The role of PI3K/AKT pathway in hypoxia-induced cell proliferation and endothelial cell differentiation of adipose-derived stem cells / 中国组织工程研究

BACKGROUND: A large number of studies have confirmed that hypoxia can promote the proliferation and differentiation of stem cells, but the pathway by which it plays its role remains unclear. OBJECTIVE: To investigate the effects of hypoxia on the proliferation of adipose-derived stem cells and their differentiation into endothelial cells and investigate the role of PI3K/Akt pathway in this process. METHODS: Passage 3 adipose-derived stem cells of Wistar rats were divided into three groups according to the different culture conditions: normoxia group, hypoxia group, and hypoxia+LY294002 group. Three groups of cells were cultured for 24 hours in an incubator containing 20% O2. Cells in the hypoxia group were cultured in an incubator containing 2% O2, and those in the normoxia group were still cultured in an incubator containing 2% O2. Cells in the hypoxia+LY294002 group were cultured in medium supplemented with 25 mmol/L PI3K/Akt pathway inhibitor LY294002 under the hypoxic culture conduction. After 24 hours of culture, p-Akt expression was detected by Western blot assay to indicate the activation of PI3K/Akt pathway. The proliferation of adipose-derived stem cells was measured by CCK-8 method. Three groups of adipose-derived stem cells were induced to differentiate into vascular endothelial cells for 10 days. The differentiation of adipose-derived stem cells into endothelial cells was detected by qRT-PCR and anti-CD31 immunofluorescence staining. RESULTS AND CONCLUSION: Adipose-derived stem cells at passage 3 exhibited elongated fibroblast-like morphology. Cytometry analysis showed most of adipose-derived stem cells at passage 3 were negative for CD 34 and CD45 and positive for CD105 and could be induced towards osteogenic and adipogenic differentiation. The expression of p-Akt was significantly increased after hypoxic culture, which was inhibited obviously after adding LY294002. CCK-8 showed that the proliferation of adipose-derived stem cells in the hypoxia group was significantly higher than that in the normoxia group and hypoxia+LY294002 group (P < 0.05). The proliferation of adipose-derived stem cells in the hypoxia+LY294002 group was significantly lower than that in the hypoxia group (P < 0.05). The results of qRT-PCR and anti-CD31 immunofluorescence staining showed that the expression of endothelial cell gene and specific protein CD31 in the hypoxia group was significantly higher than that in the normoxia and hypoxia+LY294002 groups (P < 0.05). The expression of CD31 in the hypoxia+LY294002 group was significantly lower than that in the hypoxia group (P < 0.05), but it was significantly higher than that in the normoxia group (P < 0.05). These results suggest that the PI3K/Akt pathway plays an important role in hypoxia-induced cell proliferation and vascular endothelial cell differentiation of adipose-derived stem cells.

A comparative study on autologous and allogeneic adipose derived mesenchymal stem cell transplantation in treatment of acute myocardial infarction rats / 解放军医学杂志

Objective To investigate the effects of autologous and allogeneic adipose derived mesenchymal stem cells (ADMSCs) transplantation on rat model of acute myocardial infarction (AMI) and possible mechanisms.Methods The AMI models were established with 45 male Lewis rats by ligation of left anterior descending coronary artery,and then randomly divided into 3 groups (15 each) including AMI group,allogeneic ADMSC transplantation group (Allo-ADMSC group) and autologous ADMSC transplantation group (Auto-ADMSC group).After successfully modeling,CM-Dil-labeled third-generation ADMSCs (2 × 106) were implanted into the myocardium of rats within 1 hour,and rats in AMI group were injected with equal amount of PBS.The cardiac function,immunofluorescence and Masson were identified 4 weeks after transplantation.Results Four weeks after transplantation,compared with AMI group,the left ventricular ejection fraction in Allo-ADMSC group and Auto-ADMSC group increased significantly,the left ventricular end-systolic and end-diastolic diameter decreased,the collagen deposition fraction decreased significantly (P＜0.05).Compared with Allo-ADMSC group,the left ventricular ejection fraction increased in AutoADMSC group,the number of newborn capillaries increased and the myocardial collagen deposition fraction decreased (P＜0.05).Conclusion Autologous ADMSC can promote vascular proliferation in the infarct area more better than allogeneic ADMSC,reduce the local collagen deposition,extenuate the degree of myocardial fibrosis,thereby to inhibit collagen remodeling,and repair damaged myocardial tissue and improve heart function.

Feasibility of adipose tissue-derived stem cell sheet fabricated ex vivo on temperature-responsive scaffolds as seed cells for ocular surface reconstruction / 中华实验眼科杂志

Background Development of corneal tissue engineering creates a new therapeutic method for severe corneal diseases.However,ideal seed cells and scaffold for corneal surface reconstruction have not yet been investigated well.Adipose-derived stem cells (ADSCs) are varified to have a self-renewal ability and epithelioid features,and temperature-responsive scaffolds (TRSs) can offer technical support for stem cell sheet.Objective This study was to investigate the characteristics of ADSCs cultured on TRSs and compare these features to typical oral mucosal epithelial cells (OMECs),and therefore to explore the feasibility of reconstruction of ocular surface with ADSCs as seed cells.Methods Self-made TRSs were prepared by adding isopropyl alcohol dissolved poly-Nisopropylacrylamide (PNIPAAm) to each polystyrene tissue culture dish and then irradiating using an election beam.Subcutaneious fatty tissue of rabbit neck was obtained to culture ADSCs,and 4 pieces of oral cavity mucosal tissue were digested and cultured to obtain OMECs.Then the ADSCs and OMECs were incubated on TRSs,and cell morphology,growth rate,detached duration and survival counts were compared between ADSCs and OMECs.The ADSCs sheet and OMECs sheet were stained with hematoxylin and eosin for morphological examination.Immunochemistry was used to observe the expressions of stem-cell biomakers and epithelioid-cell biomakers in the cells.The ultrastructure of cell surface was observed under the scanning electron microscope.Results Self-made TRSs were similar to ordinary culture dish in the transparancy and smoothness.The water contact angle of 4 in 5 samples were ＞10° with the effective rate upto 80％.A DSCs showed the elongated fusiform in shape,while OMECs showed a cobblestone appearance.The growth cycle,detached duration and cell number of ADSCs were 12-14 days,(46.0 ±9.6) minutes and (7.9 ±1.1)×105/sheet,and those of OMECs were 14-16 days,(91.9 ±10.9) minutes and (45.8 ±26.5)×105/sheet,respectively,showing statistically significant differences in the detached duration and cell counts between ADSCs and OMECs (P=0.002,0.028).Hematoxylin and eosin staining showed that ADSCs sheet comprised only 1-3 layer cells,while OMECs showed 4-5 layer cells.ATP-binding cassette superfamily G member 2 (ABCG2),p63 and cytokeratin 12 (CK12) were positively expressed in both ADSCs sheet and OMECs sheet.Closely packed cells and typical eithelial microvilli in the cell surface were exhibited in both ADSCs sheet and OMECs sheet under the scanning electron microscope.Conclusions Self-made TRSs can be used as scaffold of ADSCs.The ADSCs sheet on the TRSs appears to have a good cell vitality and therefore is a new seed source of ocular surface reconstruction.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Effects of Human Adipose-Tissue Derived Stem Cell Infusion on the Immunological Consequences in Skin Allograft Mice / 대한이식학회지

BACKGROUND: Many in vitro experiments have demonstrated the immunosuppressive properties of mesenchymal stem cells (MSCs). However, such properties have not yet been fully established in an in vivo setting. The purpose of this study was to determine immunosuppressive and anti-inflammatory properties of MSCs in a preclinical animal model in order to pave the way for replacement of conventional immunosuppressive therapy. METHODS: Male C57BL/6 mice and male BALB/c mice were chosen as skin graft donors and recipients, respectively. After performance of full-thickness skin transplantation on the back of mice, adipose tissue derived stem cells (1.0x10(6)/0.1 mL) stained with 4, 6-diamidino-2-phenylindole were transplanted into adipose tissue derived stem cell (ASC)-infused mice and phosphate buffered saline (PBS; 0.1 mL) was infused into PBS-infused mice. Immunological properties and graft survival were accessed and compared. RESULTS: The serum levels of proinflammatory interleukin (IL)-6 showed a decrease in ASC-infused mice compared to PBS-infused mice (P<0.005). In addition, interferon-lambda, IL-10, and tumor necrosis factor-alpha mRNA levels in the skin graft showed a decrease in ASC-infused mice, although without statistical significance. In ASC-infused mice, donor specific hyporesponsiveness was identified in a mixed lymphocyte reaction assay at 30 days after transplantation. In addition, ASC-infusion resulted in markedly prolonged skin allograft survival compared with PBS-infusion (P<0.001). CONCLUSIONS: Administration of ASC not only induced anti-inflammation and immunosuppression, but also resulted in prolonged graft survival, suggestive of their potent immunosuppressive properties. Therefore, conduct of further and more exquisite studies will be required in order to determine the role of MSC in the solid organ transplantation field in order to avoid adverse effects and toxicities caused by chemical immunosuppressive regimens.

Transplantation of adipose-derived stem cells with fibrin glue for treatment of acute myocardial infarction in rat / 中华急诊医学杂志

Objective To investigate the cell survival of the combination of fibrin glue and adiposederived stem cells (ADSCs) in rats when implanted into ischemic myocardium and the improvement of heart function.Methods The rat ADSCs were isolated from the subcutaneous adipose tissues.The surface phenotype of these cells was analyzed by flow cytometry.Myocardial infarction was induced in female rats using coronary artery ligation.One week after MI,surviving rats were randomized (random nuber) into 4 groups,control group (n =10),fibrin group (n =10),cell group (n =10) and combination group (n =10).100 μl of PBS was injected into the ischemic myocardium in control group.100 μl of Fibrin glue were injected into ischemic myocardium in fibrin group.100 μl of ADSCs labeled with DAPI were injected into the infract along the border zone in cell group.ADSCs in 100 μl of fibrin glue were injected into the infract in combination group.Four weeks after the injection the surviving rats underwent examination of heart functions by the Hemodynamics.The rats were killed and their hearts were taken out to undergo immunohistochemistry with 4,6-diamidino-2-phenylindole (DAPI) and actin and factor Ⅶ to measure the area of cardiac infarction and the capillary density.The heart infarcted size was calculated by masson trichrome staining.All data was analyzed by software SPSS 15.0,ANOVA comparison tests and the student t test were used,and P ＜ 0.05 was considered as statistically significant.Results Four weeks after the cells were transplanted,LVSP and + dp/dtmax of combination group were highest among all groups.The heart infarcted size of the combination group was (28.5 ± 3.6) ％,significantly less than those of the cell group (33.33 ± 2.3) ％ and fibrin group (35.96 ± 2.11) ％,both P ＜ 0.05.The capillary density of the combination group was (108.7 ± 11.38) /mm2,significantly greater than those of the cell group and that of the fibrin group,and greater than that of the control group.DAPI and actin double staining detected a varied increase in the number of surviving cardiomyoctyes at the heart infarcted area.Conclusions Transplantation of ADSCs with fibrin glue brings better improvement in cell survival and in restoration of heart function than either cellular or fibrin therapy alone.

Detection of embryonic stem cell markers in adult human adipose tissue-derived stem cells.

Background: Bone marrow transplantation is already an established therapy, which is now widely used in medicine to treat leukemia, lymphoma, and several inherited blood disorders. The culture of multilineage cells from easily available adipose tissue is another source of multipotent mesenchymal stem cells, and is referred to as adipose tissue-derived stem cells (ADSCs). While ADSCs are being used to treat various conditions, some lacuna exists regarding the specific proteins in these. It was therefore decided to analyze the specific proteins of embryonic cells in ADSCs. Aims: To analyze the specific protein of embryonic stem cells (ESCs) in ADSCs. Materials and Methods: Adult human adipose tissue-derived stem cells (ADSCs) were harvested from 13 patients after obtaining patients' consent. The specific markers of ESCs included surface proteins CD10, CD13, CD44, CD59, CD105, and CD166, and further nucleostemin,(NS) NANOG, peroxisome proliferator-activated receptor-gγ, collagen type 1 (Coll1), alkaline phosphate, (ALP) osteocalcin (OC), and core binding factor 1 (Cbfa1) were analyzed using by reverse transcription-polymerase chain reaction, (RT-PCR) immunofluorescence (IF), and western blot. Results: All the proteins were expressed distinctly, except CD13 and OC. CD13 was found individually with different expressions, and OC expression was discernable. Conclusions: Although the ESC with its proven self-renewal capacity and pluripotency seems appropriate for clinical use, the recent work on ADSCs suggests that these adult stem cells would be a valuable source for future biotechnology, especially since there is a relative ease of procurement.

The effects of low intensity pulsed ultrasound on the proliferation and osteogenic differentiation of rat adipose tissue-derived stem cells / 中华物理医学与康复杂志

Objective To investigate the biological effects of low intensity pulsed ultrasound (LIPUS) on the proliferation and osteogenic differentiation of adipose tissue-derived stem cells (ADSCs) in vitro.Methods Primary ADSCs were harvested from the inguinal fat pads of 4-week-old female Sprague-Dawley rats,cultured in vitro and purified by magnetic-activated cell sorting.Surface ADSC markers were identified by flow cytometry.LIPUS at 100 mW/cm2 was used to stimulate the cultured cells.Flow cytometry was performed for cell cycle analysis.Cellular proliferation was evaluated via CCK8 chromatometry,and a proliferation index was calculated.ADSCs were assigned to 4 groups:a negative control group,a LIPUS group,an osteoinduction group and a LIPUS plus osteoinduction group,and treated accordingly.Alkaline phosphatase (ALP) activity was determined at the 7th and 14th day in each group,and calcium nodes were marked by Von Kossa staining.The levels of osteogenic differentiation in the different groups were evaluated.Results The ADSCs of passage 3 expressed CD 34low,and CD29high CD44high,which was consistent with the characteristics of ADSC surface markers.Proliferation was upregulated significantly in the LIPUS group compared with the negative control group.ALP activity was also elevated significantly and it resulted in mine-ralization.The highest mineralization rate was observed in the LIPUS plus osteoinduction group.Conclusions LIPUS not only can stimulate the proliferation of rat ADSCs,it also promotes their osteogenic differentiation.

Rapid construction of engineered skin substitutes by adipose tissue-derived stem cells and fibrin glue for full thickness skin wound repair / 实用口腔医学杂志

To investigate the feasibility of construction of engineered skin substitutes with adipose tissue derived stem cells ( ADSCs)from GFP-mouse and fibrin glue. Methods: Adipose tissues were isolated from GFP-C57BL mouse groin, the fat was cut into pieces and incubated in a collagenase solution to establish ADSCs culture. The differentiation ability into adipocytes and osteoblasts of the ADSCs were investigated. The amplified cells in vitro were seeded in fibrin glue to form tissue engineered skin substitutes. Fifteen C57BL mice were randomly divided into three groups; experimental group was treated with ADSCs-fibrin glue tissue engineered active skin substitutes. Single fibrin glue and blank were served as two control groups. The materials were grafted onto total skin defects on the back of mice. The effect of wound repair was observed histologically. Results; The cultured ADSCs grew well in vitro. The cells showed characteristics of multipotent adult stem cells. ADSCs-fibrin glue shortened the wound healing time. Histological observation showed that thicker epithelium was formed and collagen fibers arranged in order in the ADSCs-fibrin glue experimental group,the number of fibroblasts was significantly increased as compared with those in single fibrin glue group or in the blank control group. Conclusion : GFP- ADSCs combined with fibrin glue can accelerate the mouse skin wound healing. It suggests that ADSCs with fibrin glue maybe an optional method of engineered skin reconstruction for wound repair.

Effect of Murine Adipose Derived Stem Cell(ADSC) on Bone Induction of Demineralized Bone Matrix(DBM) in a Rat Calvarian Defect Model

PURPOSE: Adipose tissue-derived stem cells(ADSC) has an osteoconductive potential and demineralized bone matrix(DBM) is an osteoinductive material. A combination of DBM and ADSC wound probably create osteoinductive properties. The purpose of this study is to determine the effect of the combination of DBM and ADSC mixture on healing of rat calvarial defect. METHODS: Thirty adult male Sprague-Dawley rats were randomized into 3 groups(n=10) as 1) Control, 2) DBM alone, 3) DBM with ADSC mixture. DBM with ADSC mixture group has had a 3-day preculture of ADSC from groin fat pad. An 6 mm critical size circular calvarial defect was made in each rat. Defect was implanted with DBM alone or DBM with ADSC mixture. Control defect was left unfilled. 6 and 12 weeks after the implantation, the rats were sacrificed and the defects were evaluated by histomorphometric and radiographical studies. RESULTS: Histomorphometric analysis revealed that DBM with ADSC mixture group showed significantly higher bone formation than DBM alone group(p<0.05). Although radiographs from DBM alone group and DBM with ADSC group revealed similar diffuse radiopaque spots dispersed throughout the defect. Densitometric analysis of calvarial defect revealed DBM with ADSC mixture group significantly higher bone formation than DBM alone(p<0.05). There was correlation of densitometry with new bone formation(Spearman's correlation of coefficient=0.804, 6 weeks, 0.802, 12 weeks). CONCLUSION: The DBM with ADSC mixture group showed the best healing response and the osteoinductive properties of DBM were accelerated with ADSC mixture. It will be clinically applicable that DBM and ADSC mixture in plastic and reconstructive surgery, such as alveolar cleft and congenital facial deformities that bone graft should be required.

A study on the osteogenic differentiation of adipose-derived adult stem cell

The Effects of Human Adipose Tissue Derived Mesenchymal Stem Cells on Degenerative Change of Disc in Rabbit Model

OBJECTIVE: To determine whether transplanted human adipose tissue derived stem cells (hATSCs) can survive and increase the amount of proteoglycans in degenerated intervertebral disc. METHOD: Lumbar disc degeneration was induced in thirty New Zealand white rabbits by injection of chondroitinase ABC(R). After 2 weeks, hATSCs were transplanted in degenerated disc in hATSCs group. Control group received phosphate buffered saline. The histologic grading and height of disc were measured at 2, 4, and 8 weeks after transplantation. The viability of donor cells was identified by using beta-Actin gene polymerase chain reaction (PCR). RESULTS: 4 and 8 weeks after hATSCs transplantation, the histologic grading showed significantly high score in hATSCs group (p<0.05), but the amount of proteoglycans was not significantly different between the two groups. The change of disc height was not significantly increased in hATSCs group. In the beta-Actin gene PCR analysis, positive signal in the hATSCs group was observed. CONCLUSION: hATSCs transplantation may be useful in decelerating disc degeneration in experimental models and provide new hopes for treatment of degenerative disc disease in humans

Adipose tissue derived stem cells transferred with TGF-?_1 gene and differentiated into chondrocytes / 中国矫形外科杂志

[Objective]To induce TGF-?1 gene which can increase ECM synthesis into adipose tissue derived stem cells(ADSCs) by employing gene transfer techniques and observe whether TGF-?1 gene could expresse continuously and whether type II collagen and aggrecan are synthesized in order to provide experimental data for constructing tissue-engineering cartilage. [Method]ADSCs were transferred with TGF-?1 gene ,TGF-?1,FN,ColⅡ and aggrecan were detected with RT-PCR and TGF-?1 protein was detected with Western-blot.[Result]RT-PCR demonstrated that the expression of TGF-?1,FN,ColⅡ and aggrecan in the TGF-?1 gene transferred groups increased more evidently than non-gene groups and control groups (P