Cell Tissue Res (2007) 329:301–311
DOI 10.1007/s00441-007-0417-3
REGULAR ARTICLE
Treatment of diabetic wounds with fetal murine
mesenchymal stromal cells enhances wound closure
Andrea T. Badillo & Robert A. Redden & Liping Zhang &
Edward J. Doolin & Kenneth W. Liechty
Received: 10 December 2006 / Accepted: 8 March 2007 / Published online: 24 April 2007
# Springer-Verlag 2007
Abstract Diabetes impairs multiple aspects of the wound-
healing response. Delayed wound healing continues to be a
significant healthcare problem for which effective therapies are
lacking. We have hypothesized that local delivery of mesen-
chymal stromal cells (MSC) at a wound might correct many of
the wound-healing impairments seen in diabetic lesions. We
treated excisional wounds of genetically diabetic (Db-/Db-)
mice and heterozygous controls with either MSC, CD45+
cells, or vehicle. At 7 days, treatment with MSC resulted in a
decrease in the epithelial gap from 3.2±0.5 mm in vehicle-
treated wounds to 1.3±0.4 mm in MSC-treated wounds and
an increase in granulation tissue from 0.8±0.3 mm2 to 2.4±
0.6 mm2, respectively (mean±SD, P<0.04). MSC-treated
wounds also displayed a higher density of CD31+ vessels and
exhibited increases in the production of mRNA for epidermal
growth factor, transforming growth factor beta 1, vascular
endothelial growth factor, and stromal-derived growth factor
1-alpha. MSC also demonstrated greater contractile ability
than fibroblast controls in a collagen gel contraction assay.
The effects of locally applied MSC are thus sufficient to
improve healing in diabetic mice. Possible mechanisms of
this effect include augmented local growth-factor production,
improved neovascularization, enhanced cellular recruitment
to wounds, and improved wound contraction.
A. T. Badillo : R. A. Redden : L. Zhang : E. J. Doolin :
K. W. Liechty (*)
The Center for Fetal Research at The Children’s
Hospital of Philadelphia,
The University of Pennsylvania School of Medicine,
Philadelphia, PA 19104, USA
e-mail: liechty@email.chop.edu
K. W. Liechty
Abramson Research Center,
Rm 1116E, 3615 Civic Center Blvd,
Philadelphia 19104, USA
Keywords Stromal progenitor cells .
Mesenchymal stromal cells . Mesenchymal stem cells .
Wound healing . Diabetes . Mouse (C57BKS strains)
Introduction
Diabetic wound-related morbidity is a major clinical problem
resulting in prolonged hospitalization, lost time from work,
and significant healthcare expenditure. Normal wound healing
is an intricate process involving various cell types, coordinat-
ed processes, and complex signaling interactions. In diabetic
wound healing, many of these processes are impaired, and this
intricate orchestration is disrupted. Alterations in growth-
factor production, cellular responses to inflammatory media-
tors, matrix production, angiogenesis, and wound contraction
have all been shown to contribute to the delayed healing of
diabetic wounds (Blakytny and Jude 2006). Consequently, the
effectiveness of treatments focusing on a single deficit tends
to be limited. For example, the local delivery of platelet-
derived growth factor (PDGF) requires repeated application
to achieve modest improvements in wound closure (Wieman
et al. 1998). The relative insufficiency of isolated growth-
factor delivery may also be related to a simultaneous lack of
responsive cellular targets within the wound (Falanga 2005).
Because of the multifactorial etiologies of wound-healing
impairment in these wounds, potential therapies should have
a broad impact on the wound-healing response and any
associated problems with this response.
Cell-based therapy is an attractive approach for the
treatment of wounds with multiple impairments. Cells are
biologically active and possess the machinery to provide a
wide range of support to healing wounds and the capacity to
regulate responses according to the local wound environment.
Mesenchymal stromal cells (MSC) are multipotent cells
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derived from the stroma of bone marrow and other tissues (da
Silva Meirelles et al. 2006). MSC have demonstrated a
number of properties in vitro that can promote tissue repair,
including the production of multiple growth factors, cyto-
kines, collagens, and matrix metalloproteinases (Fathke et al.
2004; Kim et al. 2005; Kinnaird et al. 2004; Parikka et al.
2005; Pittenger et al. 1999) and the ability to promote
migration of other skin cells such as keratinocytes (Akino et
al. 2005). MSC have also been implicated as participators in
normal tissue-repair processes based on their location in
areas of tissue injury, such as injured spinal cord (S. Wu et al.
2003), heart allografts in chronic rejection (G.D. Wu et al.
2003), and areas of dermal injury (Liechty et al. 2000).
Based on their in vitro properties, MSC have the potential to
improve impaired wound healing through a variety of
mechanisms; however, the effects of MSC on cutaneous
wound healing have not been extensively tested in vivo.
Until recently, the inability to isolate and culture-expand
enriched murine MSC populations has precluded the study of
the specific effects of MSC and sub-fractions of bone marrow
in established murine models of impaired wound healing.
Whole bone marrow has been used to treat wounds of patients
with diabetes and peripheral vascular disease and have
demonstrated improved wound closure (Badiavas and Falanga
2003). The precise cell type, within bone marrow, that is
responsible for this improvement has yet to be identified. We
have hypothesized that the local delivery of MSC to diabetic
wounds might correct wound-healing impairment both indi-
rectly, by reversing local growth-factor deficiencies, and
directly, by improving wound contraction through interaction
with the extracellular matrix. To test this hypothesis, we have
isolated MSC from the livers of allogeneic fetal transgenic
mice expressing green fluorescent protein (GFP). MSC have
been used here to treat wounds created in a murine model of
obesity-related diabetes and non-diabetic control mice. Wound
healing was assessed by measuring the epithelial gap, the
volume of granulation tissue, and growth-factor production.
We have also examined the location and differentiation profile
of MSC in wounds and the ability of MSC to promote
contraction of the extracellular matrix in vitro.
Materials and methods
Animal wounding model
Twelve 14-week-old female C57BKS.Cg-m+/+Leprdb/J
mice (Db-/Db-) and age-matched non-diabetic heterozy-
gous C57BKS mice (Db+/Db-) were used in wounding
experiments (Jackson Laboratories). Db-/Db- mice weighed
greater than 50 g, with blood glucose levels in excess of
400 g/dl, whereas Db+/Db- mice weighed an average of 22 g,
with blood glucose levels less than 200 g/dl. Mice were
Cell Tissue Res (2007) 329:301–311
anesthetized with inhaled isofluorane after which the dorsal
skin was shaved, depilatated, and cleaned with ethanol. Full
thickness wounds (through the panniculus carnosus) of
8 mm were created on the midback of animals by using a
dermal punch (Miltex) and then covered with a Tegaderm
dressing (3 M). Db-/Db- wounds were treated with either
1×106 MSC, 1×106 CD45+ fetal liver cells, or vehicle
(phosphate-buffered saline; PBS) alone. Non-diabetic
wounds were treated with either 1×106 MSC or vehicle
alone. Wound treatments (cells or vehicle alone) were
administered immediately after wounding and took the form
of a group of four intradermal injections spaced in a radial
pattern from the wound edge in a total volume of 40 μl
(10 μl per injection) and applied via a Hamilton syringe.
Experimental protocols were approved by the Institutional
Animal Care and Use Committee and followed guidelines
set forth in the National Institutes of Health Guide for the
Care and Use of Laboratory Animals.
Tissue processing and wound measurement
At 3, 7, and 28 days after wounding, animals were euthanized,
and the wounds were harvested. Full thickness skin was
excised and viewed under a Leica MZ16 FA fluorescent
stereomicroscope for the presence of GFP. Wounds were then
bisected. One half was fixed overnight in 10% neutral
buffered formalin at 4°C, paraffin embedded, and sectioned
at 4 μm thickness. The other half was frozen in liquid nitrogen
for RNA isolation.
Sections were stained with hematoxylin and eosin for the
measurement of the epithelial gap and the granulation tissue
area by using Spot Advanced Version 4.5 software (Diagnos-
tic Instruments) by an investigator blinded to the group and
treatment. The epithelial gap was defined as the distance
between encroaching epidermal elements (at least three cell
layers thick). The granulation tissue area was defined as the
cellular area from the lateral transition zone from the normal
epidermis to the hypertrophic epidermis, inferior to the level
of the panniculus carnosus and superior to the epithelial
basement membrane or open wound surface.
Histology
For immunoperoxidase staining, endogenous peroxidase
activity was blocked with 0.3% hydrogen peroxide in
methanol. GFP was detected with rabbit anti-GFP primary
antiserum (1:100; Invitrogen) followed by biotinylated anti-
rabbit secondary antibody (1:200) and development with
avidin-biotin complex (Vector Laboratories). Slides were
counterstained with hematoxylin. For double-immunofluores-
cent staining, sections were blocked with 1% sodium
borohydrate (Sigma) in PBS. CD31 was detected by using
rat anti-mouse CD31 antibody (1:20 dilution; Invitrogen)
Cell Tissue Res (2007) 329:301–311
followed by biotinylated rabbit anti-rat secondary antibody
and detection with the alkaline phosphatase detection
system (Vector Laboratories). GFP was detected by using the
same rabbit anti-GFP primary antiserum followed by Alexa-
Fluor-488-conjugated F(ab’)2 secondary antibodies (1:100;
Invitrogen). Slides were counterstained with 4,6-diamidino-
2-phenylindole (Vector Laboratories) and examined. Five
high-power fields per sample were counted. For alpha
smooth muscle actin (α-SMA)/GFP double-staining, anti-
α-SMA antibody directly conjugated to fluorescein isothiocya-
nate was applied (1:200 dilution; Jackson ImmunoResearch)
followed by GFP staining as previously described.
RNA isolation and polymerase chain reaction
Total cellular RNA from wounds was isolated by using
TRIzol followed by preparation of cDNA with the
SuperScript First-Strand Synthesis System (Invitrogen)
according to the manufacturer’s instructions. The polymer-
ase chain reaction (PCR) was performed with REDExtract
N-Amp PCR ReadyMix (Sigma) and primers specific for
murine epidermal growth factor (EGF), PDGF-BB, kerati-
nocyte growth factor, transforming growth factor beta-1
(TGF-β1), vascular endothelial growth factor (VEGF),
interleukin-6 (IL–6), major intrinsic protein-2 (MIP2), IL–
10, placental-derived growth factor (PlGF), stromal-derived
growth factor 1–alpha (SDF–1α), and D-glyceraldehyde-3-
phosphate dehydrogenase (GAPDH; Table 1). Aliquots of
each reaction were subjected to gel electrophoresis in a 2%
agarose gel, and densitometry for individual sample
message level was performed by using Image J analysis
software (version 1.33 u) and standardized to GAPDH.
Cell isolation and characterization
MSC were isolated from fetal livers of embryonic day-15
gestational C57BL/6TbN (act-GFP) OsbY01 transgenic mice
expressing GFP. Fetal livers were mechanically disrupted into
a single cell suspension followed by the lysis of red cells with
ammonium chloride. For MSC isolation, cells were suspended
in “complete medium” containing MesenCult Basal Medium
for Murine Mesenchymal Stem Cells and Mesenchymal Stem
Cell Stimulatory Supplements (StemCell Technologies) and
plated at a density of 7.5×105 cells/cm2 at passage 0. Medium
was changed every 3 days until cells reached 90% confluency
(approximately 2 weeks), at which point, adherent fetal cells
were detached with 0.05% trypsin (Life Technologies/
Invitrogen). Cells were then enriched for MSC by CD11b
immunodepletion by using streptavidin magnetic beads
(Dynal Biotech) coated with biotinylated antibody against
CD11b (BD Biosciences) as previously described (Tropel et
al. 2004). Depleted cells were expanded in complete growth
media for 1 week and then cryopreserved at passage 1. MSC
303
were thawed and expanded for a maximum of four passages
for use in experiments.
MSC characterization included the performance of fibro-
blast-colony forming unit (CFU-f) assays, differentiation
assays, and flow cytometric analysis of cell-surface antigen
profiles. CFU-f assays were performed in triplicate according
to the manufacturer’s instructions (StemCell Technologies).
Osteogenic and adipogenic differentiation assays were carried
out at passage 5 as previously described (Digirolamo et al.
1999; Javazon et al. 2001). Flow cytometry was performed
by using a FACS Calibur and CellQuest Pro Version 5.0.1 fl
software. Primary antibodies tested included phycoerythrin
(PE)-conjugated antibodies with specificity for the surface
antigens CD3, CD11b, CD13, CD19, CD31, CD34, CD40,
CD44, CD45, CD73, CD90, c-Kit (CD117), MHC I, MHC
II, Sca–1, and purified anti CD49a (BD Biosciences). CD49a
was detected by PE-conjugated goat anti-Armenian hamster
IgG secondary antibody (Jackson ImmunoResearch).
For CD45+ cell isolation, a single cell suspension of fetal
liver cells was created and then incubated with CD45
microbeads. MACS-positive selection for CD45 cells was
performed b using an autoMACS separator according to the
manufacturer’s instructions (Miltenyi Biotech).
Fibroblasts were isolated from age-matched fetal mice
by explant culture. Fetal back skin was harvested, placed
dermal side down onto tissue culture plastic, and grown in
Dulbecco’s Modified Eagle’s media (MEM) with 10% fetal
bovine serum (Gibco). Cells migrating onto tissue culture
plates from the dermal surface were expanded and
cryopreserved at passage 1 for later use in experiments.
Cell-populated collagen gel assay
On ice, a collagen solution (Type I from rat tail; BD
Biosciences, Bedford, Mass.) in 0.1% acetic acid was
adjusted to physiologic pH (7.6) and ionic strength with
1 N NaOH and 10× MEM respectively. A concentrated cell
suspension of MSC was incorporated to achieve a final cell
density of 2×105 cells/ml. The cell-populated solution was
added to 12-well dishes (750 μl/gel) and polymerized for
1.5 h at 37°C. The gels were released by gentle tapping
and the use of a spatula into 6-well dishes containing 3 ml
complete growth media as previously described (Redden
and Doolin 2006). The cell-populated collagen gels (CPCG)
were incubated in a humidified incubator (5% CO2, 37°C).
Digital images of the gels were taken daily with a Bio-Rad
Chemi Doc System by using Quantity One Software
(version 4.1; Bio-Rad Laboratories, Hercules, Calif.) and
an in-picture centimeter ruler for scale. The images were
imported into image-analysis software (SigmaScan Pro,
SPSS Science), and pixel-per-centimeter calibrations were
made. The gel outlines were manually traced, and gel areas
were calculated by the software in square centimeters.
304 Cell Tissue Res (2007) 329:301–311

Statistical analysis
Wound measurements are presented as the mean±SD. PCR
data were normalized to the percent of mRNA expression in
untreated control wounds and are presented as the mean±
SD. Differences between experimental and control groups
were determined by using the two-tailed Student’s t-test for
samples with unequal variance (Excel 2003, Microsoft).
Probability values of P<0.05 were used to denote statistical
significance.
Results
Effect of MSC in diabetic wound healing
To assess the early effects of MSC on diabetic wound healing,
we chose the 7-day time-point for morphologic and quantitative
analysis. At 7 days, gross examination of MSC-treated diabetic
wounds (n=10) revealed near closure with a fibrous tissue
filling of the wound base and evidence of wrinkling around
the wound edge implying wound contraction (Fig. 1a). By
comparison, vehicle-treated wounds (n=10) appeared largely
unhealed with a bare wound base and little evidence of new
tissue in-growth or wound contraction (Fig. 1b). The underside
of MSC-treated diabetic wounds revealed a visible vascular
network within the subcutaneous fat (Fig. 1c), whereas
vehicle-treated controls had no such visible vessels (Fig. 1d).
Histologic examination of MSC-treated diabetic wounds
confirmed the presence of a robust granulation tissue bed with
re-epithelialization and increased cellularity in the wound base
(Fig. 2a). In contrast, vehicle-treated control diabetic wounds
were largely devoid of granulation tissue with little evidence
of re-epithelialization and less overall cellularity (Fig. 2b). In
order to compare the effect of MSC with a cellular control, we
isolated the CD45+ fraction of fetal liver and treated wounds
with an equal number of these cells (the CD45+ fraction
represents the MSC-negative portion of fetal liver and also
serves as a hematopoietic cellular control, being the predom-
inant cell population in whole bone marrow). Quantitative
analysis revealed that the epithelial gap of MSC-treated
diabetic wounds (1.3±0.9 mm) was significantly smaller than
that of CD45+-treated wounds (4.1±0.7 mm, P<0.004) and
vehicle-treated controls (3.2±0.5 mm, P<0.001, Fig. 2g).
Indeed, the epithelial gap of CD45+-treated wounds was
greater than that of vehicle-treated wounds, but this was not
statistically significant (P=0.06). The granulation tissue area
of MSC-treated diabetic wounds (2.4±0.6 mm2) was also
significantly higher than that of wounds administered vehicle
alone (0.8±0.3 mm2, P<0.05, Fig. 2h). There was no
significant difference, however, in the volume of granulation
tissue between MSC- and CD45+-treated wounds.

Fig. 1 Gross appearance

of MSC versus vehicle-treated
wounds at 7 days. Representa-
tive gross photographs of
wounds were taken at the time
of wound-tissue harvest.
a MSC-treated wound
exhibiting wrinkled skin around
the wound and fibrous tissue
growth filling the wound base.
b Largely unhealed wound
treated with vehicle alone.
c Underside of MSC-treated
wound demonstrating growing
vessels. d Vehicle-treated
wound; no vessels are visible in
the subcutaneous fat underlying
the wound base. Bars 5 mm
Cell Tissue Res (2007) 329:301–311
Fig. 2 Histologic analysis of MSC-, vehicle-, or CD45+-treated diabetic
wounds. An 8-mm excisional diabetic wound was immediately treated
with an intradermal injection of 1×106 MSC, 1×106 CD45+ fetal liver
cells, or PBS vehicle alone, placed radially around the wound. Wounds
were harvested at 7 days and stained with hematoxylin and eosin.
a MSC-treated wound. b Vehicle-treated wound. c CD45+-treated wound.
The edge of the encroaching epithelium is indicated by black arrow-
heads. Bars1 mm. d-f Corresponding higher power views of the right
Effect of MSC on wound healing of non-diabetic mice
In order to assess the effect of MSC on normal wound
healing, non-diabetic (Db+/Db-) heterozygous mice were
wounded in the same fashion and treated with MSC or PBS
vehicle alone. At 7 days post-wounding, MSC-treated (n=
10) and vehicle-treated (n=10) wounds had a similar
histologic appearance, and there was no significant differ-
ence in the epithelial gap (MSC 3.7±0.5 mm vs PBS 4.0±
0.8 mm, P=0.6) or granulation tissue area (MSC 3.9±
1.6 mm2 vs PBS 2.6±1.4 mm2, P=0.3).
Localization and differentiation of MSC in wounds
To determine the persistence of MSC, we analyzed wounds
for MSC content, both grossly and histologically, by using
GFP as a cell marker. At 7 days post-wounding, fluorescent
stereomicroscopy of samples immediately after harvesting
demonstrated GFP expression both in the wound base and at
the wound margin (Fig. 3b). Immunoperoxidase staining was
305
epithelial margin (boxed areas) demonstrating the encroaching epithelial
tongue over top of cellular granulation tissue. g Epithelial gap of diabetic
wounds treated with MSC (n=10; solid bar, MSC), CD45+ (n=10;
striped bar, CD45), or vehicle alone (n=10; open bar, PBS). *P<0.001
vs vehicle, †P<0.004 vs CD45+. h Granulation tissue area (GTA) of
diabetic wounds treated with MSC (n=10; solid bar, MSC), CD45+ (n=
10; striped bar, CD45), or vehicle alone (n=10; open bar, PBS). ‡P<0.05
vs vehicle
used to confirm the presence GFP in the wound histologically
and to allow for a more precise determination of MSC location
within the wounds. MSC were located within the dermis and
wound base. No GFP+ cells were seen in the epidermis
(Fig. 3c), and no GFP signal was detected in vehicle-treated
control wounds either grossly or histologically (Fig. 3e,f).
To determine any potential differentiation of MSC in
wounds, we performed double-immunofluorescent staining
for GFP and either CD31, an endothelial cell marker, or α-
SMA, a marker of myofibroblast differentiation. MSC-treated
diabetic wounds demonstrated higher levels of CD31 staining
(Fig. 4a) compared with vehicle-treated wounds (Fig. 4b).
Quantitative comparison revealed a significant increase in the
number of vessels per high-powered field in MSC versus
vehicle-treated wounds at 7 days (MSC 30.8±7.8 versus PBS
9.2±4.6, P<0.0005). In addition, GFP+ MSC were seen in
close association with CD31+ vessels, but no double-positive
cells were identified (Fig. 4a). MSC in diabetic wounds,
however, did demonstrate double-positive staining for GFP
and α-SMA, consistent with myofibroblast differentiation
306
Fig. 3 Presence of GFP+ cells in treated diabetic wounds at 7 days.
MSC and vehicle treated wounds were harvested at 7 days and
visualized by using fluorescent stereomicroscopy and immunohisto-
chemistry. a Representative gray-scale image of MSC-treated wound
(8 mm long) demonstrating near closure. b Corresponding fluorescent
image demonstrating GFP signal at the wound edge. c Immunoperox-
(Fig. 4c), whereas CD45+ cells stained positively for GFP
only (Fig. 4d).
Analysis of wound closure
MSC have the potential for induced differentiation into
other cell types of the mesenchymal lineage in vitro. To
exclude the possibility of late-onset differentiation of MSC
into other cell types of the mesenchymal linage, we ana-
lyzed the histology of wounds at 28 days post-wounding.
Based on the morphologic appearance of healed wounds
stained with hematoxylin and eosin, both MSC-treated
diabetic wounds and untreated diabetic control wounds
were completely re-epithelialized, with an absence of
epidermal appendages, and typical scar formation with the
resolution of granulation tissue and increased connective
tissue content compared with 7-day wounds (Fig. 5). In all
healed MSC-treated wounds examined, no histologic
evidence of bone or cartilage formation was noted (n=5,
5 sections examined per animal).
Production growth factors and cytokines in MSC-treated
wounds
To determine the contribution of MSC treatment to growth-
factor production during the early proliferative phase of
Cell Tissue Res (2007) 329:301–311
idase staining for GFP confirms the presence of GFP-expressing MSC
in the wound margin located exclusively within the dermis. Bar100 μm.
d-f Corresponding images of a vehicle-treated wound (8 mm long)
demonstrating minimal healing at 7 days and an absence of GFP signal
by fluorescent stereomicroscopy and immunoperoxidase staining
wound healing, wounds were harvested 3 days after
wounding, and mRNA production for various growth
factors was analyzed. MSC-treated diabetic wounds
exhibited increased mRNA production for EGF, TGFβ–
1, and VEGF compared with vehicle-treated controls and
with CD45+-treated wounds (n=6 per group, P<0.05),
whereas levels of PDGF-BB were not significantly
different (Fig. 6a). Because of the enhanced cellularity of
MSC-treated wounds, we also examined mRNA produc-
tion for factors important in cellular recruitment. Of the
factors tested, there was a two-fold increase in SDF–1α
mRNA. Levels of PlGF and the inflammatory cytokines
MIP2 and IL–6 were not significantly elevated. In
contrast, CD45+-treated wounds demonstrated levels of
chemotactic factors that were below those of controls
(Fig. 6b).
To determine the level of SDF–1α mRNA expression
during normal wound healing, we analyzed SDF-1α mes-
sage levels at 3 days in non-diabetic heterozygous animals
(Db+/Db-). Relative to GAPDH, SDF–1α mRNA xpression
was significantly higher in wounded Db+/Db- (1.5±0.2-fold)
compared with Db-/Db- animals (1.1±0.2-fold, P<0.05). In
diabetic wounds treated with MSC, SDF-1α production was
similar (at 1.7±0.1-fold higher than GAPDH) to SDF–1α
production in non-wound-healing-impaired non-diabetic het-
erozygote animals.
Cell Tissue Res (2007) 329:301–311
Fig. 4 MSC differentiation in wounds. To assess for endothelial
differentiation, wounds were harvested at 7 days and stained for both
GFP (green, white arrowheads) and CD31 (red, white arrows).
Representative images were taken of the wound base of MSC and
vehicle-treated control wounds. a MSC-treated wound demonstrating
increased vascularity, with GFP cells located in close proximity to
vessels. No double-positive cells were identified to indicate endothelial
cell differentiation by MSC. b Vehicle-treated wound in comparison
Collagen gel contraction
To determine potential direct mechanisms by which MSC
might improve diabetic wound healing, we utilized the free-
floating CPCG contraction assay. Since fibroblasts are
Fig. 5 Histology of healed wounds at 28 days post-wounding.
Wounds were harvested from MSC- and vehicle-treated animals at
28 days post-wounding and stained with hematoxylin and eosin. The
area of the healed wound was identified by the absence of epidermal
appendages. MSC-treated wounds (a) demonstrated complete re-
307
exhibiting fewer CD31+ vessels. Bars50 μm. To assess for myofibro-
blast differentiation, sections were stained for α-SMA (green) and
GFP (red). c Double-staining for α-SMA and GFP (yellow, open
arrowheads) in an MSC-treated wound, demonstrating myofibroblast
differentiation. d In CD45+-treated wounds, cells stained positive for
GFP only (red, white arrowheads). Other wound cells stained for α-
SMA only (green, white arrow). No double-positive cells were
identified. Bars 100 μm
thought to mediate wound contraction, we chose age-
matched fetal fibroblasts for the control cell population in
the gel contraction assay. At all time-points, MSC were
more efficient than dermal fibroblasts with regard to
contraction of the collagen matrix (P<0.05, Fig. 7).
epithelialization and typical scar formation with no epidermal
appendages and an increased connective tissue content similar to that
of vehicle-treated wounds (b). No evidence of bone or cartilage
formation was seen in MSC-treated wounds. Bars 50 μm
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Fig. 6 Cytokine production in MSC versus CD45+-treated wounds at
3 days. Total cellular mRNA was isolated from wounds at 3 days post-
wounding, standardized to GAPDH for each sample, and analyzed by
semi-quantitative PCR for the expression of various cytokines. mRNA
levels from MSC-treated (black bars) and CD45+-treated (open bars)
wounds are presented as the percent expression of vehicle-treated
control wounds (dotted line control level of expression). a MSC-
treated wounds demonstrated an elevation in message levels for
epidermal growth factor (EGF), transforming growth factor beta 1
(TGF-β1), and vascular endothelial growth factor (VEGF) compared
with CD45+-treated wounds at P<0.05 (PDGF-BB platelet-derived
growth factor). b MSC-treated wounds also exhibited a significant
elevation in stromal-derived growth factor 1 alpha (SDF–1α) mRNA
(IL-6 interleukin-6, MIP2 major intrinsic protein-2, PLGF placental-
derived growth factor)
MSC isolation and characterization
MSC are identified by a combination of functional criteria
and by their cell-surface antigen profile. To date, no
specific MSC marker has been identified.
In order to test the adequacy of our MSC enrichment and
the reproducibility of our isolation protocol, we performed
CFU-f assays (a quantitative measure of mesenchymal
progenitor content) and differentiation assays and charac-
terized the cell-surface antigen profile of cells at each
passage (n=10). With our isolation protocol, the value of
CFU-f per 100 cells plated increased from 1.7×10−3% pre-
depletion to 2% immediately following CD11b depletion, a
1,000-fold enrichment.
Myeloid cells, identified as CD11b+, are the largest
contaminating cell population in MSC cultures. Following
CD11b depletion, the percentage of CD11b+ cells was less
than 2%. The cell-surface antigen profile of the remaining
cell population was CD31−, CD34−, and CD45−, but
strongly positive for CD44, CD90.2, Sca–1, and CD49a
(data not shown). Short-term culturing further selected for
Cell Tissue Res (2007) 329:301–311
Fig. 7 MSC-mediated extracellular matrix interactions. MSC were
observed in a free-floating cell-populated collagen gel contraction
assay and compared with fibroblast-seeded control gels. a Gel area
was calculated daily from digital images. At the same cell density,
MSC-seeded gels contracted faster then fibroblast-populated gels.
b At individual time-points, the percent gel area of MSC-seeded gels
was less than that with an equal density of fibroblasts, indicating
greater overall contraction of MSC-seeded gels. *P<0.05
the expansion of MSC yielding a dramatic increase in
CD49a levels with >90% of cells expressing CD49a by
passage 5. MSC also demonstrated multipotent capacity for
induced osteogenic and adipogenic differentiation and
spontaneous myofibroblast differentiation as defined by
the expression of α-SMA.
Discussion
We have demonstrated the improved healing and closure of
diabetic wounds following the local application of MSC.
This improvement is characterized by enhanced epithelial-
ization, granulation tissue production, and neovasculariza-
tion. These dramatic wound-healing effects might be
mediated through a combination of direct and indirect
mechanisms, including myofibroblast differentiation and
promotion of wound contraction, augmented growth-factor
production, and enhanced cellular recruitment.
In the wound environment, MSC differentiation is
restricted to the mesenchymal lineage. MSC differentiation
into myofibroblasts may contribute to the contracted smaller
appearance of MSC-treated wounds compared with untreated
diabetic wounds as early as 7 days post-wounding. This is a
significant change from the typical pattern of Db-/Db- wound
closure since, unlike normal rodent wound healing, Db-/Db-
wound healing relies more on new tissue production than does
Cell Tissue Res (2007) 329:301–311
wound contraction to achieve wound closure (Sullivan et al.
2004). In addition to these in vivo observations, the
contractile abilities of MSC have been specifically tested
and compared with that of fibroblasts in a contraction assay.
In collagen gels, MSC demonstrate not only their ability to
interact directly with extracellular matrix components, but
also their greater contractile capacity than fibroblasts.
Fibroblasts are generally regarded as the principal cellular
mediators of wound contraction in response to growth
factors and alterations in mechanical forces within the
wound (Broughton et al. 2006). Therefore, the ability of
MSC to generate contractile forces on the local wound
matrix may be superior to that of fibroblasts. In human
diabetic ulcers, the failure of timely wound contraction is
known to increase the risk of infection, to prolong inflamma-
tion, and to exacerbate wound-healing difficulties (Singer and
Clark 1999). MSC-mediated decreases in wound size
through contraction alone might have the potential to offset
diabetes-related wound-healing impairment significantly.
In contrast to other reports, we have not observed
epidermal or endothelial cell differentiation following
MSC treatment. To our knowledge, epidermal transdiffer-
entiation has only been described for human embryonic
mesenchymal stem cell populations (M. Wu et al. 2006). In
our study, MSC are distinct from the epidermal layer. To
assess for endothelial differentiation, we have chosen a
time-period with robust granulation tissue and high vessel
density but have failed to observe transdifferentiation
events. Although transduced progenitor cells might down-
regulate GFP expression with terminal differentiation, our
cell population is derived from transgenic animals, and
therefore, GFP expression should be maintained in differ-
entiated cell types. Previous reports of endothelial differ-
entiation come from in vitro observations, which are of
unclear relevance to their actual differentiation potential in
vivo (Gang et al. 2006; Oswald et al. 2004). Claims of such
transdifferentiation in vivo are extremely rare events in the
absence of severe tissue injury and selection pressures
(Nagaya et al. 2004) and are unlikely to make a significant
contribution to the therapeutic benefit of MSC observed in
this study.
MSC may also influence healing indirectly through local
increases in the production of VEGF, EGF, TGFβ–1, and
SDF–1α. Growth factors are important in modulating the
wound-healing response; however, the effect of a growth
factor is highly dependent on the concentration and timing of
its delivery. Theoretically, a possible explanation for the
success of MSC in our study is their persistence in the wound
during early wound healing and their potential for regulated
delivery of multiple growth factors in response to local
environmental cues without the need for re-administration.
Whereas MSC have been shown to produce a variety of
growth factors in vitro, the cellular origin of growth factors
309
in MSC-treated wounds might not be MSC, but rather
endogenous cells that are induced by MSC to increase the
production of growth-factor mRNA. Regardless of the
cellular source of growth factor, inappropriate growth-factor
delivery can contribute to the establishment of a chronic
inflammatory state. Therefore, it is important to note that all
MSC-treated wounds healed completely in our experiments,
with the full resolution of inflammation. MSC-mediated
increases in local growth-factor levels may provide a
powerful therapeutic tool for the improvement of diabetes-
related healing impairments.
Interestingly, MSC-treated wounds demonstrate signifi-
cant increases in SDF–1α, one of the most important
factors in the cellular recruitment of stem and progenitor
cells to tissues (Dar et al. 2006). MSC and inflammatory
cells have been shown to migrate in response to SDF–1α
(Bhakta et al. 2006). More recently, SDF–1α has also been
shown to have a role in dermal wound healing and to be
down-regulated by inflammatory cytokines (Fedyk et al.
2001). We have found a deficiency of SDF–1α in diabetic
wounds. These differences in SDF–1α production suggest
that the impairment in wound healing may be attributable in
part to a defect in progenitor cell recruitment and/or
function. The deficiency of SDF–1α seen in diabetic
wounds may be explained by the down-regulation of SDF
in response to protracted inflammation in the wound. Of
interest, MSC does not significantly improve the rate of
wound healing in non-diabetic mice. Perhaps, wound
healing in these animals proceeds at a maximal rate, and
MSC do not have a detectable effect on healing in the
setting of intact host-progenitor-cell recruitment and mobi-
lization to wounds. MSC-mediated increases in SDF–1α
production may facilitate the mobilization and recruitment
of cells to healing wound for their contribution to tissue
repair; however, since no specific MSC marker exists, we
have been unable to determine the proportions of these cells
that represent endogenous progenitor cells or other inflam-
matory cells.
The observed improvements in wound closure seen with
placement of MSC in the periphery of the wound is
supportive of the importance of growth-factor production
and paracrine-based mechanisms of MSC wound-healing
effects. In our study, we have used intradermal application
of MSC, since this mode of delivery results in better cell
persistence than topical cell application in the wound base
(data not shown). Since MSC have been shown to migrate
in response to an SDF–1α gradient, and since the
concentration of SDF–1α is likely to be the highest at the
site of tissue injury (Ceradini et al. 2004), we would expect
that any migration of MSC would be toward the area of
maximal tissue injury within the wound. Delivery of cells
within a prefabricated biologic matrix is an attractive
approach to improving the retention of cells in the wound
310
base. However, the use of such scaffolds also has the
potential to alter MSC properties and to elicit host
inflammatory reactions (Pandit et al. 1999; Sung et al.
2004). Further studies are required to determine the
influence of different scaffolds on MSC properties and
any overall wound-healing benefit.
Bone marrow cells have been hypothesized to be
recruited to wounds and to participate in tissue repair, but
the specific type of bone marrow cell responsible for such
repair remains unidentified. Indirect evidence suggests the
presence of a non-hematopoietic population of cells with
tissue-repair functions (Badiavas et al. 2003; Fathke et al.
2004; Opalenik and Davidson 2005). Identification of this
tissue-repair population has been limited by an inability to
isolate and characterize purified subpopulations from
hematopoietic organs for subsequent transplantation stud-
ies. To address this question, we have compared the effect
of MSC with that of the hematopoietic fraction of fetal
liver. We have chosen fetal liver as our tissue source
because of the higher frequency of MSC and the ability to
eliminate hematopoietic cell contamination by early immuno-
depletion. Of note, our fetal liver MSC population is
uniformly stromal and entirely devoid of hematopoietic
contamination, unlike early-passage adult murine bone-
marrow-derived MSC. We have observed that MSC-treated
wounds heal better than wounds treated with the CD45+
hematopoietic cell fraction of fetal liver. Indeed, CD45+-
treated wounds demonstrate a trend toward lower growth-
factor production and epithelialization compared with controls.
MSC also tend to persist in wounded skin, whereas CD45+
cells are cleared. This comparison directly supports the role
of stromal non-hematopoietic progenitor cells as being the
principal effectors of tissue repair within hematopoietic
organs.
Of note, MSC properties may potentially vary depending
on the tissue of derivation. However, the only differences in
differentiation properties have been seen in MSC isolated
from adult spleen, umbilical cord blood, and adult liver (in ’t
Anker et al. 2003; Kern et al. 2006). In addition, a direct
comparison of growth-factor profiles between human adult
bone-marrow-derived and human adult adipose-derived
MSC has failed to show any differences (Wang et al.
2006). To date, no formal comparisons have been made
between bone-marrow-derived and other tissue-derived
MSC preparations with regard to their tissue-repair proper-
ties. Such a comparison might be helpful in identifying the
optimal tissue source for MSC-based wound-healing
applications.
MSC offer several additional advantages over other cell-
based therapies. Cultured keratinocytes and fibroblasts have
been incorporated into scaffolds and bioengineered for use
as wound coverage and vehicles for growth-factor delivery
(Dinh and Veves 2006; Veves et al. 2001), although the
Cell Tissue Res (2007) 329:301–311
creation of such constructs can be complicated and time-
consuming. Our isolation protocol produces a defined
reproducible MSC population in just 2 weeks. For our
study, we have chosen a fetal liver source because of the
species-related difficulty in the isolation of MSC. Human
MSC, by comparison, are easily isolated in sufficient
numbers from donor and from autologous bone marrow.
Autologous derivation has the advantage of avoiding the
transmission of infectious diseases and the need for
immunosuppression. The autologous isolation of fibro-
blasts, however, requires the creation of a new wound to
obtain tissue in a patient with impaired healing. Such
practices have the potential to result in additional wound
morbidity.
Locally applied MSC can overcome many of the
pathophysiologic abnormalities in diabetic wounds. This
improvement appears to be mediated through direct and
indirect mechanisms, including promotion of contraction
through myofibroblast differentiation, direct interaction
with the extracellular matrix, and augmentation of growth-
factor production. Together, these mechanisms contribute to
enhanced neovascularization and cellular recruitment.
These cells may also be responsible for the tissue-repair
properties observed with regard to cell preparations from
hematopoietic organs. Recognition of the impact of MSC
on impaired wound healing has significant implications for
the development of novel therapeutic strategies to facilitate
wound closure, and further studies are needed to optimize
this therapy and to compare directly its efficacy with that of
other cell-based therapies.
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