Search

Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: “network” and “cluster”. Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures.

The potential role of brown and beige adipose tissue against obesity has been recognized. Browning, or beiging of white adipose tissue (WAT) is associated with the remodeling of adipocytes and the improvement of their metabolic and secretory functions. Here, palmitoylethanolamide (PEA) restore the plasticity of brown and white adipocytes impaired in mice on a high-fat diet (HFD). Young male C57Bl/6J mice were fed with control (STD) diet or HFD for 12 weeks. Ultramicronized PEA (30 mg/kg/die p.o.) was administered for an additional 7 weeks, together with HFD. PEA recovered interscapular brown fat morphology and function, increasing UCP1 positivity, noradrenergic innervation, and inducing the mRNA transcription of several specialized thermogenic genes. PEA promotes the beige-conversion of the subcutaneous WAT, increasing thermogenic markers and restoring leptin signaling and tissue hormone sensitivity. The pivotal role of lipid-sensing peroxisome proliferator-activated receptor (PPAR)-α in PEA effects was determined in mature 3T3-L1. Moreover, PEA improved mitochondrial bioenergetics in mature adipocytes measured by a Seahorse analyzer and induced metabolic machinery via AMPK phosphorylation. All these outcomes were dampened by the receptor antagonist GW6471. Finally, PEA induced adipogenic differentiation and increased AMPK phosphorylation in human adipose-derived stromal cells (ASCs) obtained from subcutaneous WAT of normal-weight patients and patients with obesity. We identify PEA and PPAR-α activation as the main mechanism by which PEA can rewire energy-storing white into energy-consuming brown-like adipocytes via multiple and converging effects that restore WAT homeostasis and metabolic flexibility.

Vasculogenesis and angiogenesis are the processes by which new blood vessels are formed. We have developed a serum-free human adipose stromal cell and umbilical cord vein endothelial cell based vasculogenesis/angiogenesis test. In this study, the test was validated in our GLP laboratory following the OECD Guidance Document 34 [1] using erlotinib, acetylic salicylic acid, levamisole, 2-methoxyestradiol, anti‐VEGF, methimazole, and D-mannitol to show its reproducibility, repeatability, and predictivity for humans. The results were obtained from immunostained tubule structures and cytotoxicity assessment. The performance of the test was evaluated using 26 suspected teratogens and non-teratogens. The positive predictive value was 71.4% and the negative predictive value was 50.0%, indicating that inhibition of vasculogenesis is a significant mechanism behind teratogenesis. In conclusion, this test has great potential to be a screening test for prioritization purposes of chemicals and to be a test in a battery to predict developmental hazards in a regulatory context. [ABSTRACT FROM AUTHOR]

Abstract: Stem cells offer significant therapeutic promise for the treatment of ischemic disease. However, stem cells transplanted into ischemic tissue exhibit limited therapeutic efficacy due to poor engraftment in vivo. Several strategies for improving the survival and engraftment of stem cells in ischemic tissue have been developed including transplantation in combination with growth factor delivery, genetic modification of stem cells, and the use of cell-transplantation scaffolds. Here, we demonstrate that human adipose-derived stromal cells (hADSCs) cultured and grafted as spheroids exhibit improved therapeutic efficacy for ischemia treatment. hADSCs were cultured in monolayer or spheroids. Spheroid cultures were more effective in preconditioning hADSCs to a hypoxic environment, upregulating hypoxia-adaptive signals (i.e., stromal cell-derived factor-1α and hypoxia-inducible factor-1α), inhibiting apoptosis, and enhancing secretion of both angiogenic and anti-apoptotic factors (i.e., hepatocyte growth factor, vascular endothelial growth factor, and fibroblast growth factor 2) compared to monolayer cultures. Moreover, cell harvesting following spheroid cultures avoided damage to extracellular matrices due to harsh proteolytic enzyme treatment, thereby preventing anoikis (apoptosis induced by a lack of cell–matrix interaction). Following intramuscular transplantation to ischemic hindlimbs of athymic mice, hADSC spheroids showed improved cell survival, angiogenic factor secretion, neovascularization, and limb survival as compared to hADSCs grafted as dissociated cells. Taken together, spheroid cultures precondition hADSCs to a hypoxic environment, and grafting hADSCs as spheroids to ischemic limbs improves therapeutic efficacy for ischemia treatment due to enhanced cell survival and paracrine effects. Spheroid-based cell delivery could be a simple and effective strategy for improving stem cell therapy for ischemic diseases, eliminating the need for growth factor delivery, biomaterial scaffolds or genetic modification. [Copyright &y& Elsevier]

The potential role of brown and beige adipose tissue against obesity has been recognized. Browning, or beiging of white adipose tissue (WAT) is associated with the remodeling of adipocytes and the improvement of their metabolic and secretory functions. Here, palmitoylethanolamide (PEA) restore the plasticity of brown and white adipocytes impaired in mice on a high-fat diet (HFD). Young male C57Bl/6J mice were fed with control (STD) diet or HFD for 12 weeks. Ultramicronized PEA (30 mg/kg/die p.o.) was administered for an additional 7 weeks, together with HFD. PEA recovered interscapular brown fat morphology and function, increasing UCP1 positivity, noradrenergic innervation, and inducing the mRNA transcription of several specialized thermogenic genes. PEA promotes the beige-conversion of the subcutaneous WAT, increasing thermogenic markers and restoring leptin signaling and tissue hormone sensitivity. The pivotal role of lipid-sensing peroxisome proliferator-activated receptor (PPAR)-α in PEA effects was determined in mature 3T3-L1. Moreover, PEA improved mitochondrial bioenergetics in mature adipocytes measured by a Seahorse analyzer and induced metabolic machinery via AMPK phosphorylation. All these outcomes were dampened by the receptor antagonist GW6471. Finally, PEA induced adipogenic differentiation and increased AMPK phosphorylation in human adipose-derived stromal cells (ASCs) obtained from subcutaneous WAT of normal-weight patients and patients with obesity. We identify PEA and PPAR-α activation as the main mechanism by which PEA can rewire energy-storing white into energy-consuming brown-like adipocytes via multiple and converging effects that restore WAT homeostasis and metabolic flexibility. [ABSTRACT FROM AUTHOR]

Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: "network" and "cluster". Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures. [ABSTRACT FROM AUTHOR]

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limbgirdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4-6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies. [ABSTRACT FROM AUTHOR]

Background/Aim: Although transplantation of MSC derived from bone marrow or adipose tissues has been shown in proangiogenic action in hindlimb ischemia model of nude mice, little information is available regarding comparison of the angiogenic potency between human adipose stromal cells (hADSC) and bone marrow stromal cells (hBMSC). We compared their therapeutic potential by transplantation of equal numbers of hADSC or hBMSC in a nude mice model of hindlimb ischemia. Methods and Results: One day after creating hindlimb ischemia, mice were randomized to receive hADSC transplantation (hADSC group), hBMSC transplantation (hBMSC group), or vehicle transplantation (Control group). Two weeks after transplantation, the laser Doppler perfusion index was significantly higher in the hADSC group and hBMSC group than in the control group. Comparison between hADSC and hBMSC group showed better recovery of blood flow in hADSC group than in hBMSC group. Conditioned media from hADSC (hADSC-CM) showed better in vitro tube formation of hADSC than conditioned media from hBMSC (hBMSC-CM). hADSC showed higher expression of MMP3 and MMP9 than hBMSC. A MMP inhibitor, GM6001, and the transfection of MMP3 or MMP9 siRNA oligonucleotides inhibited in vitro tube formation of hADSC. Transplantation of MMP3 or MMP9 siRNA oligonucleotieds-transfected hADSC showed lower blood flow recovery and higher tissue injury than control oligonucelotide-transfected cells. Conclusion: This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease in terms of efficacy, accessibility and available tissue amounts. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Adult mesenchymal stem cells (MSCs) are promising tools for such applications as tissue engineering and cellular therapy. It is not clear how stem cells exposed to unfavorable conditions (e.g., hypoxia or inflammation) respond to signals of danger after in vivo transplantation. Toll-like receptors (TLRs) play a major role in the immune system, participating in the initial recognition of microbial pathogens and pathogen-associated components. This study was designated to determine the role of TLRs in human MSCs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry analysis demonstrated that MSCs derived from human adipose tissue and bone marrow express TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, and TLR-9. We investigated induction of the differentiation and proliferation of human adipose tissue stromal cells (hADSCs) by TLR agonists, including flagellin, peptidoglycans (PGN), lipopolysaccharide (LPS), the synthetic double-stranded RNA analog poly(I:C), and synthetic CpG oligodeoxydinucleotide (CpG-ODN). None of these agonists, except ODN, affected the proliferation of hADSCs. LPS and PGN increased osteogenic differentiation, but CpG-ODN decreased it. Poly(I:C) itself did not affect adipogenic or osteogenic differentiations, but exerted a synergistic effect on LPS- or PGN-induced osteogenic differentiation. RT-PCR analysis demonstrated that LPS and PGN induce osteogenic markers in hADSCs. TLR agonists affected the expression of chemokines and cytokines differentially. Furthermore, hADSCs affected the expression of specific TLRs in vitro under hypoxic conditions. These data provide evidence of a nonimmune role for TLR signaling on MSCs and may provide clues to the behavior of transplanted MSCs in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Flavonoids, which have been detected in a variety of foods, have been repeatedly reported to affect bone metabolism. However, the effects of flavonoids on osteoblastogenesis remain a matter of some controversy. In this study, the effects of quercetin on the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) were determined. Quercetin was found to increase osteogenic differentiation in a dose-dependent manner. Other flavonoids, chrysin and kaempferol, were also shown to increase the osteogenic differentiation of hADSC, but this stimulatory effect was weaker than that associated with quercetin. Quercetin pretreatment administered prior to the induction of differentiation also exerted stimulatory effects on the osteogenic differentiation of hADSC. RT-PCR and real time PCR analysis showed that quercetin treatment induced an increase in the expression of osteopontin, BMP2, alkaline phosphatase and Runx2. Quercetin inhibited the proliferation of hADSC, but did not affect their survival. The pretreatment of quercetin increased ERK phosphorylation during osteogenic differentiation, although it did not increase ERK activity in control culture condition. ICI182780, an specific estrogen receptor antagonist, failed to inhibit the effects of quercetin on osteogenic differentiation. Quercetin-pretreated hADSC showed better bone regenerating ability in skull defect model of nude mice than naive cells. Our findings indicate that quercetin enhances osteogenic differentiation via an independent mechanism from estrogen receptor (ER) activation, and prove useful for in vivo bone engineering, using human mesencymal stem cells (hMSC). [Copyright &y& Elsevier]

Background/Aim: It has been reported that adipose tissue contain progenitor cells with angiogenic potential and that therapy based on adipose tissuederived progenitor cells administration may constitute a promising cell therapy in patients with ischemic disease. In this study we evaluated the effect of culture-expanded mesenchymal stem cells (MSC) derived from adipose tissue on neovascularization and blood flow in an animal model of limb ischemia in immunodeficient mice. Methods: MSC were cultured from human adipose tissue by collagenase digestion. Hindlimb ischemia was created by ligating the proximal femoral artery of male nude mice. Human adipose tissue stromal cells (hADSC) were transplanted one day or 7 days after ligation. Results: During culture expansion of hADSC CD34 expression was downregulated. The laser Doppler perfusion index was significantly higher in the CD34(-), Flk-1(-), CD31(-) ADSC-transplanted group than in the control group, even when cells were transplanted 7days after hindlimb ischemia. Histological examination showed that hADSC transplantation recovered muscle injury and increased vascular density, compared with the control group. The effect of hADSC was correlated with the number of transplanted cells, but not with the ratio of CD34 expression. In vitro, hADSC can form vessel-like structure and express von Willibrand Factor. Conditioned media from hADSC increased proliferation and inhibited apoptotic cell death in of human aortic endothelial cells. Conclusion: This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease. [ABSTRACT FROM AUTHOR]

Besides being an energy storage, adipose tissue is an endocrine organ closely associated with vascular system. Human relevant in vitro models are needed to study adipose tissue and related diseases. Vasculature plays a central role in the development and inhibition of adipose tissue related diseases. Here, adipocyte culture was established from hASC (human adipose stromal cells), and a vascularized adipose tissue model was established from hASC and HUVEC (human umbilical cord vein endothelial cell) co-culture, utilizing the same differentiation procedure. Using these models together allowed analysis of the effect of vascularization on adipocytes. Adipocyte culture and Vascularized adipose tissue model were characterized on gene (adipocyte and vasculature-related), protein (von Willebrand factor, CollagenIV, CD140b and CD144, secretion of leptin, adiponectin and FABP4) and functional (triglyceride accumulation, glucose uptake and lipolysis) levels. Additionally, vascularized adipose tissue model was exposed to chemicals with known effects on adipogenesis and angiogenesis (rosiglitazone, chlorpyrifos, prochloraz, mancozeb, butylparaben, 15-deoxy-δ12,14-prostaglandin j2, bisphenol a, bis-(2-ethylhexyl) phthalate, tributyltin chloride) to compare their effects to the literature. The in vitro vascularized adipose tissue model showed presence of functional adipocytes and extensive vascular network. Adipocytes and the vasculature showed relevant gene and protein markers. Insulin induced glucose uptake, inhibited lipolysis and influenced vasculature-related genes. The results showed that vasculature led to faster insulin response in lipolysis inhibition and modulated responses to chemicals. This novel thoroughly characterized vascularized adipose tissue model is a promising new tool for studying adipose tissue as well as effect of chemicals on adipogenesis and angiogenesis in adipose tissue.

The objective of this study was to investigate whether the co-injection of human adipose stromal cells (hASCs) and rhBMP-2/fibrin gel into the bone tunnels of anterior cruciate-ligament reconstructions can effectively enhance tendon graft osteointegration. We performed bilateral reconstructions using autologous semitendinosus tendons in 45 New Zealand rabbits, which we divided into three groups. We injected the bone tunnels with fibrin gel for group 1, rhBMP-2 (1 μg/ml)/fibrin gel for group 2, and hASCs wrapped in rhBMP-2 (1 μg/ml)/fibrin gel for group 3. We sacrificed five rabbits (two for histological assessment and three for biomechanical tests) from each group at 2, 4, and 8 weeks post surgery. At 2 and 4 weeks post surgery, histological analysis showed that fibro-cartilage had appeared in the tendon-bone interface in group 2. At 4 weeks post surgery, mature bone cells could be seen in group 3. There was new bone formed between the host bone and the graft in groups 2 and 3 at 8 weeks post surgery. Biomechanical testing showed that at 4 and 8 weeks post surgery, the ultimate failure loads in group 3 were significantly higher than those in groups 1 and 2 (both P=0.01). The tendon stiffness in group 3 was significantly higher than that in the other groups at 4 weeks post surgery (P=0.01). Our results indicate that co-injection of hASCs and rhBMP-2/fibrin gel has the potential to promote tendon-bone healing after anterior cruciate-ligament reconstruction., Competing Interests: None.

Aging is accompanied by loss of subcutaneous adipose tissue. This may be due to reduced differentiation capacity or deficiency in DNA damage repair (DDR) factors. Here we investigated the role of SNEV hPrp19/hPso4 , which was implicated in DDR and senescence evasion, in adipogenic differentiation of human adipose stromal cells (hASCs). We showed that SNEV is induced during adipogenesis and localized both in the nucleus and in the cytoplasm. Knockdown of SNEV perturbed adipogenic differentiation and led to accumulation of DNA damage in hASCs upon oxidative stress. In addition, we demonstrated that SNEV is required for fat deposition in Caenorhabditis elegans. Consequently, we tested other DDR factors and found that WRN is also required for adipogenesis in both models. These results demonstrate that SNEV regulates adipogenesis in hASCs and indicate that DDR capacity in general might be a pre-requisite for this process., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)