Adipose tissue-derived stromal/stem cells + cholecalciferol: a pilot study in recent-onset type 1 diabetes patients

ABSTRACT Objective: Adipose tissue-derived stromal/stem cells (ASCs) and vitamin D have immunomodulatory actions that could be useful for type 1 diabetes (T1D). We aimed in this study to investigate the safety and efficacy of ASCs + daily cholecalciferol (VIT D) for 6 months in patients with recent-onset T1D. Materials and methods: In this prospective, dual-center, open trial, patients with recent onset T1D received one dose of allogenic ASC (1 x 106 cells/kg) and cholecalciferol 2,000 UI/day for 6 months (group 1). They were compared to patients who received chol-ecalciferol (group 2) and standard treatment (group 3). Adverse events were recorded; C-peptide (CP), insulin dose and HbA1c were measured at baseline (T0), after 3 (T3) and 6 months (T6). Results: In group 1 (n = 7), adverse events included transient headache (all), mild local reactions (all), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), scotomas (n = 2), and central retinal vein occlusion at T3 (n = 1, resolution at T6). Group 1 had an increase in basal CP (p = 0.018; mean: 40.41+/-40.79 %), without changes in stimulated CP after mixed meal (p = 0.62), from T0 to T6. Basal CP remained stable in groups 2 and 3 (p = 0.58 and p = 0.116, respectively). Group 1 had small insulin requirements (0.31+/- 0.26 UI/kg) without changes at T6 (p = 0.44) and HbA1c decline (p = 0.01). At T6, all patients (100%; n = 7) in group 1 were in honeymoon vs 75% (n = 3/4) and 50% (n = 3/6) in groups 2 and 3, p = 0.01. Conclusions: Allogenic ASC + VIT D without immunosuppression was safe and might have a role in the preservation of β-cells in patients with recent-onset T1D. ClinicalTrials.gov: NCT03920397.

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.

Adipose Tissue-Derived Stem Cell Therapy for Cavernous Nerve Injury-Induced Erectile Dysfunction in the Rat Model: A Systematic Review and Meta-Analysis Using Methodological Quality Assessment

BACKGROUND AND OBJECTIVES: Few studies were evaluated the effect of blindness on outcome in animal models, though a potential effect of blinding has been reported in clinical trials. We evaluated the effects of adipose tissue-derived stem cells (ADSCs) on cavernous nerve injury (CNI)-induced erectile dysfunction (ED) in the rat and examined how proper blinding of the outcome assessor affected treatment effect. METHODS AND RESULTS: We searched in Pubmed, EMBASE, Cochrane and Web of Science databases from inception to January 2019. We included CNI animal model, randomized controlled experiments, and ADSC intervention. Erectile function and structural changes were assessed by intracavernous pressure and mean arterial pressure (ICP/MAP) ratios, neuronal nitric oxide synthase (nNOS) levels, cavernous smooth muscle and collagen (CSM/collagen) ratios, and cyclic guanosine monophosphate (cGMP). RESULTS: Nineteen studies were included in the final meta-analysis. The ICP/MAP ratio of the ADSC treatment group increased compared to the control group (SMD=1.33, 95%CI: 1.11~1.56, I²=72%). The nNOS level (SMD=2.29, 95%CI: 1.74~2.84, I²=75%), CSM/collagen (SMD=2.57, 95%CI: 1.62~3.52; I²=85%), and cGMP (SMD=2.96, 95%CI: 1.82~4.10, I²=62%) were also increased in the ADSC treatment group. Preplanned subgroup analysis was conducted to explore the source of heterogeneity. Five studies with blinded outcome assessment were significantly less effective than the unblinded studies (SMD=1.33, 95%CI: 0.86~1.80; SMD=1.81, 95%CI: 1.17~2.46, respectively). CONCLUSIONS: ADSCs might be effective in improving erectile function and structural change in CNI-induced ED. However, non-blinded outcome assessors might cause detection bias and overestimate treatment efficacy. Therefore, the ADSC efficacy must be further evaluated with a rigorous study design to avoid bias.

Adipose tissue-derived mesenchymal stromal cells for treating chronic kidney disease: A pilot study assessing safety and clinical feasibility

BACKGROUND: Chronic kidney disease (CKD) is a growing public health concern, and available treatments are insufficient in limiting disease progression. New strategies, including regenerative cell-based therapies, have emerged as therapeutic alternatives. Results from several groups, including our own, have reported evidence of a supportive role for mesenchymal stromal cells (MSCs) in functional recovery and prevention of tissue damage in murine models of CKD. Prompted by these data, an open pilot study was conducted to assess the safety and efficacy of a single injection of autologous adipose tissue-derived MSCs (AT-MSCs) for treatment of CKD. METHODS: AT-MSCs were infused intravenously into six CKD patients at a dose of 1 million cells/kg. Patients were stabilized and followed for one year prior to MSC infusion and one year following infusion. RESULTS: No patients presented with adverse effects. Statistically significant improvement in urinary protein excretion was observed in AT-MSCs transplanted patients, from a median of 0.75 g/day (range, 0.15–9.57) at baseline to 0.54 g/day (range, 0.01v2.66) at month 12 (P = 0.046). The glomerular filtration rate was not significantly decreased post-infusion of AT-MSCs. CONCLUSION: Findings from this pilot study demonstrate that intravenous infusion of autologous expanded AT-MSCs into CKD patients was not associated with adverse effects and could benefit patients already undergoing standard medical treatment.

Experimental research of adipogenic differentiative ability of adipose tissue-derived stromal cells in patients with type 2 diabetes mellitus / 重庆医学

Objective To investiagte the adipogenic differentiative ability of adipose tissue-derived stromal cells(ADSCs) between the patients with type 2 diabetes mellitus(T2DM) and healthy persons.Methods The adipose tissues were taken from the adipose tissue in T2DM patients and healthy persons for separating and culturing ADSCs.The cells of third generation were taken for inoculation.The difference in cellular phenotype and growth speed were compared between the two groups.Adding adipogenesis inducing fluid,the adipogenic differentiative situation was observed in the two groups.The oil red O was added on 14 d for conducting the cell staining and observation.The oil red O was extracted by isopropanol,and the cellular absorbances were compared between two groups.Meanwhile,the expression of PPAR-γ,C/EBP-α and C/EBP-β on 14 d of adipogenic differentiation were compared between two groups by using qPCR method.Results The cellular phenotype and growth speed of ADSCs had no statisticat difference between T2DM patients and healthy persons.On 14 d of adipogenic differentiation,the oil red O absorbance value of AD-SCs in T2DM patients was significantly higher than that in the healthy persons,and the expression of PPAR-γ,C/EBP-α and C/EBP-β were significantly higher than those in the healthy persons.Conclusion The adipogenic differentiative ability of ADSCs in T2DM patients is obviously higher than that in healthy persons,which may be one of causes easy to be obese in T2DM patients.

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.

Impact of SPK1 Modified ADSC on Osteogenesis of Tissue Engineered Bone / 华中科技大学学报(医学版)

Objective To investigate the impact of sphingosine kinase 1 (SPK1) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A595nm and A490 nm.In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7)expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4％ vs.94.9％ respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was (0.73±0.10) vs.(1.29±0.17)(P＜0.05).The A value of CON and SPK1 group at 595 nm was (0.20±0.02) vs.(0.41±0.01) (P＜0.05),respectively.The A value of CON and SPK1 group at 490 nm was (0.72±0.01) vs.(0.51±0.02)(P＜0.05),respectively.The expression level of ALP in CON and SPK1 group was (1.42±-0.09) vs.(2.68±0.09) (P＜0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect.The expression level of BMP7 in CON and SPK1 group was (1.13±0.16) vs.(4.46±0.23)(P＜0.05),respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.

Effects of non-surgical periodontal treatment on level of vaspin and TNF-α in gingival crevicular fluid of type 2 diabetic patients with chronic periodontitis / 口腔疾病防治

Objective @#To investigate the changes of vaspin and TNF-α levels in gingival crevicular fluid (GCF) of type 2 diabetic (T2DM) patients with chronic periodontitis (CP) after non-surgical periodontal treatment.@*Methods@#60 subjects were divided into 4 groups: DM-CP group (patients with both T2DM and CP, n=15); CP group (CP patients without T2DM, n=15); DM group (T2DM patients without CP, n=15), and CTRL group (systemically and periodontally healthy individuals, n=15). The clinical parameters of periodontal tissue and GCF were measured before and 8 weeks after non-surgical periodontal treatment. @*Results@#The levels of vaspin and TNF-α were measured by ELISA. The levels of vaspin and TNF-α in CP group were significantly higher than those in CTRL group (P < 0.05), while the levels of vaspin and TNF-α in CP group were significantly decreased after treatment (P < 0.05). There was a statistically significant positive correlation between the total amount of vaspin and the total amount of TNF-α, the level of HbA1c, gingival index (GI) and probing depth (PD) (P < 0.05). @*Conclusion @#The results shows that vaspin and TNF-α are greatly decreased in periodontitis after non-surgical periodontal treatment. It suggests that vaspin and TNF-α in GCF may serve as inflammatory markers for the diagnosis and prognosis of diabetes and periodontitis.

Extensive characterization of feline intra-abdominal adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Effect of combined use of autologous adipose-derived stem cells and sterile biological films on chronic wound / 解放军医学杂志

Objective To analyze and evaluate the effectiveness of combined use of autologous adipose-derived stem cells (ADSCs) and sterile biological films on chronic wound. Methods Sixty patients of chronic wound were selected from the General Hospital of Chinese People’s Armed Police Forces, and randomly divided into three groups (20 each): the conventional treatment group (group A), the sterile biological films group (group B) and ADSCs combined with sterile biological films group (group C). The wound healing time and healing rate of the 3 groups on 7, 21 and 40d after treatment were observed; The proliferation of the basilar membrane cells of wound epithelium in the 3 groups were observed before and 7, 14d after treatment, and the epithelization on 50d after treatment was also observed. The neonatal microvessel density (MVD) in epidermal basal layer was calculated before and 7 days after treatment. Results On wound healing, the best result was shown in group C, manifested as the minor inflammatory response, the good formation of granulation tissue and faster speed in epithelial growth; and the result was better in group B than in group A. On wound healing time, the result was shown as group A > group B > group C, and the difference was statistically significant (P<0.05). On wound healing rate, cell proliferation and MVD, group C showed the best result in the 3 groups, group B was better than group A, and the differences were statistically significant (P<0.05). Conclusion ADSCs combined with sterile biological films in treatment of chronic wound healing may significantly improve the proliferation of repaired cells, promote wound vascular regeneration, improve the local growth environment and accelerate the wound healing.

Coculture Effects on the Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Cell-based bone regeneration is generally pursued based on single cell type approaches, for which human adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used, owing to their easy accessibility and relatively large yield. In view of multiple cell types involved in physiological bone regeneration, this study aimed to evaluate the osteogenic differentiation of AT-MSCs upon co-culture with endothelial cells or macrophages in a direct or indirect in vitro co-culture set-up. Our hypotheses were that 1) endothelial cells and macrophages stimulate AT-MSCs proliferation and osteogenic differentiation and that 2) these two cell types will more profoundly affect osteogenic differentiation of AT-MSCs in a direct compared to an indirect co-culture set-up, because of the possibility for both cell-cell interactions and effects of secreted soluble factors in the former. Osteogenic differentiation of AT-MSCs was stimulated by endothelial cells, particularly in direct co-cultures. Although initial numbers of AT-MSCs in co-culture with endothelial cells were 50% compared to monoculture controls, equal levels of mineralization were achieved. Macrophages showed a variable effect on AT-MSCs behavior for indirect co-cultures and a negative effect on osteogenic differentiation of AT-MSCs in direct co-cultures, the latter likely due to species differences of the cell types used. The results of this study demonstrate potential for cell combination strategies in bone regenerative therapies.

Isolation, Culturing, Characterization and Aging of Adipose Tissue-derived Mesenchymal Stem Cells: A Brief Overview

ABSTRACT The aim of this review was to describe the current state-of-the-art regarding isolation, characterization and aging of adipose tissue-derived mesenchymal stem cells (ADSCs). Mesenchymal stem cells (MSCs) have recently received widespread attention because of their potential use in tissue-engineering applications. Various studies have indicated that MSCs with a fibroblast-like morphology migrate to the sites of injury and help to regenerate damaged tissue. Over the past few years, it has been recognized that fat is not only an energy supply, but also a rich source of multipotent stem cells that can be easily harvested, isolated and selected as compared with other tissues. ADSCs are particularly interesting because of their rapid proliferation and multidirectional differentiation potential.

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.

Effect of serum-derived albumin scaffold and canine adipose tissue-derived mesenchymal stem cells on osteogenesis in canine segmental bone defect model

Composite biological and synthetic grafts with progenitor cells offer an alternative approach to auto- or allografts for fracture repair. This study was conducted to evaluate osteogenesis of autologous serum-derived albumin (ASA) scaffolds seeded with canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) in a canine segmental bone defect model. ASA scaffold was prepared with canine serum using cross-linking and freeze-drying procedures. Beta-tricalcium phosphate (beta-TCP) was mixed at the cross-linking stage. Ad-MSCs were seeded into the scaffold and incubated for one day before implantation. After 16 weeks, the grafts were harvested for histological analysis. The dogs were divided into five groups: control, ASA scaffolds with and without Ad-MSCs, and ASA scaffolds including beta-TCP with and without Ad-MSCs. ASA scaffolds with Ad-MSCs had a significantly larger area of increased opacity at the proximal and distal host cortex-implant interfaces in radiographs 16 weeks after implantation compared to the groups with beta-TCP (p < 0.05). Histomorphometric analysis showed that ASA scaffolds with Ad-MSCs had significantly greater new bone formation than other groups (p < 0.05). These results suggest that Ad-MSCs seeded into ASA scaffolds enhanced osteogenesis in the bone defect model, but that beta-TCP in the ASA scaffold might prevent penetration of the cells required for bone healing.

Concise Review: Differentiation of Human Adult Stem Cells Into Hepatocyte-like Cells In vitro

Adult stem cells (ASCs) are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues, which are the powerful sources for cell therapy and tissue engineering. Bone marrow-derived mesenchymal stem cells (BMSCs), adipose tissue-derived mesenchymal stem cells (ADSCs), and peripheral blood monocytes (PBMCs) are the common ASCs, and many studies indicated that ASCs isolated from various adult tissues could be induced to hepatocyte-like cells in vitro. However, the isolation, culture protocols, characterization of ASCs and hepatocyte-like cells are different. This review aims to describe the isolation and culture procedures for ASCs, to summarize the molecular characterization of ASCs, to characterize function of hepatocyte-like cells, and to discuss the future role of ASCs in cell therapy and tissue engineering.

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.

Influence of adipose tissue derived mesenchymal stem cells in combination with injectable bone substitute on osteoclastogenesis in osteoporotic rats.

The present study was designed to evaluate the influence of adipose tissue derived mesenchymal stem cell (ASCs) with or without calcium phosphate composite on osteoclastogenesis in osteoporotic rats. Mesenchymal stem cells (MSCs) were harvested from adipose tissue of both the omentum and the inguinal fat pad of male rats, as the sex mismatch, to track the MSCs fate and to ensure their homing to the injured females' femurs. The isolated ASCs were characterized via the morphological appearance, multilineage potential and the PCR detection of CD29, CD44, CD106, CD14, CD34 and CD45 surface markers. Fifty adult female albino rats were enrolled in the current study. The rats were classified into five groups: group 1 was the gonad intact control, group 2 served as untreated ovariectomized (OVX) rats, group 3 was OVX rats treated with ASCs, group 4 was OVX rats treated with ASCs with injectable bone substitute (IBS) and group 5 was OVX rats treated with IBS. The serum levels of osteoprotegerin (OPG) and receptor activator of NF-қβ ligand (RANKL) were assayed using ELISA procedure. In addition, nuclear factor-κβ (NF-κβ) gene expression level was estimated in femur bones using real time –PCR. The isolated ASCs proved their MSCs identity via their morphological appearance and multilineage potential. In addition, the isolated ASCs showed positive expression for CD29, CD45, CD44 as well as CD106 and negative expression for CD34 and CD14. Besides, the positive expression of the Y-chromosome (sry) gene detected in the ASCs treated groups indicated that the systemically delivered single dose of undifferentiated ASCs was able to home at the females' femur bones. Adipose tissue derived mesenchymal stem cells (ASCs) injection with or without calcium phosphate composite in OVX rats reversed the effect of ovariectomy on the studied biomarkers causing significant increase in serum OPG level accompanied with significant decrease in serum RANKL level. Also, significant down regulation of NF-κβ gene expression in femur bones was detected in the treated groups compared with untreated OVX group. These results clarified the good influence of ASCs against osteoclastogenesis. In addition the combination of ASCs injection with osteoinductive material injectable calcium phosphate composite (IBS), may be useful to achieve the significant antiosteoporotic effects.

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.

Adipose-tissue-derived Stem Cells Enhance the Healing of Ischemic Colonic Anastomoses: An Experimental Study in Rats

PURPOSE: This experimental study verified the effect of adipose-tissue-derived stem cells (ASCs) on the healing of ischemic colonic anastomoses in rats. METHODS: ASCs were isolated from the subcutaneous fat tissue of rats and identified as mesenchymal stem cells by identification of different potentials. An animal model of colonic ischemic anastomosis was induced by modifying Nagahata's method. Sixty male Sprague-Dawley rats (10-week-old, 370 +/- 50 g) were divided into two groups (n = 30 each): a control group in which the anastomosis was sutured in a single layer with 6-0 polypropylene without any treatment and an ASCtreated group (ASC group) in which the anastomosis was sutured as in the control group, but then ASCs were locally transplanted into the bowel wall around the anastomosis. The rats were sacrificed on postoperative day 7. Healing of the anastomoses was assessed by measuring loss of body weight, wound infection, anastomotic leakage, mortality, adhesion formation, ileus, anastomotic stricture, anastomotic bursting pressure, histopathological features, and microvascular density. RESULTS: No differences in wound infection, anastomotic leakage, or mortality between the two groups were observed. The ASC group had significantly more favorable anastomotic healing, including less body weight lost, less ileus, and fewer ulcers and strictures, than the control group. ASCs augmented bursting pressure and collagen deposition. The histopathological features were significantly more favorable in the ASC group, and microvascular density was significantly higher than it was in the control group. CONCLUSION: Locally-transplanted ASCs enhanced healing of ischemic colonic anastomoses by increasing angiogenesis. ASCs could be a novel strategy for accelerating healing of colonic ischemic risk anastomoses.