2.1.

Animals, Antibodies, and Reagents

Male Lewis (LEW) rats $\left(250\text{to}280\mathrm{g}\right)$ were purchased from Charles River Japan, Incorporated (Yokohama, Japan). LacZ-Tg LEW rats (ROSA/LacZ-LEW)^{18, 19} and luciferase (luc)-Tg LEW rats (ROSA/Luciferase-LEW)²⁰ previously generated were used as donors ( $8\text{to}12\text{weeks}$ old). All rats had free access to standard chow and drinking water, and were maintained on a $12\text{-}\mathrm{h}$ light/dark cycle. All animal experiments in this study were performed in accordance with the Jichi Medical University Guide for Laboratory Animals.

Fluorescent isothiocyanate (FITC)-conjugated antirat CD29 monoclonal antibody (mAb, clone Ha2/5), (FITC)-conjugated antirat CD90 (Thy-1, clone HIS51) mAb, and phycoerythrin (PE)-conjugated antirat CD45 mAb (clone OX-1) were purchased from BD Pharmingen (San Diego, California). PE-conjugated antirat CD34 mAb (clone ICO115, sc-7324) was purchased from Santa Cruz Biotechnology (Santa Cruz, California). Isotype-matched IgG controls were purchased from BD Pharmingen. Mouse antivascular endothelial growth factor (VEGF) mAb (clone C1, sc-7269, Santa Cruz), rabbit anti-CD31 affinity-purified IgG (sc-1506-R, Santa Cruz), mouse IgG (sc-3879, Santa Cruz), and mouse antihypoxia inducible factor- $1\alpha$ (HIF- $1\alpha$ ) mAb (clone, H1alpha67, MAB5382, Chemicon, Temecula, California) were used as the primary Ab for immunohistochemistry, followed by biotinylated antirabbit (BA-1000) and antimouse IgG (BA-2001, Vector Laboratories, Burlingame, California), respectively.

Streptozotocin (STZ) was purchased from Wako Pure Chemicals (Osaka, Japan). Terudermis was used as an artificial dermis and kindly provided by Olympus Terumo Biomaterials Corporation (Tokyo, Japan).

2.2.

Isolation and Culture of Mesenchymal Stromal Cells

Rat BMCs were harvested by flushing femurs and tibiae with ice-cold phosphate-buffered saline (PBS) as previously described.²¹ Cells were filtered through a $70\text{-}\mu \mathrm{m}$ nylon mesh and plated in $\mathrm{T}75\text{-}{\mathrm{cm}}^2$ or $\mathrm{T}225\text{-}{\mathrm{cm}}^2$ flasks with DMEM/F-12 (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cultures were kept in a humidified atmosphere containing 5% $\mathrm{C}{\mathrm{O}}_2$ and 95% air at $37°\mathrm{C}$ . Nonadherent cells were removed after $24\mathrm{h}$ . Adherent cells were trypsinized with 0.25% trypsin-EDTA (Gibco), harvested, and then plated into new flasks at every 90% confluency. Adherent cells from passage 2 were frozen in liquid nitrogen for future use. Early passage cells were examined for their capacity to differentiate in culture (Fig. 1 ).

Fig. 1

Schematic of mesenchymal stromal cells (MSCs) purification. Bone marrow cells (BMCs) were harvested by flushing femurs and tibiae with ice-cold PBS. Cells were cultivated in flasks containing DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

2.3.

In-Vitro Differentiation Assay

Passage 4 MSCs were tested for their ability to differentiate into osteocytes and adipocytes. For adipocyte differentiation, cells $(2\times {10}^5)$ were cultured with Differentiation Media BulletKits-Adipogenic (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. MSCs were cultured in six-well plates with MSCs culture medium until they reached confluency. Cells were then exposed to three cycles of adipogenic induction media alternating with adipogenic maintenance media. Following three complete cycles of induction/maintenance, the MSCs were cultured for 7 more days in supplemented adipogenic maintenance medium. Cell differentiation to adipocytes was confirmed by Oil Red O (Muto Chemicals, Tokyo, Japan) staining. For osteogenic differentiation, cells $(2\times {10}^4)$ were plated, and the culture medium was replaced with Differentiation Media BulletKits-Osteogenic (Lonza) until confluence. Cells were stained with alizaline red S (Wako Pure Chemicals).

2.4.

Diabetic Wound Model and Cell Transplantation

To induce diabetes mellitus, streptozotocin (STZ) in a $0.1\text{-}\mathrm{M}$ citrate buffer (pH 4.3) was injected intravenously into the penile vein of normal LEW rats ( $60\mathrm{mg}∕\mathrm{kg}$ body weight). Rat blood glucose levels were then assessed using a glucometer after $3\text{days}$ , and individual rats with glucose levels greater than $250\mathrm{mg}∕\mathrm{dl}$ were classified as diabetic and immediately utilized for further experimentation.

STZ-induced diabetic rats were anesthetized with pentobarbital $(40\mathrm{mg}∕\mathrm{kg})$ and full-thickness skin defects $(2\times 2\mathrm{cm})$ were made on the scalp.²² Two hundred microliters of PBS containing fresh BMCs $(1\times {10}^7)$ or MSCs $(1\times {10}^7)$ was added to the inner layer of the same-sized artificial dermis for $30\sec$ at room temperature.²³ The wound was covered with the impregnated artificial dermis and sutured using 4-0 nylon. Artificial dermis impregnated with PBS was grafted onto rats that formed the control group.

The wounds were photographed every week in a standard prone position, and were monitored by planimetry using Scion image software (Scion Image Alpha 4.0.3.2, Scion Corporation, Maryland). The percent of wound area was calculated as the ratio of the nonhealing wound area to the original wound area.

2.5.

In-Vivo Bioluminescent Imaging

In-vivo luminescent imaging was obtained using the noninvasive bioimaging system IVIS™ (Xenogen, Alameda, California) and analyzed using Igor (WaveMetrics, Lake Oswego, Oregon) and IVIS Living Image (Xenogen) software packages. To detect luminescence from luciferase-expressing cells, D-luciferin (potassium salt, Biosynth, Postfach, Switzerland) was injected intravenously into the penile vein or subcutaneously into the head skin of rats anesthetized ( $30\text{-}\mathrm{mg}∕\mathrm{kg}$ body weight) with isoflurane. The signal intensity was quantified as photons flux in units of photons/ $\sec ∕{\mathrm{cm}}^2$ /steradian in the region of interest.

2.6.

X-Gal Staining and Immunohistochemistry

Rats were euthanized and the systemic circulation was flushed through the left ventricle with PBS followed by 4% paraformaldehyde (PFA). Specimens were divided and fixed in 4% PFA, and embedded in either paraffin or Tissue Tec OCT Compound (Sakura Finetechnical Company, Limited, Tokyo, Japan). Thin sections $\left(4\text{to}7\mu \mathrm{m}\right)$ were stained with hematoxylin and eosin, or were used for immunohistochemistry.

To visualize lacZ expression, X-gal staining was performed as previously described.¹⁸ Briefly, sections were washed three times with PBS containing $2\text{-}\mathrm{mM}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , 0.01% sodium deoxycholate, and 0.02% Nonidet-P40. Specimens were treated with a $\beta$ -gal staining solution ( $1\mathrm{mg}∕\mathrm{ml}$ of 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, $2\text{-}\mathrm{mM}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , $5\text{-}\mathrm{mM}$ potassium hexacyanoferrate [III], and $5\text{-}\mathrm{mM}$ potassium hexacyanoferrate [II] trihydrate) at $37°\mathrm{C}$ for $1\text{to}2\mathrm{h}$ .

For immunohistochemistry, sections were probed with anti-CD31, anti-VEGF, and anti-HIF- $1\alpha$ antibodies. Specimens were blocked with 1% BSA in PBS and incubated overnight at $4°\mathrm{C}$ with anti-CD31 (diluted 1:50, $4\mu \mathrm{g}∕\mathrm{ml}$ ), anti-VEGF (diluted 1:100, $100\mu \mathrm{g}∕\mathrm{ml}$ ), and anti-HIF- $1\alpha$ antibody (diluted 1:200, $5\mu \mathrm{g}∕\mathrm{ml}$ ), in 0.1% BSA, respectively. Biotinylated antirabbit IgG (Vector Laboratories, Burlingame, California) or biotinylated antimouse IgG (Vector Laboratories) was used as the secondary antibody (diluted 1:200 in 0.1% BSA for $1\mathrm{h}$ at room temperature). The labeled sections were incubated with horseradish peroxidase (HRP)-conjugated streptavidin (Vector Laboratories), and followed by diaminobenzidine $(0.5\mu \mathrm{g}∕\mathrm{ml})$ for color visualization. Specimens were counterstained with hematoxylin. In sections stained with anti-CD31 antibody, CD31-positive tubular structures within the wound were considered as blood vessels. In ten similar sections, CD31-positive vessels were counted and normalized to $1\mathrm{mm}$ .²⁴

2.7.

Statistical Analysis

The Student’s t-test, Tukey’s HSD test, and U-test were used for the statistical analyses. The SPSS 11.0 system (SPSS Incorporated, Chicago, Illinois) was used for the statistical analysis. Differences between groups were considered significant when $\mathrm{P}<0.05$ .

3.1.

Characteristics of Bone-Marrow-Derived Mesenchymal Stromal Cells from Transgenic Rats

To define the isolated MSCs from adult transgenic rats, the expression of cell surface markers was examined using FACS analysis [Fig. 2a ]. As previously described,^{5, 10} isolated MSCs from lacZ-transgenic rats^{18, 19} were CD29 and CD90 (Thy-1) positive, and CD34 and CD45 negative. Moreover, the MSCs rapidly proliferated, formed colonies, and retained an adherent spindle-shaped morphology [Fig. 2b]. These cells were capable of differentiating into adipocytes [Fig. 2c] and osteocytes [Fig. 2d]. All of these phenotypes were equivalent to those of wild-type rats, and remained unchanged even in luciferase transgenic rats (data not shown). Thus, these results demonstrate that MSCs from transgenic rats possess appropriate differentiation potential. To avoid loss of multipotentiality for differentiation through passage in culture, MSCs derived from no more than a fourth passage were used for all experiments.

Fig. 2

Characteristics of MSCs. (a) Flow cytometric histogram analysis of culture-expanded MSCs was performed as detailed in Sec. 2. Expression profiles for CD29, CD34, CD45, and Thy-1 are depicted. Cells were uniformly negative for CD34 and CD45, and positive for CD29, Thy-1 markers associated with MSCs. Specific antibody profile (gray) versus isotype-matched antibody control (black line) is shown. (b) Colony formation of MSCs from wild-type LEW rats. Colonies were stained with crystal violet. Adherent spindle-shaped cells proliferated to form colonies. $\text{Bar}=500\mu \mathrm{m}$ . Under appropriate differentiation conditions, MSCs were capable of differentiating into (c) adipocytes (stained with Oil Red O for lipid droplets; $\text{Bar}=50\mu \mathrm{m}$ ) and (d) osteocytes (stained with alizaline red for mineral deposition).

To visualize the fate of MSCs, luminescence sensitivity was examined in luc-Tg-derived MSCs. As shown in Fig. 3a , at least $5\times {10}^4$ MSCs from luc-Tg rats are required for substantial detection over the background in vitro, even though a linear dose-dependent output of light was maintained in the presence of D-luciferin. Although the MSCs comprised approximately 0.001% freshly isolated BMCs (data not shown), both expanded MSCs in culture and freshly isolated BMCs exhibited equivalent photon counts at the same cell number $(1\times {10}^6)$ [Fig. 3b].

Fig. 3

Characteristics of MSCs from luciferase Tg LEW rats. (a) Luciferase activity of MSCs from luciferase Tg LEW rats. MSCs from luciferase Tg rats were plated onto 96-well plates at the indicated number. Luciferase activity (photon intensity) was evaluated in the presence of D-luciferin. Data represent the mean luciferase activity from two independent experiments (black squares and gray diamonds represent two different experimental trials). (b) Luciferase-expressing BMCs and MSCs possessed stronger luciferase activity than wild-type BMCs.

3.2.

Topical Administration of Mesenchymal Stromal Cells Promotes Skin Wound Healing in Diabetic Rats

To investigate whether skin wound healing could be impaired in the diabetic condition, streptozotocin (STZ) was intravenously injected into the penile vein of male rats, and full-thickness skin defects $(2\times 2\mathrm{cm})$ were then made on the scalp of diabetic rats (greater than $250\mathrm{mg}∕\mathrm{dl}$ of blood glucose). The skin defect was covered with an artificial dermis. The wound area of diabetic rats was significantly smaller than that of control rats [Figs. 4a and 4b ]. Skin wounds of control rats were almost totally restored at $3\text{weeks}$ , while those of diabetic rats remained unhealed. These findings indicate impaired wound healing in diabetic rats.

Fig. 4

Delayed wound healing studies in rats. A square area $(2\times 2\mathrm{cm})$ was surgically excised down to muscle fascia on the head of streptozotocin (STZ)-induced diabetes mellitus (DM) LEW rats or normal LEW rats ( $\mathrm{n}=22$ animals in total). (a) The wounds were photographed every week, followed by planimetry analysis using Scion image software. $\text{Bar}=1\mathrm{cm.}$ 1 and $2\text{weeks}$ after the operation, DM rats healed significantly more slowly (b) than normal rats ( ${}^{*}\mathrm{p}<0.05$ and ${}^{**}\mathrm{p}<0.01$ , respectively). The skin wounds of normal rats were totally restored at $3\text{weeks}$ . Data expressed as means $\pm \mathrm{SD}$ . DM stands for streptozotocin-induced diabetes mellitus LEW rats. Normal is for normal LEW rats.

In an effort to address the therapeutic effect of MSCs on skin wounds in diabetic rats, skin defects of diabetic rats were treated with either MSCs or BMCs that were impregnated within an artificial dermis. Since an artificial dermis provides a scaffold for tissue regeneration and is amenable to impregnation with various cellular materials, it was used as a scaffold for the MSCs and BMCs. As shown in Figs. 5a and 5b , the wound area of MSC-treated diabetic rats was strikingly smaller than that of BMC-treated diabetic rats $1\text{week}$ after treatment. Notably, retention of MSCs and BMCs in the wound site was difficult without the artificial dermis (data not shown). These data demonstrate that topical administration of MSCs promotes skin wound healing in diabetic rats at an early stage. The artificial dermis promoted wound healing at 2 and $3\text{weeks}$ after treatment, although the artificial dermis when used with BMCs or MSCs was not significantly smaller than when it was used alone.

Fig. 5

Healing effect of MSCs on diabetic wounds. (a) Groups comprising $\mathrm{MSCs}+\text{artificial}$ dermis (AD), $\mathrm{BMCs}+\mathrm{AD}$ , AD-only, and defects were formed using DM rats ( $\mathrm{n}=58$ animals in total) and photographed $1\text{week}$ after transplantation. $\text{Bar}=1\mathrm{cm}$ . (b) Percent of wound area was calculated using Scion image software, and values for the $\mathrm{MSCs}+\mathrm{AD}$ group were significantly lower than those for the $\mathrm{BMCs}+\mathrm{AD}$ , the AD-only and defect groups at $1\text{week}$ after transplantation ( ${}^{*}\mathrm{p}<0.05$ , ${}^{**}\mathrm{p}<0.01$ , and ${}^{***}\mathrm{p}<0.001$ , respectively). Data expressed as means $\pm \mathrm{SD}$ .

3.3.

Fate of Topically Administered Mesenchymal Stromal Cells on the Skin Wound of Diabetic Rats

To examine the fate of MSCs administered topically onto the wound area of diabetic rats, either MSCs or BMCs from luc-Tg rats were used as the therapeutic cell source with an artificial dermis. As shown in Fig. 6a , MSC-derived photons were obtained during the whole period of wound healing (for $21\text{days}$ ), although photon counts decreased within a few days [to 40%, Fig. 5b]. BMC-derived photons decreased considerably to background levels within $7\text{days}$ [Fig. 6b]. These results demonstrate that MSCs administered topically were retained to a greater extent than BMCs at the wound area of diabetic rats.

Fig. 6

Transplantation of artificial dermis-impregnated luciferase-expressing bone-marrow-derived cells. Groups comprising $\mathrm{MSCs}+\mathrm{AD}$ , $\mathrm{BMCs}+\mathrm{AD}$ , and AD-only were formed using DM rats ( $\mathrm{n}=15$ animals in total). D-luciferin was injected locally into the head skin of rats and luminescence was detected using the IVIS system. (a) Luminescence was recorded 0, 7, 14, and $21\text{days}$ post-transplantation. (b) The time-course rate of luminescence is depicted. The $\mathrm{MSCs}+\mathrm{AD}$ group had significantly higher luminescence than the $\mathrm{BMCs}+\mathrm{AD}$ group $({}^{*}\mathrm{p}<0.05)$ .

3.4.

Mesenchymal Stromal Cells Accelerate Angiogenesis during Diabetic Wound Healing

Since the wound area of MSC-treated diabetic rats improved $1\text{week}$ after treatment, specimens were stained with hematoxylin and eosin. As shown in Fig. 7a , an increased number of vessel structures was observed in the MSC specimen, suggesting neoangiogenesis. Neoangiogenesis is a critical step in wound healing, and the CD31 molecule represents a major marker for vascular endothelial cells. Therefore, we set out to determine whether the administered MSCs affected the CD31-positive vessel number at the wound site of diabetic rats. In contrast with BMC treatment, MSC treatment showed that CD31-positive adventitial vessels significantly increased in number at $1\text{week}$ posttreatment $(\mathrm{p}<0.001)$ [Fig. 7a]. Furthermore, MSCs from LacZ-Tg rats were used to assess the relationship between the presence of MSCs and vascular endothelial growth factor 1 (VEGF-1) expression using immunohistochemistry [Fig. 7b]. Specimens were stained using anti-VEGF antibodies and X-gal $1\text{week}$ following cell transplantation. In comparison with BMC treatment, MSC transplantation increased VEGF expression with LacZ-expressing cells. Expression of hypoxia-inducible factor $1\alpha$ (HIF- $1\alpha$ ), whose transcription factor tightly regulates VEGF expression under hypoxic conditions, was also well correlated with VEGF expression [Fig. 7b]. These results suggest that HIF-1-mediated VEGF expression contributes to the increased number of vessels at the wound site.

Fig. 7

Transplantation of artificial dermis-impregnated LacZ-expressing bone-marrow-derived cells. (a): Upper panels show hematoxylin and eosin (HE) staining at $1\text{week}$ after the transplantation, and lower panels show CD31 immunostaining. $\text{Bar}=20\mu \mathrm{m}$ . The $\mathrm{MSCs}+\mathrm{AD}$ group had many microvessels (arrows) as shown by HE staining, and many CD31 positive vessels (arrows) as shown by CD31 immunostaining. The total vessel number was determined and expressed as vessel number/ ${\mathrm{mm}}^2$ . The $\mathrm{MSCs}+\mathrm{AD}$ group had significantly more vessels than the $\mathrm{BMCs}+\mathrm{AD}$ and AD-only groups $({}^{*}\mathrm{p}<0.001)$ . Data expressed as means $\pm \mathrm{SD}$ . (b) Specimens were stained for VEGF (red arrows), HIF- $1\alpha$ (yellow arrows), and mouse IgG (control) after X-gal staining (blue arrows). $\text{Bar}=20\mu \mathrm{m}$ . The $\mathrm{MSCs}+\mathrm{AD}$ group expressed considerable levels of VEGF and HIF- $1\alpha$ , and double-positive cells were identified.