Apoptosis Of Adipose-derived Stem Cells Research Articles

Effects of EGF on apoptosis of adipose derived stem cells by regulating miRNA-21

Abstract Objective The miroRNA-21 (MiR-21) plays a vital role in the process of proliferation and apoptosis in different types of cells. EGF could increase the miR-21 primary transcript during adipose derived stem cells (ADSCs) differentiation, however, the underlying molecular mechanism is unclear. We hypothesized that EGF affects the proliferation and apoptosis of ADSCs by regulating miRNA-21. Methods Gain or loss of function strategy was used through transfecting ADSCs with chemically synthesized oligonucleotides complementary to miR-21. Profiles of post-transfection miR-21 caused by EGF were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Apoptosis of ADSCs was evaluated with flow cytometry. The expression levels of miR-21's targeted genes PTEN and FASLwere determined by Western blot assay. Results In response to miR-21 mimics or inhibitor, the apoptosis and cytoactivity of ADSCs changed correspondingly. Change of miR-21 expression level was checked and an inverse correlation of EGF was observed with PTEN and FASL expression in ADSCs. Conclusion Our results indicated that miR-21 could influence EGF expression and furtherly inhibits the apoptosis of ADSCs by downregulating PTEN and FASL. These findings may provide clues toward regulating biological activity of ADSCs with miR-21.

In vitro effects of tamoxifen on adipose-derived stem cells.

In breast reconstructive procedures, adipose-derived stem cells (ASCs) that are present in clinical fat grafting isolates are considered to play the main role in improving wound healing. In patients following chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. However, it is unclear if tamoxifen (TAM) as the most widely used hormonal therapeutic agent for breast cancer treatment, affects the ASCs and ultimately wound healing. This study evaluated whether TAM exposure to in vitro human ASCs modulate cellular functions. Human ASCs were isolated and treated with TAM at various concentrations. The effects of TAM on cell cycle, cell viability and proliferation rates of ASCs were examined by growth curves, MTT assay and BrdU incorporation, respectively. Annexin V and JC-1 Mitochondrial Membrane Potential assays were used to analyze ASC apoptosis rates. ASCs were cultured in derivative-specific differentiation media with or without TAM (5 uM) for 3 weeks. Adipogenic and osteogenic differentiation levels were measured by quantitative RT-PCR and histological staining. TAM has cytotoxic effects on human ASCs through apoptosis and inhibition of proliferation in dose- and time-dependent manners. TAM treatment significantly down-regulates the capacity of ASCs for adipogenic and osteogenic differentiation (p<0.05 vs. control), and inhibit the ability of the ASCs to subsequently formed cords in Matrigel. This study is the first findings to our knowledge that demonstrated that TAM inhibited ASC proliferation and multi-lineage ASC differentiation rates. These results may provide insight into the role of TAM with associated poor soft tissue wound healing and decreased fat graft survival in cancer patients receiving TAM.

Vascular regeneration effect of adipose-derived stem cells with light-emitting diode phototherapy in ischemic tissue.

The objective of this study was to investigate the effects on the vascular regeneration of adipose-derived stem cells (ASCs) by using red light-emitting diode (LED) irradiation in ischemic hind limbs. Low-level light therapy (LLLT) has been shown to enhance proliferation and cytokine secretion of a number of cells. ASCs are an attractive cell source for vascular tissue engineering. This approach is hindered because transplanted ASCs decline rapidly in the recipient tissue. Ischemic hind limbs were treated with LLLT from an LED array (660nm) at an irradiance of 50mW/cm(2) and a radiant exposure of 30J/cm(2). LLLT, ASC transplantation, and ASC transplantation with LLLT (ASC + LLLT) were applied to ischemic limbs, and cell survival and differentiation, and secretion of vascular endothelial growth factor and basic fibroblast growth factor of the ASCs were evaluated by immunostaining and Western blot analyses. Vascular regeneration was assessed by immunostaining and hematoxylin and eosin staining. In the ASC + LLLT group, the survival of ASCs was increased due to the decreased apoptosis of ASCs. The secretion of growth factors was stimulated in this group compared with ASCs alone. The ASC + LLLT group displayed improved treatment efficacy including neovascularization and tissue regeneration compared with ASCs alone. In particular, quantitative analysis of laser Doppler blood perfusion image ratio showed that blood perfusion was enhanced significantly (p < 0.05) by ASC + LLLT treatment. These data suggest that LLLT is an effective biostimulator of ASCs in vascular regeneration, which enhances the survival of ASCs and stimulates the secretion of growth factors in ischemic limbs.

Inhibition of oxygen-glucose deprivation-induced apoptosis of human adipose-derived stem cells by genetic modification with antiapoptotic protein bcl-2.

Adipose-derived stem cells (ADSCs) have become a promising tool for a wide range of cell-based therapies. However, transplanted ADSCs do not survive well under ischemic conditions. In this study we aimed to inhibit oxygen-glucose deprivation (OGD)-induced apoptosis of human ADSCs by genetic modification with antiapoptotic protein Bcl-2. After isolation and culture, the phenotypes of human ADSCs at passage 3 were analyzed by flow cytometry. Then, genetic modification of ADSCs with Bcl-2 was carried out. Bcl-2 gene transfection was verified by Western blot analysis and multipotent differentiation properties were evaluated in Bcl-2-modified ADSCs (Bcl-2-ADSCs). Apoptosis was evaluated by a TUNEL assay under ischemic conditions induced by OGD. Apoptotic nuclei were also assessed and quantified by Hoechst staining. The cultured ADSCs expressed stem cell-associated markers CD29, CD34, CD44, and CD90, but not fibroblast marker HLA-DR or hematopoietic stem cell marker CD133. The Bcl-2 gene was transferred into ADSCs efficiently, and Bcl-2-ADSCs differentiated into adipocytes, chondrocytes, and osteoblasts. In addition, Bcl-2 overexpression reduced the percentage of apoptotic Bcl-2-ADSCs by 38 % under OGD. Our results indicate that Bcl-2 overexpression through gene transfection inhibits apoptosis of ADSCs under ischemic conditions. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

A combination of insulin and ubiquitin A20 promotes osteocalcin expression in adipose-derived stem cells

Osteocyte generation can be used in bone defect repair; the generation efficiency needs to be further improved. This study aims to evaluate the role of ubiquitin A20 (A20) in facilitating the expression of osteocalcin in adipose-derived stem cells (ADSCs). In this study, adipose tissue was obtained from 10 healthy human subjects; ADSCs were isolated from the adipose samples. The ADSCs were transfected with core binding factor alpha 1 (Cbfa1) and/or insulin-like growth factor-1 receptor (IGF-1R). Expression of osteocalcin, A20 in ADSCs was assessed by quantitative RT-PCR (qRT-PCR) and Western blotting. Apoptosis of ADSCs was analyzed by flow cytometry. The results showed that after the gene transfection and stimulation of insulin, the ADSCs expressed high levels of osteocalcin. However, apoptotic ADSCs were induced by the activation of IGF-1R. Exposure to insulin down-regulated the expression of Bcl-xL and A20, and increased Bax, in ADSCs. The addition of exogenous A20 prevented the ADSC apoptosis. We conclude that activation of IGF-1R can induce apoptosis in ADSCs, which can be prevented by addition of exogenous A20.

The Effect of Lipoaspirates Cryopreservation on Adipose-Derived Stem Cells

Autologous fat grafting has gained popularity, particularly with the discovery of adipose-derived stem cells (ADSC). The possibility of freezing lipoaspirates (LA) for later use has intriguing clinical potential. However, the effect of LA cryopreservation on ADSC is unclear. The authors explore the effect of LA cryopreservation on ADSC viability. Human LA (n = 8) were harvested using a standard technique. Lipoaspirate samples were either processed immediately as fresh LA (A) or stored at -20°C and then at -80°C for 30 days with (B) or without (C) freezing medium. Stromal vascular fraction (SVF) was separated from adipocytes and either cultured to obtain purified ADSC or processed for the isolation of 3 distinct ADSC subpopulations (CD90(+)/CD45(-), CD105(+)/CD45(-), and CD34(+)/CD31(-)). Apoptosis and necrosis were determined by an annexin V/propidium iodide assay and quantified by flow cytometry. The capability of ADSC for long-term proliferation and differentiation was also examined. There were no significant differences in the apoptosis and necrosis of adipocytes, SVF, or ADSC between groups A and B. However, cell viability in SVF and ADSC was significantly compromised in group C as compared with group B (P < .01) due to higher ADSC apoptosis but not necrosis. The viable ADSC isolated from fresh or frozen LA were cultured for more than 20 passages and demonstrated similar patterns and speed of proliferation with strong capability to differentiate, evidenced by cell doubling time and positive staining with Oil Red O (Sigma-Aldrich, St Louis, Missouri) and alkaline phosphatase. Lipoaspirates cryopreservation had a significant impact on ADSC apoptosis but not on ADSC necrosis, proliferation, or differentiations. Freezing medium provides significant protection against ADSC apoptosis.

Commentary on: The Effect of Lipoaspirates Cryopreservation on Adipose-Derived Stem Cells

In the study entitled “The Effect of Lipoaspirates Cryo-preservation on Adipose-Derived Stem Cells,” Wang et al1 conducted a thorough investigation to evaluate the potential effect of lipoaspirates (LA) cryopreservation on adipose-derived stem cells (ADSC). The authors have chosen what they believe is a reasonably good cryopreservation protocol for “banking” of LA harvested by standard liposuction techniques. The study appears to be quite well conducted, and the methods are standard and scientifically sound for the study of ADSC. On the basis of the study, the authors concluded that LA cryopreservation had no effect on adipocyte viability. It had a significant impact on ADSC apoptosis but not on ADSC necrosis, proliferation, or differentiation. The cryoprotective agent may provide adequate protection against ADSC apoptosis. In addition, CD34+/CD31– ADSC have a significantly lower rate of apoptosis and necrosis compared with other subpopulations of ADSC, such as CD105+/CD45−.1 I believe that the study is an important contribution to the scientific literature of fat grafting research because of ADSC and their therapeutic potential in the near future. In addition, the study itself represents a continued effort by plastic surgery investigators to explore the possibility of whether cryopreservation of LA could become a clinical reality for subsequent fat grafting procedures and ADSC-directed cell therapy after long-term preservation for our patients in the future. I would agree with the authors that there has been controversy in terms of the effect of LA cryopreservation on ADSC. Some studies show that there are negative effects of LA cryopreservation on ADSC for their proliferation and differentiation.2,3 Other studies show that …

GW24-e3607 Pretreatment of ADSCs with exendin-4 improve therapeutic efficiency following myocardial infarction via enhanced adhesion

ObjectivesExendin-4 has the strong anti-oxidant andanti-inflammatory properties. Reactive oxygen species (ROS) produced in ischaemic niche play a pivotal role in stem cells detachment from the impairedmyocardium. To improve the poor...

Influence of epidermal growth factor on proliferation, migration and apoptosis of adipose-derived stem cells in vitro

Objective To investigate the effects of epidermal growth factor (EGF) on the proliferation , migration and apoptosis of adipose-derived stem cells (ADSCs) in vitro. Methods Starved cell model and FasL-induced apoptosis model were established in serum-free media. The effects of 10 nmol/L and 100 nmol/L of EGF on the proliferation, migration and apoptosis of ADSCs in vitro were observed. The exrpessions of signal pathway proteins like phospholipaseC-γ(PLC-γ) , extracellular regulated kinase (ERK) and AKT were also detected. Results The proliferation, migration and anti-ap-optosis of ADSCs were promoted by 10 nmol/L or 100 nmol/L of EGF, and the expressions of PLC-'y, ERK and AKT were up-regulated. Conclusion EGF can promote the proliferation, migration and an-ti-apoptosis of adipose-derived ADSCs in vitro. Key words: Epidermal growth factors; Adipose-derived stem cells; Apoptosis

Adenoviral transfer of PKGIα; attenuates apoptosis and necrosis in adipose derived stem cells

Adenoviral transfer of protein kinase G-Iα (PKGIα) protects myocytes from ischemia but the effect of PKGIα on adipose derived stem cells (ASCs) remains unknown. We hypothesize that PKGIα will attenuate necrosis and apoptosis of ASCs in an ischemic environment. ASCs were infected with adenoviral vectors containing PKGIα or a mutant then subjected to simulated ischemia (SI) and reoxygenation (RO) for necrosis or apoptosis studies. To determine the role of PKG overexpression in vivo, adult male ICR mice underwent permanent left coronary artery occlusion before receiving treatment of 2x10^5 cells overexpressing PKGIα or mutant. 2 weeks after surgery, transthoracic echocardiography (TTE) was performed to assess left ventricular (LV) function. PKGIα overexpression reduced necrosis as seen by a decrease in trypan blue positive cells and LDH release when compared to SI/RO control (Fig 1 A, B). TUNEL revealed that apoptosis was also significantly reduced in cells overexpressing PKGIα (5.1± 0.47%) as compared to SI/RO control (12.8±1.07%). ASCs infected with the inactive PKGIαK390A mutant lacked protection. TTE results showed reduced LV dilatation and preservation of ejection fraction with PKG overexpression (Fig 1 C, D). Therefore, adenoviral gene transfer of PKGIα may provide a novel strategy for protecting ASCs against ischemia which could translate to a favorable function in hearts following ischemia. Figure 1Open in figure viewerPowerPoint