Comparison of surface markers of mesenchymal stem cells from different sources / 解剖学报

Objective To investigate the expression difference of surface markers of human umbilical cord mesenchymal stem cells (HUCMSCs), human adipose mesenchymal stem cells (ADSCs) and human fetal blood source endometrial mesenchymal stem cells (MenSCs) and the changes of surface markers with the culture generation. Methods HUCMSCs, ADSCs and MenSCs were cultured to passage 3, 6, 9 and 12. Five MSC-specific markers, CD29, CD44, CD73, CD90 and CD 105, and one HSC markers, CD45, were assessed by flow cytometry and immunofluorescence. Results The cultured MenSCs and HUCMSCs were spindle-shaped, and the ADSCs forms were mainly spindle-shaped and multiple-angular. The results of flow cytometry showed that the MSC-positive markers including CD29, CD44, CD73 and CD105 were highly expressed by at least 95% in the passage 3 HUCMSCs, ADSCs and MenSCs cells and CD45 negative expression. Specifically, CD90 levels of MenSCs of passage 3 was (72. 43 ± 0. 7 6) %, which was lower than that in HUCMSCs (99.67±0. 12)% and ADSCs (99. 70 ± 0. 15)% (P 0. 05). The results of immunofluorescence was consistent with those of flow cytometry. Conclusion With the increase of culture time, HUCMSCs, ADSCs and MenSCs are stable in high expression of CD29, CD44, CD73, CD90 and CD 105, which does not express CD45, while the CD90 expression rate of MenSCs is lower than that in HUCMSCs and ADSCs.

Effects of different storage media on the activity of human adipose-derived stem cell / 中华医学美学美容杂志

Objective To determine the proper storage environment by comparing the activity of adipose-derived stem cell (ADSC) at 10％ PRP,10％ HS and 0.9％ NaCl.Methods ADSC was isolated and cultured.Then the ADSC was stored in 10％ PRP,10％ HS,and 0.9％ Nacl at room temperature (RT,about 26 ℃C) for 2 hours.After that the proliferation ability,survival rate and differentiation ability of the cells were detected.Finally storage media of ADSC was determined.Results Compared with 0.9％ NaCl,ADSC showed a better proliferation and higher differentiation ability at 10％ PRP and 10％ HS;besides,the survival rates of two groups were higher than the group of 0.9％Nacl.Among these groups,the survive rate of 10％ HS and 10％ PRP were 78.08％ and 75.68％ respectively,while it was 62.34％ in 0.9％ NaCl group.Conclusions 10％ HS is more suitable for short-term preservation of ADSC.

Effects of human adipose-derived mesenchymal stem cells and platelet-rich plasma on healing of wounds with full-thickness skin defects in mice / 中华烧伤杂志

Objective@#To investigate the effects of human adipose-derived mesenchymal stem cells (ADSCs) and platelet-rich plasma (PRP) on healing of wounds with full-thickness skin defects in mice.@*Methods@#ADSCs were isolated from the lumbar and abdominal fat donated voluntarily by a healthy woman undergoing liposuction in the Department of Plastic Surgery of Guangzhou General Hospital of Guangzhou Military Area Command, and the cells were cultured and identified. ADSCs of the second passage were used in the following experiments. The venous blood of the volunteer was taken, and PRP was obtained by secondary centrifugation. Thirty-six C57BL/6 mice were divided into simple injury group (n=12), simple ADSCs treatment group (n=12), and ADSCs+ PRP treatment group (n=12) according to the random number table. Each mouse was inflicted with a 1 cm×1 cm wound with full-thickness skin defect on the back. Immediately after injury, the wounds of mice in simple injury group were subcutaneously injected with 1 mL normal saline, the wounds of mice in simple ADSCs treatment group were subcutaneously injected with 1 mL phosphate buffer solution-blended ADSCs suspension (with concentration of 5×105 /mL, the same below), and the wounds of mice in ADSCs+ PRP treatment group were subcutaneously injected with 1 mL mixture of PRP and ADSCs (1∶2 volume ratio). Three mice in each group were taken on post injury day (PID) 3, 5, 7, and 14 to observe the gross condition of wound, and the wound healing rate was calculated. On PID 3, 5, and 7, the non-healing wound tissue and 0.5 cm normal skin tissue around the wound margin were taken after gross observation. The inflammation, re-epithelialization, and angiogenesis of tissue were observed by hematoxylin and eosin staining, and the re-epithelialization rate was calculated. The collagen synthesis of tissue was observed by masson staining. Immunohistochemistry was used to observe the expression of macrophages of tissue samples collected on PID 3 and 5. Data were processed with analysis of variance of factorial design and Least-Significant Difference test.@*Results@#(1) On PID 3, the wounds of mice in ADSCs+ PRP treatment group were with granulation tissue regeneration, redness, and swelling, and the wounds of mice in the other two groups were ruddy and with effusion. On PID 5, the wounds of mice in ADSCs+ PRP treatment group had less redness and swelling, which were dry with obvious scab, and wounds of mice in the other two groups were obviously red and swollen. On PID 7, scab formed basically on wounds of mice in the three groups. On PID 14, the wounds of mice in the three groups basically healed, and their crusts were off. On PID 3, 5, 7, and 14, the wound healing rates of mice in ADSCs+ PRP treatment group were obviously higher than those of the other two groups (P<0.05 or P<0.01). On PID 5 and 7, the wound healing rates of mice in simple ADSCs treatment group were obviously higher than those of simple injury group (P<0.01). (2) On PID 3, granulation tissue regeneration of wounds in ADSCs+ PRP treatment group was more than that in the other two groups. On PID 5, inflammatory reaction of wounds of mice was mild in ADSCs+ PRP treatment group, which was severe in the other two groups. On PID 7, the re-epithelialization process of wounds of mice was almost completed in ADSCs+ PRP treatment group, and the number of new vessels was more in ADSCs+ PRP treatment group than in the other two groups. The migration distance of regenerated epithelia around the wound edge in simple injury group and simple ADSCs treatment group was short. On PID 3, 5, and 7, the re-epithelialization rates of wounds of mice in ADSCs+ PRP treatment group were (37.6±4.5)%, (59.1±1.3)%, and (89.2±4.3)%, respectively, significantly higher than (25.7±1.5)%, (34.5±4.4)%, and (50.8±2.7)% in simple injury group and (29.1±0.8)%, (42.6±2.9)%, and (72.9±3.0)% in simple ADSCs treatment group (P<0.01). On PID 5 and 7, the re-epithelialization rates of wounds of mice in simple ADSCs treatment group were significantly higher than those in simple injury group (P<0.05 or P<0.01). (3) On PID 3 and 5, a quite large number of new collagen fibers appeared in granulation tissue of wounds of ADSCs+ PRP treatment group, while the collagen fibers in the other two groups were less. On PID 7, the granulation tissue of mice in ADSCs+ PRP treatment group decreased, and a large number of new collagen fibers appeared. The collagen fibers in wounds tissue of mice in simple ADSCs treatment group increased, while the collagen fibers deposited in wounds tissue of mice in simple injury group was still less. (4) On PID 3 and 5, the numbers of macrophages in wounds tissue of mice in simple ADSCs treatment group were 4.7±0.6 and 5.3±0.6 respectively, obviously lower than 6.3±0.6 and 7.7±0.6 in injury group (P<0.05 or P<0.01); the numbers of macrophages in wounds tissue of mice in ADSCs+ PRP treatment group were 3.0±1.1 and 2.7±0.5, significantly lower than those in the other two groups (P<0.05 or P<0.01).@*Conclusions@#Human PRP and ADSCs are involved in the early inflammation, metaphase of tissue proliferation, and re-epithelialization and shaping process of late stage of wounds with full-thickness skin defects in mice. The combination of ADSCs and PRP may be a comparatively good combination to improve the speed and quality of wound healing.

Flow cytometric immunophenotyping of canine adipose-derived mesenchymal stem cells (ADMSCs) and feline ADMSCs using anti-human antibodies / 대한수의학회지

Various trials have been conducted to develop therapies for serious untreatable diseases. Among these, those using stem cells have shown great promise, and adipose-derived mesenchymal stem cells (ADMSCs) are easier to obtain than other types of stem cells. Prior to clinical trials, characterization of ADMSCs with monoclonal antibodies should be performed. However, it is difficult to use species-specific antibodies for veterinarians. This study was conducted to confirm the panel of human antibodies applicable for use in immunophenotypic characterization of canine adipose-derived stem cells and feline ADMSCs extracted from subcutaneous adipose tissue collected during ovariohysterectomy. For flow cytometric immunophenotyping, the third passages of canine ADMSC and feline ADMSC and human CD31, CD34, CD42, CD44, CD62 and CD133 antibodies were used. Of these, CD133 reacted with canine cells (3.74%) and feline cells (1.34%). CD133 is known as a marker related with more primitive stem cell phenotype than other CD series. Because this human CD133 was not a species-specific antibody, accurate percentages of immunoreactivity were not confirmed. Nevertheless, the results of this study confirmed human CD133 as a meaningful marker in canine and feline ADMSCs.

Effects of Adipose-derived Mesenchymal Stem Cell Exosomes on Corneal Stromal Fibroblast Viability and Extracellular Matrix Synthesis / 中华医学杂志(英文版)

<p><b>Background</b>Corneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.</p><p><b>Methods</b>ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.</p><p><b>Results:</b>ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40-200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = -82.742, P < 0.01; collagen II: t = -72.818, P < 0.01; collagen III: t = -104.452, P < 0.01; collagen IV: t = -133.426, P < 0.01, and collagen V: t = -294.019, P < 0.01; and fibronectin: t = -92.491, P < 0.01, respectively).</p><p><b>Conclusion:</b>The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.</p>

Repairing mechanism of adipose-derived mesenchymal stem cell for acute kidney injury in rats / 中华泌尿外科杂志

Objective To study the mechanism of adipose-derived mesenchymal stem cell (ADMSC) in treating rats ischemia/reperfusion (I/R) induced acute kidney injury (AKI).Methods From June 2011 to March 2012,AKI was induced in 40 male SD rats (weight 180-220 g) by clamping bilateral renal pedicles for 40 minutes.Another 8 SD rats (weight 60-80 g) were used to obtain the primary ADMSC from inguinal fat tissue.After being transferred by lentivirus,those cells were cultured for transplantation until passage two.Animals with AKI were then randomly treated by intraparenchymally injecting ADMSC/PBS solutions (ADMSC/PBS group,n =20) or PBS solutions only (PBS group,n =20) (2× 106 cells,100 μl).During injection,the solutions were injected into the upper,middle and lower part of left kidney.The HE staining from 5 rats in each group was used to detect the histological injury at 3 days and 4 weeks after injection,respectively.The apoptosis and proliferation of host renal cells were evaluated using TUNEL staining and PCNA staining.The serum levels of SCr and BUN in animals were measured at day 1,3,14 and 28 after injection.Results HE staining showed ADMSC/PBS group got a lower injury score compared with that in PBS group at 3 days and 4 weeks,respectively (2.0 vs.3.4,1.3 vs.2.6,P＜0.05).In the result of TUNEL staining,the mean number of apoptosis cells was 30 in ADMSC/PBS group and 55 in PBS group.In terms of PCNA staining,the mean number of proliferative cells was 35 in ADMSC/PBS group and 10 in PBS group.All those results suggested that ADMSC could significantly reduce apoptosis and increase proliferation of renal cell (P＜0.05,repectively).The levels of serum SCr in ADMSC/PBS group were lower than those in PBS groups at day 1,3,14 and 28,after injection,respectively (40 vs.70 μmol/L,32 vs.58 μmol/L,26 vs.38 μ mol/L,26 vs.37 μmol/L; P＜0.05).The similar results were shown in the levels of serum BUN between the 2 groups (15 vs.24 mmol/L,13 vs.20 mmol/L,10 vs.13 mmol/L,7 vs.10 mmol/L; P＜0.05).Conclusion ADMSC could repair AKI after acute I/R injury.

Therapeutic effect of adipose-derived mesenchymal stem cells on radiation enteritis / 中华放射医学与防护杂志

Objective To evaluate the therapeutic effect of adipose-derived mesenchymal stem cells on radiation enteritis.Methods A total of 52 male Sprague-Dawley rats were used in the present study.Herein,46 rats were randomly selected and irradiated with a dose of 15 Gy at their abdomens.Two hours post-irradiation,23 rats were randomly selected and infused intraperitoneally with adipose-derived mesenchymal stem cells in passage 6 from young-female donor.The other 23 rats were intraperitoneally infused with PBS.The rest 6 rats were set as normal control.During the first 10 days post-irradiation,peripheral blood-samples from irradiated rats were harvested for testing the levels of IL-10 in serum using ELISA assay.Additionally,after isolating the thymic cells and peripheral blood mononuclear cells,the percentages of CD4/CD25/Foxp(3)-positive regulatory T cells in thymus and peripheral blood were tested by flow-cytometry.Finally,infiltration of inflammatory cells and deposition of collagens within irradiated small intestine were analyzed by H&E staining and Masson Trichrome staining,respectively.Based on the MPO-immunohistochemistry staining,the type of infiltrated cells was identified.The Kaplan-Meier method was used for analyzing the survival rate of irradiated rats.Results During a period of 30 days post-irradiation,the irradiated rats receiving adipose-derived mesenchymal stem cells survived longer than those receiving PBS (t =4.53,P ＜ 0.05).Compared to the irradiated rats with PBS-treatment,adipose-derived mesenchymal stem cells could elevate the level of IL-10 in serum (7 d:t =13.93,P ＜ 0.05) and increase the percentages of CD4/CD25/Foxp(3)-positive regulatory T cells in both peripheral blood (3.5 d:t =7.72,7 d:t=11.11,10 d:t =6.99,P ＜0.05) and thymus (7 d:t =16.17,10 d:t =12.12,P＜ 0.05).Moreover,infiltration of inflammatory cells and deposition of collagens within irradiated small intestine were mitigated by adipose-derived mesenchymal stem cells.Conclusions Adipose-derived mesenchymal stem cells were capable of curing radiation enteritis.