An acute hindlimb ischemia mice model was established according to published protocols of Ngoc Bich Vu et al. (2012) using 3-5-month-old immunosuppressed mice Pham et al., 2014aVu, 2013. All procedures involving animals were approved by the Animal Welfare Committee of the Stem Cell Research and Application Laboratory, University of Science, VNUHCM, VN. Briefly, mice were anesthetized by ketamine-xylazine, and were fixed to trays. Hairy limb was shaved and thigh skin was cut along approximately 1 cm. Femoral artery and vein were separated from muscle, and then ligated at 2 sites, one at the femoral triangle and the other at the popliteal artery. An incision was performed between the 2 ligations. Damaged tissue recuperation was evaluated using graded morphological scales at the area of muscle necrosis, following the guidelines of Takako Goto et al. (2006) Goto et al., 2006 and our previous studies Pham et al., 2014bVu et al., 2015. The damage of limb was classified as Grade 0 (G0), if no change; GI, if necrosis in nail and toes; GII, if necrosis in feet; GIII, if necrosis in knee; and GIV, if total leg necrosis.

hADSCs were isolated according to our previous study Van Pham et al., 2013, with the following 3 criteria: (1) hADSCs maintained the differentiation potential to form chondrocyte and adipocyte (2) possessed plastic adherent ability and fibroblastic-like appearance and (3) expressed CD44, CD73, and CD90 and did not express CD14, CD34, and CD45. hADSCs were cultured in MSCcult medium containing DMEM/F12 supplemented with 10% fetal bovine serum, 1% antibiotic, 100× antimycotic, 10 ng/mL EGF, and 10 ng/mL bFGF (Sigma, USA) in a humidified incubator with 5% atmospheric CO2 at 37°C. On reaching 70-80% confluence, hADSCs were detached by treating with 0.25% trypsin/EDTA and subcultured in fresh medium.

GFP lentivirus-transduced hADSCs were used for labeling the cells to assess the role of the transplanted cell in the host. copGFP control lentiviral particles (Santacruz, USA) are lentiviral particles containing a copGFP coding construct for copGFP expression in mammalian cells after transduction. The transduction of lentiviral-activated particles was carried out according to the manufacturer’s instructions. Briefly, 1.5 × 10⁵ – 2.5 × 10⁵ cells were seeded in a 6-well tissue culture flask. Polybrene (8 μg/mL) (Sigma, USA) was added after approximately 24 h. After one day, fresh medium without polybrene was replaced and copGFP lentiviral particles were supplemented into the medium. GFP lentivirus-transduced cells were cultured for 7 days. The cells were further subcultured and medium replenished, if needed.

Cells stably expressing copGFP were isolated from MSCcult medium, supplemented with puromycin (8 μg/mL) (Sigma), and observed under fluorescence microscopy to ensure that gene transduction was successful.

Six-to-twenty-week-old acute hind limb ischemia mice were injected with GFP-transduced hADSCs (GFPhADSCs) with a dose of 106 cells/100 μL phosphate buffer saline (PBS) at the ligature blood vessel.

To evaluate the transplanted cell presentation at ischemic hindlimb, the mice were anesthetized and scanned by iBox Explorer Imaging Microscope system. The GFP-fluorescence signals in the ischemic hindlimb were imaged under UV light until 8 days after cell transplantation. The images were recorded and analyzed by Vision WorksLS Image Acquisition and Analysis Software.

The survival rate of the transplanted cells at the ischemic hindlimb was assessed by flow cytometry. Thigh muscle tissue of GFP-hADSC transplanted mice was collected. Muscle tissue was then separated to 3 parts: the cell injection site (IS), the opposite of the injection site (OIS), and the lateral gastrocnemius site (LGS)  Figure 5C . The muscle tissue was finely cut and trypsinized using 0.5% Trypsin/EDTA to detach single cells. The rate of GFP-positive cells was analyzed by CellQuest Pro software (BD Biosciences). These single cells were also evaluated by analyzing the expression of the human angiogenic marker in the mouse by labeling with anti VEGFR2-PE and CD31-PE (BD Biosciences), and incubated at room temperature for 15 min. Finally, labeled cell population was analyzed by flow cytometer and CellQuest Pro software.

Muscle tissues were fixed in 4% paraformaldehyde for 24 h. Then, the muscle tissues were transferred to 30% sucrose until they sink to the bottom. Tissue sections were frozen, then cut into 10-γm-thick section and mounted on a slide. Slides were stained with hematoxylin and eosin. Tissue structure was assessed under the microscope.

Damaged tissue recuperation was evaluated by using graded morphological scales representing an area of muscle necrosis following the guidelines of Takako Goto et al. (2006) Goto et al., 2006. Briefly, the damage of limb was classified as Grade 0 (G0), if no change; GI, if necrosis in nail and toes; GII, if necrosis in feet; GIII, if necrosis in knee; and GIV, if total leg necrosis.

All the results were analyzed by using the GraphPad Prism 6.0 software and Microsoft Office 2011. Differences were considered significant at p ≤ 0.05.

The morphology of GFP-hADSCs was similar to that of fibroblasts Figure 1A . GFP-hADSCs were bright green under the fluorescence microscope Figure 1B and the percentage of GFP-positive hADSCs was over 97% ( Figure 1C ).

On the other hand, expression analysis of specific factors on MSC surface showed that ADSC was positive to VEGFR2 (100%) ( Figure 1E ), but negative to CD31 (1.48% ± 0.11% positive) ( Figure 1F ).

At 90 min after a GFP-hADSCs injection into the ischemic hindlimb, 100% of transplanted cells in the mouse exhibited green fluorescence at the injected location, when exposed to fluorescent light. Beyond that time, the percentage decreased from 93.75% on day 1 to 6.25% on day 7 Figure 2A . On the other hand, both the intensity and area of fluorescence emission decreased. Stem cell-transplanted site exhibited strong luminous intensity, which was displayed in red ( Figure 2B ) over a large area, immediately after transplantation. After that, luminous intensity became weaker and was displayed by the yellow ( Figure 2C ) and green area ( Figure 2D ). Fluorescent signal was reduced and not found on day 8 ( Figure 2E ).

The survival rate of transplanted cell at the IS was 3.62% ± 2.06% (n=90 after 1 day and reduced to 3.03 ± 0.92% (n=11) on day 4. On the eighth day, GFP-hADSCs survival was significantly decreased by 13-fold, compared to day 4 (n=13; p>0.05). Interestingly, the presentation of GFP-hADSCs at the OIS was significantly higher than that at the IS (p<0.05), approximately 15.71% ± 12.29% (n=5) on the first day. However, this rate significantly reduced to 0.18% ± 0.09% on the eighth day (n=14; Figure 3 ).

The rate of VEGFR2 positive cells was 3.57% ± 1.79% at IS and 6.69% ± 3.44% at LGS on the first day post transplantation, but not found at OIS (n=6). Then, it had the highest expression at 4 days post transplantation, approximately 5.46% ± 2.13% (n=10) at the IS; 9.12% ± 7.17% at the OIS (n=9), and 7.22% ± 4.59% at the LGS (n=9). After 14 days, it significantly decreased (p<0,05) ( Figure 4A-C ) to 2.07% ± 1.49% (n=9), 1.39% ± 0.67% (n=9), and 0.98% ± 0.94% (n=7) at the IS, OIS, and LGS, respectively. After 14 days, VEGFR2 expression was reduced to zero. This showed that hADSC could assist in angiogenesis via the VEGF signaling pathway.

CD31 is a specific marker of differentiation of hADSC to endothelial cell. This study investigated endothelial differentiation of GFP-hADSCs by estimating the percentage of human CD31-positive cells in mouse. When GFP-hADSCs were transplanted in the mouse with acute hindlimb ischemia, the percentage of CD31-positive cells increased to the highest on day 4 at IS, approximately 0.8% ± 1.60% (n=13). However, it increased to the highest on day 8 at LGS and OIS, approximately 1.56% ± 0.44% (n=9) and 1.17% ± 1.69% (n=1), respectively. On the other hand, it was highest at LGS on day 1 after transplantation, and reached 1.33% ± 2.05% (n=8), but not significantly different, when compared to IS and OIS. Cells in the hindlimb expressed CD31 at all sites on day 8, and reached 0.66% ± 0.57% (n=13) at IS, 1.17% ± 1.68% (n=10) at OIS, and 1.56% ± 1.44% (n=9) at LGS. On day 14, the rate of CD31 positive cells was decreased, compared to day 8 after transplantation ( Figure 4D-F ). Therefore, GFP-hADSCs could take part in endothelial differentiation of angiogenesis in mouse.

GFP-hADSCs stimulated the new blood vessel formation

New blood vessels were observed in GFP-hADSCsinjected acute hindlimb ischemic mice. The tiny blood vessels could be observed visually. New blood vessels had appeared in all the three areas Figure 5C . However, the density of new blood vessels at the LGS was higher than that at the OIS and IS. On the other hand, the density of blood vessels was higher in the GFP-hADSCs group as compared with the PBS group ( Figure 5B ) and normal mice ( Figure 5A ). Thus, GFPhADSCs contributed in the formation of new blood vessels in mouse with acute hindlimb ischemia.

GFP-hADSCs participated in restoring tissue structure better than no treatment

In a normal tissue, the skeletal muscle cells are arranged into bundles, and blood vessels (Yellow narrow) are scattered in the bundles ( Figure 6 ). In this study, the muscle bundles had broken structures, and muscle cells were incoherently arranged on day 3 in both the PBS and GFP-hADSCs-injected ischemic tissue. However, adipocyte formation was observed in the PBS group (Black narrow) from day 15 to 30, but new muscle cell (Green narrow) was found growing in the GFP-hADSCs group. On the other hand, there was new blood vessel formation in both the groups; however, the high density of small vessels was identified in the GFP-hADSCs group. This showed that GFP-hADSCs played an important role in remodeling damaged tissue, as in angiogenesis.

One day post transplantation, approximately 60% of mice had signs of tenderness, swelling, and skin crimson. The mice’s rate of recovery from limb ischemia and necrosis with GI was 57.78% (n=18). However, 22.23% of mice had serious injury with GII, GIII, and GIV. There were approximately 66.66% of mice without any damage or necrosis with GI after day 14 in the PBS injected-ischemic mice and about 33.34% of mice from GII to GIV (n=18). Thus, the recovery of GFP-hADSCs - transplanted acute ischemic hindlimb was better than non-treated limb.