryonic stem cell markers. a–c Cultured omental stromal cells
stained positively for adult stem markers WT–1 (nuclear), CXCR4
(nuclear), and SDF–1α (cytoplasmic). As seen in intact omental tissue
(Fig. 1), the cultured cells were strongly immunopositive for Nanog
(d, nuclear) and Oct–4 (e, cytoplasmic). Cell staining was negative in
the absence of the primary antibody (f, control). Unlike the omental
tissue, the cultured omental stromal cells were negative for SSEA–1
(not shown) suggesting that, even though the cultured cells largely
maintained their stem cell property, pluripotency was partially lost
upon culture. Bar 50 μm
Table 1 Synthesis rate of VEGF in cultured omental stromal cells
compared with other cultured cells
Cultured cell type VEGF synthesis rate
(pg/h per million cells)
Omental cells 322±22
Glomerular epithelial cellsa 17.6±2.4
Primary mesangial cellsb 32.1±3.8
a Glomerular epithelial cell line originally obtained form Kreisberg
et al. (1978)
b VEGF synthesis rate was determined from our previous work (Singh
et al. 2007a, b)
Cell Tissue Res (2008) 332:81–88 85

uncontrollable growth and tumor induction (Chambers and
Smith 2004; Andrews et al. 2005). By contrast, adult cells
with stem cell properties are safer and have greater practical
use, having been employed for over 50 years to replace
bone marrow in leukemias and non-hematological diseases
without causing malignant transformation (Hoffman 2005).
However, the challenge remains to determine the conditions
(specific chemical stimuli and cues from other cells and
extracellular matrix) that will differentiate these cells into
the appropriate parenchymal cells once they engraft into an
injured organ. Such adult cells with stem cell properties
have previously been identified in the bone marrow
mesenchyme, skin, hair, dental pulp, kidney, and even
peripheral blood (Jiang et al. 2002; Lee et al. 2006; Grove
et al. 2004; Taylor et al. 2000; Oshima et al. 2001; Kerkis
et al. 2006; Oliver et al. 2004; Zhao et al. 2007). Omental
stromal cells, however, are more easily obtainable in large
quantities and can be harvested from the patient’s own
omentum, thus obviating the need for immunosuppressive
therapy. As these cells can be passaged in culture without
loss of pluripotent markers (Oct–4, Nanog), they can be
frozen in large numbers for long-term storage and later use.
Some investigators have confirmed “stem cellness” by
demonstrating the ability of cells to transform themselves
into bone, cartilage, and fat cells when cultured in specially
formulated media (Jiang et al. 2002; Lu et al. 2005). Others
have labeled cells with identifiable markers (such as lacZ)
and, after injecting them into the blastocyst of a nude
mouse, re-implanted them into the uterus of the mother. The
incorporation of labeled cells into the various formed
tissues of the developing mouse is regarded as evidence
of pluripotency (Jiang et al. 2002; Grove et al. 2004). We
have tested the “stem cellnes” of cultured omental cells by
showing that they engraft into injured sites. Such engraft-
ment is characteristic of cells with stem cell properties,
because they express a receptor (CXCR4) that binds to
SDF–1α released by injured organs (Togel et al. 2005;
Hatch et al. 2002; Kayali et al. 2003; Peled et al. 1999). We
have used two injury models and found that the omental
cells migrate to injured but not to uninjured sites.
Fig. 5 Migration of cultured omental stromal cells to injured kidney.
a Labeled omental stromal cells (green) were injected systemically
into a rat with unilateral ischemic injured kidney (3 days after injury).
Labeled cells migrated and possibly attached to injured tubules. The
morphology of the injected cells also appeared to change from round
to more elongated, suggesting their participation in the healing
process. b The non-ischemic contralateral kidney, on the other hand,
did not contain green fluorescent cells suggesting that omental stromal
cells had not migrated to the uninjured kidney. For contrast, tissues
were counterstained with ethidium bromide to stain cell nuclei red.
Bar 100 μm
Fig. 4 Migration of cultured omental stromal cells to a wound-
healing site. a Suspension of cultured omental stromal cells uniformly
labeled with a vital fluorescent dye (green) before injection into rats. b
Cryo-section of granulation tissue 24 h after injection of fluorescein-
labeled cultured omental stromal cells in the vicinity of granulation
tissue. Note the migration and engraftment of the injected omental
stromal cells in the granulation tissue. c Cryo-sections of granulation
tissue 24 h after injection of fluorescein-labeled kidney cells in the
vicinity of granulation tissue. Labeled adult kidney cells remain at the
injection site without migrating to the granulation tissue. Bar 100 μm
86 Cell Tissue Res (2008) 332:81–88

In this new and emerging field of stem cell science, the
establishment of whether adult stem cells indeed exist
remains controversial (Dor et al. 2004). Strictly speaking,
an adult stem cell should be able to regenerate a tissue in
vivo, and so far this has not been shown convincingly for
any adult-derived tissue cells. Nor are the criteria for even
the presumptive recognition of adult stem cells well
established, largely because of a lack of consensus
concerning the surface markers (CD markers) and the
functional criteria of such cells. Indeed, only recently, as a
result of the large body of work on human mesenchymal
stem cells (now to be called mesenchymal stromal cells), a
“position paper” has stated that such cells should meet at
least three criteria, namely, the cells should have the ability
(1) to attach to plastic, (2) to differentiate to adipogenic,
osteogenic, and chondrogenic phenotypes in culture (after
incubation in specially formulated media), and (3) to
express a minimum set of specific positive and negative
CD markers (Dominici et al. 2006). Unfortunately, no such
clear guidelines exist for non-human tissues, which actually
form the bulk of the research literature.
This problem of classifying adult stem cells is com-
pounded because the properties usually assigned to stem
cells (such as migration to sites of injury and the presence
of pluripotent cell surface markers) have also been found in
adult cells (such as monocytes (Kuwana et al. 2003; Ruhne
et al. 2005) and cultured fibroblasts (Sudo et al. 2007)
normally considered to be devoid of stem cell properties.
Moreover, the pluripotent stem cell markers used to identify
embryonic cells (such as Oct–4, Nanog, SSEA–1) may not
be suitable for identifying adult stem cells, because such
marker proteins may exert pluripotent functions in embry-
onic stem cells but have a different role in normal adult
cells. However, we believe that, until broad agreement is
reached regarding adult stem cell characteristics, cells such
as the omental stromal cells described here as showing
important stem cell markers, secreting high levels of growth
factors, and engrafting to injured sites could be considered
as potential stem cells.
Why should the omentum harbor stem cells? Could it
be that the omentum, being a vestigial organ and having
the power to repair injured tissues, may have retained
remnants of embryonic or multipotent cells (Fatima et al.
2005). The presence of such multipotent cells would
endow it with the plasticity and ability to expand and form
complex tissue with new blood vessels, interstitial and
stromal cells. Because the omentum can be expanded by
injury and by foreign bodies, we further hypothesize that
the release of inflammatory factors activates the omentum
and triggers its resident stem cells to proliferate and
differentiate. Whether these cells will respond differently
to diverse kinds of stimuli is a question worthy of further
exploration.
In conclusion, we have cultured and propagated omental
stromal cells for several passages without loss of stem cell
markers. Based on the well-known healing property of the
omentum, stem cell marker characterization, secretion of
high amounts of VEGF, and the ability to recognize injured
sites, cultured omental stromal cells could qualify as
potential stem cells from the adult. If so, the omentum
would be a convenient source of adult stem cells that could
be used to repair and possibly regenerate damaged tissues.
Acknowledgements The authors thank Ms. Linda Wanna for culture
work and Dr. Lev Rappoport for help with histological processing and
immunostaining of tissues.
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