Stimulation Of Adipose-derived Stem Cells Research Articles

The Effect of Ionizing Irradiation on the Autotaxin-Lysophasphatidic Acid Axis and Interleukin-6/8 Secretion in Different Breast Cancer Cell Lines.

The Autotaxin (ATX)-lysophosphatidic acid (LPA) axis is involved in decreasing radiation sensitivity of breast tumor cells. This study aims to further elucidate the effect of irradiation on the ATX-LPA axis and cytokine secretion in different breast cancer cell lines to identify suitable breast cancer subtypes for targeted therapies. Different breast cancer cell lines (MCF-7 (luminal A), BT-474 (luminal B), SKBR-3 (HER2-positive), MDA-MB-231 and MDA-MB-468 (triple-negative)) and the breast epithelial cell line MCF-10A were irradiated. The influence of irradiation on LPA receptor (LPAR) expression, ATX expression, and Interleukin (IL)-6 and IL-8 secretion was analyzed. Further, the effect of IL-6 and IL-8 on ATX expression of adipose-derived stem cells (ADSC) was investigated. Irradiation increased ATX and LPAR2 expression in MDA-MB-231 cells. Additionally, IL-6 secretion was enhanced in MDA-MB-231, and IL-8 secretion in MDA-MB-231 and MDA-MB-468. Stimulation of ADSC with IL-6 and IL-8 increased ATX expression in ADSC. Targeting ATX or its downstream signaling pathways might enhance the sensitivity of triple-negative breast cancer cells to radiation. Further exploration of the interplay between irradiation, the ATX-LPA axis, and inflammatory cytokines may elucidate novel pathways for overcoming radioresistance and improving individual treatment outcomes.

Harnessing adipose‑derived stem cells to release specialized secretome for the treatment of hepatitis B.

Mesenchymal stem cells (MSCs) have the function of repairing damaged tissue, which is known to be mediated by the secretome, the collection of secretory materials shed from MSCs. Adjusting the culture conditions of MSCs can lead to a significant difference in the composition of the secretome. It was hypothesized that pre-sensitization of MSCs with specific disease-causing agents could harness MSCs to release the therapeutic materials specialized for the disease. To validate this hypothesis, the present study aimed to generate a 'disease-specific secretome' for hepatitis caused by hepatitis B virus using hepatitis BX antigen (HBx) as a disease-causing material. Secretary materials (HBx-IS) were collected following the stimulation of adipose-derived stem cells (ASCs) with 100-fold diluted culture media of AML12 hepatocytes that had been transfected with pcDNA-HBx for 24 h. An animal model of hepatitis B was generated by injecting HBx into mice, and the mice were subsequently intravenously administered a control secretome (CS) or HBx-IS. Compared with the CS injection, the HBx-IS injection significantly reduced the serum levels of interleukin-6 and tumor necrosis factor-α (pro-inflammatory cytokines). Western blot analysis and immunohistochemistry of the liver specimens revealed that the HBx-IS injection led to a higher expression of liver regeneration-related markers, including hepatocyte growth factor and proliferating cell nuclear antigen, a lower expression of pro-apoptotic markers, such as cleaved caspase 3 and Bim in mouse livers, and a lower expression of pro-inflammatory markers (F4/80 and CD68) compared to the CS injection. HBx-IS exhibited higher liver regenerative, anti-inflammatory and anti-apoptotic properties, particularly in the mouse model of hepatitis B compared to CS. This suggests that the secretome obtained by stimulating ASCs with disease-causing agents may have a more prominent therapeutic effect on the specific disease than the naïve secretome.

Electrical Stimulation of Adipose-Derived Stem Cells in 3D Nanofibrillar Cellulose Increases Their Osteogenic Potential.

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.

Ex-Vivo Stimulation of Adipose Stem Cells by Growth Factors and Fibrin-Hydrogel Assisted Delivery Strategies for Treating Nerve Gap-Injuries.

Peripheral nerve injuries often result in lifelong disabilities despite advanced surgical interventions, indicating the urgent clinical need for effective therapies. In order to improve the potency of adipose-derived stem cells (ASC) for nerve regeneration, the present study focused primarily on ex-vivo stimulation of ASC by using growth factors, i.e., nerve growth factor (NGF) or vascular endothelial growth factor (VEGF) and secondly on fibrin-hydrogel nerve conduits (FNC) assisted ASC delivery strategies, i.e., intramural vs. intraluminal loading. ASC were stimulated by NGF or VEGF for 3 days and the resulting secretome was subsequently evaluated in an in vitro axonal outgrowth assay. For the animal study, a 10 mm sciatic nerve gap-injury was created in rats and reconstructed using FNC loaded with ASC. Secretome derived from NGF-stimulated ASC promoted significant axonal outgrowth from the DRG-explants in comparison to all other conditions. Thus, NGF-stimulated ASC were further investigated in animals and found to enhance early nerve regeneration as evidenced by the increased number of β-Tubulin III+ axons. Notably, FNC assisted intramural delivery enabled the improvement of ASC’s therapeutic efficacy in comparison to the intraluminal delivery system. Thus, ex-vivo stimulation of ASC by NGF and FNC assisted intramural delivery may offer new options for developing effective therapies.

Generation of induced secretome from adipose-derived stem cells specialized for disease-specific treatment: An experimental mouse model.

Recently, the exclusive use of mesenchymal stem cell (MSC)-secreted molecules, named as the secretome, have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages. To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials. We collected the secretory materials (named as inducers) released from AML12 hepatocytes that had been pretreated with thioacetamide (TAA) and generated the TAA-induced secretome (TAA-isecretome) after stimulating adipose-derived stem cells with the inducers. The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy. TAA-isecretome infusion showed higher therapeutic potential in terms of (1) restoring disorganized hepatic tissue to normal tissue; (2) inhibiting proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α); and (3) reducing abnormally elevated liver enzymes (aspartate aminotransferase and alanine aminotransferase) compared to the naïve secretome infusion in mice with TAA-induced hepatic failure. However, the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice. Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome. In addition, peroxiredoxin-1, a potent antioxidant protein, was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury. Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins, especially peroxiredoxin-1, with higher antioxidant activity. Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen. This approach is expected to open a new way of developing various specific therapeutics based on the high plasticity and responsiveness of MSCs.

Combinatorial conditioning of adipose derived-mesenchymal stem cells enhances their neurovascular potential: Implications for intervertebral disc degeneration.

ABSTRACTBackgroundMesenchymal stem cells (MSCs) are becoming an increasingly attractive option for regenerative therapies due to their availability, self‐renewal capacity, multilineage potential, and anti‐inflammatory properties. Clinical trials are underway to test the efficacy of stem cell‐based therapies for the repair and regeneration of the degenerate intervertebral disc (IVD), a major cause of back pain. Recently, both bone marrow‐derived MSCs and adipose‐derived stem cells (ASCs) have been assessed for IVD therapy but there is a lack of knowledge surrounding the optimal cell source and the response of transplanted cells to the low oxygen, pro‐inflammatory niche of the degenerate disc. Here, we investigated several neurovascular factors from donor‐matched MSCs and ASCs that may potentiate the survival and persistence of sensory nerve fibers and blood vessels present within painful degenerate discs and their regulation by oxygen tensions and inflammatory cytokines.MethodsDonor‐matched ASCs and MSCs were conditioned with either IL‐1β or TNFα under normoxic (21% O2) or hypoxic (5% O2) conditions. Expression and secretion of several potent neurovascular factors were assessed using qRT‐PCR and human magnetic Luminex assay.ResultsASCs and MSCs expressed constitutive levels of key neurotrophic factors; and stimulation of ASCs with hypoxia triggered increased secretion of both angiogenic factors (Ang‐2 and VEGF‐A) and neurotrophic (NGF and NT‐3) compared to MSCs. We also report increased transcriptional regulation of pain‐associated neuropeptides in hypoxia stimulated ASCs compared to those in normoxic conditions. We demonstrate transcriptional and translational upregulation of NGF, NT‐3, Ang‐1, and FGF‐2 in response to cytokines in ASCs in 21% and 5% O2.ConclusionsThis work highlights fundamental differences between the neurovascular secretome of donor‐matched ASCs and MSCs, demonstrating the importance of cell‐selection for tissue specific regeneration to reduce ectopic sensory nerve and blood vessel survival and improve patient outcomes.

Udenafil Induces the Hair Growth Effect of Adipose-Derived Stem Cells.

Udenafil, which is a PDE5 inhibitor, is used to treat erectile dysfunction. However, it is unclear whether udenafil induces hair growth via the stimulation of adipose-derived stem cells (ASCs). In this study, we investigated whether udenafil stimulates ASCs and whether increased growth factor secretion from ASCs to facilitate hair growth. We found that subcutaneous injection of udenafil-treated ASCs accelerated telogen-to-anagen transition in vivo. We also observed that udenafil induced proliferation, migration and tube formation of ASCs. It also increased the secretion of growth factors from ASCs, such as interleukin-4 (IL-4) and IL12B, and the phosphorylation of ERK1/2 and NFκB. Furthermore, concomitant upregulation of IL-4 and IL12B mRNA levels was attenuated by ERK inhibitor or NFκB knockdown. Application of IL-4 or IL12B enhanced anagen induction in mice and increased hair follicle length in organ culture. The results indicated that udenafil stimulates ASC motility and increases paracrine growth factor, including cytokine signaling. Udenafil-stimulated secretion of cytokine from ASCs may promote hair growth via the ERK and NFκB pathways. Therefore, udenafil can be used as an ASC-preconditioning agent for hair growth.

Minoxidil Promotes Hair Growth through Stimulation of Growth Factor Release from Adipose-Derived Stem Cells.

Minoxidil directly promotes hair growth via the stimulation of dermal papilla (DP) and epithelial cells. Alternatively, there is little evidence for indirect promotion of hair growth via stimulation of adipose-derived stem cells (ASCs). We investigated whether minoxidil stimulates ASCs and if increased growth factor secretion by ASCs facilitates minoxidil-induced hair growth. Telogen-to-anagen induction was examined in mice. Cultured DP cells and vibrissae hair follicle organ cultures were used to further examine the underlying mechanisms. Subcutaneous injection of minoxidil-treated ASCs accelerated telogen-to-anagen transition in mice, and increased hair weight at day 14 post-injection. Minoxidil did not alter ASC proliferation, but increased migration and tube formation. Minoxidil also increased the secretion of growth factors from ASCs, including chemokine (C-X-C motif) ligand 1 (CXCL1), platelet-derived endothelial cell growth factor (PD-ECGF), and platelet-derived growth factor-C (PDGF-C). Minoxidil increased extracellular signal–regulated kinases 1/2 (ERK1/2) phosphorylation, and concomitant upregulation of PD-ECGF and PDGF-C mRNA levels were attenuated by an ERK inhibitor. Subcutaneous injection of CXCL1, PD-ECGF, or PDGF-C enhanced anagen induction in mice, and both CXCL1 and PDGF-C increased hair length in ex vivo organ culture. Treatment with CXCL1, PD-ECGF, or PDGF-C also increased the proliferation index in DP cells. Finally, topical application of CXCL1, PD-ECGF, or PDGF-C with 2% minoxidil enhanced anagen induction when compared to minoxidil alone. Minoxidil stimulates ASC motility and increases paracrine growth factor signaling. Minoxidil-stimulated secretion of growth factors by ASCs may enhance hair growth by promoting DP proliferation. Therefore, minoxidil can be used as an ASC preconditioning agent for hair regeneration.

Mechanical Stimulation of Adipose-Derived Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain.

It is critical that human adipose stem cell (hASC) tissue-engineering therapies possess appropriate mechanical properties in order to restore function of the load bearing tissues of the musculoskeletal system. In an effort to elucidate the hASC response to mechanical stimulation and develop mechanically robust tissue engineered constructs, recent research has utilized a variety of mechanical loading paradigms including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter describes methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.

Lysophosphatidic acid increases the proliferation and migration of adipose‑derived stem cells via the generation of reactive oxygen species.

Phospholipid derivatives, such as lysophosphatidic acid (LPA), exhibit mitogenic effects on mesenchymal stem cells; however, the molecular mechanism underlying this stimulation has yet to be identified. The aims of the present study were as follows: To evaluate the stimulatory effects of LPA on the proliferation and migration of adipose‑derived stem cells (ASCs); to study the association between reactive oxygen species (ROS) and LPA signaling in ASCs; and to investigate the microRNAs upregulated by LPA treatment in ASCs. The results of the present study demonstrated that LPA increased the proliferation and migration of ASCs, and acted as a mitogenic signal via extracellular signal‑regulated kinases 1/2 and the phosphoinositide 3‑kinase/Akt signaling pathways. The LPA1 receptor is highly expressed in ASCs, and pharmacological inhibition of it by Ki16425 significantly attenuated the proliferation and migration of ASCs. In addition, LPA treatment generated ROS via NADPH oxidase 4, and ROS were able to function as signaling molecules to increase the proliferation and migration of ASCs. The induction of ROS by LPA treatment also upregulated the expression of miR‑210. A polymerase chain reaction array assay demonstrated that the expression levels of adrenomedullin and Serpine1 were increased following treatment with LPA. Furthermore, transfection with Serpine1‑specific small interfering RNA attenuated the migration of ASCs. In conclusion, the present study is the first, to the best of our knowledge, to report that ROS generation and miR‑210 expression are associated with the LPA‑induced stimulation of ASCs, and that Serpine1 mediates the LPA‑induced migration of ASCs. These results further suggest that LPA may be used for ASC stimulation during stem cell expansion.

Functional regulation of adipose-derived stem cells by PDGF-D.

Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-β are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation.

Primary Involvement of NADPH Oxidase 4 in Hypoxia-Induced Generation of Reactive Oxygen Species in Adipose-Derived Stem Cells

We have previously demonstrated that hypoxia stimulates adipose-derived stem cells (ASCs) through the generation of reactive oxygen species (ROS). However, the precise mechanism involved in the ROS generation by ASCs is not well understood. We sought to investigate in this work: (1) which subtype of NADPH oxidase (Nox) is primarily expressed in ASCs; (2) where Nox4 is localized in ASCs; and (3) whether silencing of Nox4 attenuates hypoxia-enhanced function of ASC. We used 2',7'-dichlorofluorescin diacetate (DCF-DA) as an indicator of ROS generation and found that the fluorescence intensity of DCF-DA was significantly increased after hypoxia exposure (2% oxygen). In addition, hypoxia enhanced the proliferation and migration of ASCs and upregulated the mRNA expression of Oct4 and Rex1. Quantitative analysis of mRNA expression of Nox family in ASCs demonstrated that Nox4 is primarily expressed in ASCs, while immunofluorescence assay showed that Nox4 is mainly localized in the perinuclear region and overlaps with Mitotracker, a mitochondria marker. Silencing of Nox4 by siRNA treatment downregulated the RNA and protein expression of Nox4, which significantly reduced the ROS generation under hypoxia. In addition, Nox4 silencing significantly reduced the proliferation and migration of ASCs and downregulated the mRNA expression of Oct4 and Rex1. Phosphorylation of platelet-derived growth factor receptor-β, AKT, and ERK1/2 also diminished following Nox4 silencing. In a nutshell, these results suggest that Nox4 is primarily expressed in ASCs and plays a pivotal role in the hypoxia-enhanced stimulation of ASCs.

Adipose-Derived Stem Cells Promote Lymphangiogenesis in Response to VEGF-C Stimulation or TGF-β1 Inhibition

Recent studies have demonstrated that augmentation of lymphangiogenesis and tissue engineering hold promise as a treatment for lymphedema. The purpose of this study was to determine whether adipose-derived stem cells (ASCs) can be used in lymphatic tissue-engineering by altering the balance between pro- and anti-lymphangiogenic cytokines. ASCs were harvested and cultured in media with or without recombinant VEGF-C for 48 h. ASCs were then implanted in mice using Matrigel plugs. Additional groups of animals were implanted with ASCs transfected with a dominant-negative TGF-β1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-β1 has been shown to have potent antilymphangiogenic effects. Lymphangiogenesis, lymphatic differentiation and cellular proliferation were assessed. Stimulation of ASCs with VEGF-C in vitro significantly increased expression of VEGF-A, VEGF-C and Prox-1. ASCs stimulated with VEGF-C prior to implantation induced a significant (threefold increase) lymphangiogenic response as compared with control groups (unstimulated ASCs or empty Matrigel plugs; p < 0.01). This effect was significantly potentiated when TGF-β1 signaling was inhibited using the dominant-negative TGF-β1 receptor-II virus (4.5-fold increase; p < 0.01). Stimulation of ASCs with VEGF-C resulted in a marked increase in the number of donor ASCs (twofold; p < 0.01) and increased the number of proliferating cells (sevenfold; p < 0.01) surrounding the Matrigel. ASCs stimulated with VEGF-C expressed podoplanin, a lymphangiogenic cell marker, whereas unstimulated cells did not. Short-term stimulation of ASCs with VEGF-C results in increased expression of VEGF-A, VEGF-C and Prox-1 in vitro and is associated with a marked increase lymphangiogenic response after in vivo implantation. This lymphangiogenic response is significantly potentiated by blocking TGF-β1 function. Furthermore, stimulation of ASCs with VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express podoplanin, a lymphangiogenic cell marker.

The Pivotal Role of Reactive Oxygen Species Generation in the Hypoxia-Induced Stimulation of Adipose-Derived Stem Cells

Adipose-derived stem cells (ASCs) offer a potential alternative for tissue repair and regeneration. We have recently shown that hypoxia stimulates ASCs and enhances the regenerative potential of ASCs, which is beneficial for ASC therapy. In the present study, we further investigated a key mediator and a signal pathway involved in the stimulation of ASC during hypoxia. Culturing ASC in a hypoxic incubator (2% oxygen tension) increased the proliferation and migration, and this was mediated by Akt and ERK pathways. To determine the generation of reactive oxygen species (ROS), 2',7'-dichlorofluorescin diacetate intensity was detected by fluorescence-activated cell sorting. Hypoxia significantly increased the dichlorofluorescin diacetate intensity, which was greatly reduced by N-acetyl-cysteine and diphenyleneiodonium treatment. Likewise, the hypoxia-induced proliferation and migration of ASCs were reversed by N-acetyl-cysteine and diphenyleneiodonium treatment, suggesting the involvement of ROS generation in ASC stimulation. Further, we examined the activation of receptor tyrosine kinases and observed that hypoxia stimulated the phosphorylation of platelet-derived growth factor receptor-β. In summary, the ROS produced by ASCs in response to hypoxia was mostly likely due to NADPH oxidase activity. The increased cellular ROS was accompanied by the phosphorylation of platelet-derived growth factor receptor-β as well as by the activation of ERK and Akt signal pathways. Our results suggest a pivotal role for ROS generation in the stimulation of ASCs by hypoxia.