Adipose-derived Stem Research Articles

Adipose stem cell-derived extracellular matrix represents a promising biomaterial by inducing spontaneous formation of prevascular-like structures by mvECs.

Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self-assembled formation of prevascular-like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose-derived stem cells (ASCs) or adipogenically differentiated ASCs, with the classical cell-based FL. All cell-derived ECM (cdECM) substrates enabled mvEC growth with high viability. Prevascular-like structures were visualized by immunofluorescence staining of endothelial surface protein CD31 and could be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenically differentiated ECM, longer and higher branched structures could be found compared with stem cell cdECM. An increased concentration of proangiogenic factors was found in cdECM substrates and FL approaches compared with controls. Finally, the expression of proteins associated with tube formation (E-selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for adipose tissue engineering by inducing the spontaneous formation of prevascular-like structures by mvECs.

Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells

Effective engineering approaches for cartilage regeneration involve a combination of cells and biomaterial scaffolds. Multipotent mesenchymal stem cells (MSCs) are important sources for cartilage regeneration. Atelocollagen provides a suitable substrate for MSC attachment and enhancing chondrogenic differentiation. Here, we assessed the chondrogenic potential of adipose tissue derived human MSCs (hMSCs) mixed with atelocollagen gel. We observed cell attachment, viability, and microstructures by electron microscopy over 21 days. The levels of Sox9, type II collagen, aggrecan, type I collagen, Runx2, type X collagen, ALP, Osterix, and MMP13 were measured by RT-qPCR. Cartilage matrix-related proteins were assessed by enzyme-linked immunosorbent assay (ELISA), histology, and immunohistochemistry. hMSCs of all groups exhibited well-maintained cell survival, distribution and morphology. Abundant type II collagen fibers developed on day 21; while Sox9, type II collagen, and aggrecan expression increased over time in the atelocollagen group. However, type I collagen, RUNX2, type X collagen (CoL10A1), Osterix, and ALP were not expressed. These results corroborated the protein expression detected by ELISA. Further, histological analysis revealed lacunae-like structures, while staining demonstrated glycosaminoglycan accumulation. Cumulatively, these results indicate that atelocollagen scaffolds improve hMSC chondrogenic differentiation and are a potential approach for cartilage regeneration.

A New Selective PPARγ Modulator Inhibits Triglycerides Accumulation during Murine Adipocytes' and Human Adipose-Derived Mesenchymal Stem Cells Differentiation.

Understanding the molecular basis of adipogenesis is vital to identify new therapeutic targets to improve anti-obesity drugs. The adipogenic process could be a new target in the management of this disease. Our aim was to evaluate the effect of GMG-43AC, a selective peroxisome proliferator-activated receptor γ (PPARγ) modulator, during adipose differentiation of murine pre-adipocytes and human Adipose Derived Stem Cells (hADSCs). We differentiated 3T3-L1 cells and primary hADSCs in the presence of various doses of GMG-43AC and evaluated the differentiation efficiency measuring lipid accumulation, the expression of specific differentiation markers and the quantification of accumulated triglycerides. The treatment with GMG-43AC is not toxic as shown by cell viability assessments after the treatments. Our findings demonstrate the inhibition of lipid accumulation and the significant decrease in the expression of adipocyte-specific genes, such as PPARγ, FABP-4, and leptin. This effect was long lasting, as the removal of GMG-43AC from culture medium did not allow the restoration of adipogenic process. The above actions were confirmed in hADSCs exposed to adipogenic stimuli. Together, these results indicate that GMG-43AC efficiently inhibits adipocytes differentiation in murine and human cells, suggesting its possible function in the reversal of adipogenesis and modulation of lipolysis.

Adipose mesenchymal stem cell transplantation alleviates spinal cord injury-induced neuroinflammation partly by suppressing the Jagged1/Notch pathway

BackgroundThe therapeutic effects of adipose-derived mesenchymal stem cell (ADSC) transplantation have been demonstrated in several models of central nervous system (CNS) injury and are thought to involve the modulation of the inflammatory response. However, the exact underlying molecular mechanism is poorly understood. Activation of the Jagged1/Notch signaling pathway is thought to involve inflammatory and gliotic events in the CNS. Here, we elucidated the effect of ADSC transplantation on the inflammatory reaction after spinal cord injury (SCI) and the potential mechanism mediated by Jagged1/Notch signaling pathway suppression.MethodsTo evaluate the therapeutic effects of ADSC treatment and the potential inhibitory effects of ADSCs on Notch signaling, mice were subjected to contusion SCI, and GFP-labeled ADSCs were injected into the lesion site immediately after the injury. Locomotor function, spinal cord tissue morphology, and the levels of Notch-related proteins and proinflammatory transcripts were compared between groups. To validate the hypothesis that the therapeutic effects of ADSCs are partly due to Notch1 signaling inhibition, a Jagged1 small interfering RNA (siRNA) was injected into the spinal cord to knock down Jagged1/Notch signaling. Neuronal staining and analyses of microglia/macrophage activation and signaling pathways were performed.ResultsWe demonstrated that ADSCs survived in the injured spinal cord for at least 28 days without differentiating into glial or neuronal elements. ADSC treatment resulted in significant downregulation of proinflammatory mediator expression and reduced ionized calcium-binding adapter molecule 1 (IBA1) and ED-1 staining in the injured spinal cord, eventually improving functional recovery. The augmentation of the Jagged1/Notch signaling pathway after SCI was suppressed by ADSC transplantation. The inhibition of the Jagged1/Notch signaling pathway by Jagged1 siRNA resulted in decreases in SCI-induced proinflammatory cytokines and the activation of microglia and an increase in the survival of neurons. Furthermore, Jagged1 knockdown suppressed the phosphorylation of JAK/STAT3 in astrocytes following SCI.ConclusionThe results of this study demonstrated that the therapeutic effects of ADSCs in SCI mice were partly due to Jagged1/Notch signaling pathway inhibition and a subsequent reduction in JAK/STAT3 phosphorylation in astrocytes.

The safety of a therapeutic product composed of a combination of stem cell released molecules from adipose mesenchymal stem cells and fibroblasts.

Aim:We sought to determine the safety profile of a therapeutic candidate composed of the released molecules from a combination of human adipose-derived mesenchymal stem cells and fibroblasts. Although stem cells, their progenitor cells and the molecules that are released from these cells have some demonstrated therapeutic value, much more needs to learn about the efficacy, mechanism of action and the safety profiles of these stem cell-based therapeutics.Methods:A number of cellular, in vitro, in vivo and human studies were performed to analyze cellular, tissue and systemic safety profiles of the combinatorial product.Results:At the levels tested in this study, ranging from demonstrated therapeutic doses to supratherapeutic doses, the combinatorial product demonstrated an excellent safety profile in all in vitro, cellular, tissue and systemic studies.Conclusions:We found evidence that a therapeutic candidate composed of the molecules released from human adipose-derived mesenchymal stem cells and human fibroblasts has an excellent safety profile, and that the product warrants further studies for safety and efficacy where dosing may include topical application, injection and oral application.

Mesenchymal stem cells combined with liraglutide relieve acute lung injury through apoptotic signaling restrained by PKA/\u03b2-catenin

BackgroundARDS and ALI are life-threatening diseases with extremely high mortality in patients. Different sources of MSCs could mitigate the symptoms of ALI from diverse mechanisms. Liraglutide is an activator of glucagon-like peptide-1 receptor (GLP-1R) that activates anti-apoptotic pathways and exerts anti-inflammatory effects. We mainly compared the effects of human chorionic villus-derived mesenchymal stem cells (hCMSCs), human bone marrow-derived mesenchymal stem cells (hBMSCs), and human adipose-derived mesenchymal stem cells (hAMSCs) on the treatment of ALI and explored the apoptosis mechanism of combination MSCs of liraglutide.MethodsThe proliferation of MSCs was detected by MTT assay. Western blot and RT-qPCR were used to detect the expression of GLP-1R, SPC, Ang-1, and KGF in MSCs stimulated by LPS and liraglutide. By using flow cytometry and TUNEL assay to compare the apoptosis of three MSCs under the action of LPS and liraglutide, we selected hCMSCs as the target cells to study the expression of apoptotic protein through the PKA/β-catenin pathway. In ALI animal models, we observed the effects of liraglutide alone, MSCs alone, and MSCs combined with liraglutide by H&E staining, cell counting, immunohistochemistry, and ELISA assay.ResultsWe demonstrated that LPS attenuates the proliferation of the three MSCs and the expression of GLP-1R. Liraglutide could reverse the effects of LPS; increase the expression of SPC, Ang-1, and KGF; and can reduce the apoptosis of three MSCs through the PKA/β-catenin pathway. In the LPS-induced ALI model, MSCs combined with liraglutide showed a significant therapeutic effect, and hCMSCs combined with liraglutide have advantages in the treatment of ALI.ConclusionsThe therapeutic effect of combination MSCs of liraglutide on ALI was higher than that of MSCs alone or liraglutide alone, and liraglutide could alleviate the symptoms of ALI by reducing MSCs apoptosis.

Electrical stimulation confers pre-chondrogenic differentiation by modulating TGF-β1 levels in canine adipose-derived mesenchymal stem cells

Background & Aim Background Osteoarthritis (OA) is closely related to the extent of defective articular cartilage, and the targeting of this loss of chondrocytes has emerged as a promising therapeutic strategy. Aim Here, we investigate that electrical stimulation (ES) can drive direct reprogramming of adipose mesenchymal stem cells (MSCs) into hyaline chondrogenic cells. Methods, Results & Conclusion Methods Canine primary adipose-derived mesenchymal stem cells were formed in micromass for 90 min and stimulated under the electrical field for 3 days. Enzymatic digested ES mass and their 3D control were analyzed through western blot and flow cytometry using chondrogenic markers. In addition, secreted TGF-β1 and TGF-β1 inhibition assay were performed in ES samples. Results We found that TGF-beta1, a key requirement for the in vitro chondrogenic differentiation of MSCs, is highly expressed in micromass of MSCs under ES. TGF-beta1 is also an important regulator of specific cartilage genes during chondrogenesis. Notably, Sox9, a key transcriptional regulator of chondrogenesis, distinctively upregulated in ES-micromass in TGF-beta1 dependent manner. Conclusions We demonstrated that ES-micromass showed the features closely resembling those of chondrocytes in articular cartilage. Further understanding of these insights could serve as the basis for broadening the electrotherapeutic scope for cartilage repair using MSCs.

Exploitation of fibrin-based signaling niche for deriving progenitors from human adipose-derived mesenchymal stem cells towards potential neural engineering applications

Adipose-derived mesenchymal stem cells (hADMSC) retaining proliferation and multi-differentiation potential may support the central nervous system (CNS) regeneration. Multipotency of MSC may result in both desirable and undesirable cells, post-transplantation. A better strategy to attain desired cells may be in vitro commitment of hADMSCs to uni-/bi- potent neural progenitor cells (NPCs), prior to transplantation. Derivation of stable NPCs may require a suitable niche eliciting proliferation and differentiation signals. The present study designed a biomimetic niche comprising insoluble fibrin supported adhesion matrix and exogenously added growth factors (GFs) for deriving different neural cells and established the role of Notch and Wnt signals for proliferation and differentiation of hADMSCs, respectively. The stable transformation of hADMSCs into neurospheres (NS) comprising Nestin+ve NPCs was achieved consistently. Slight modifications of niche enable differentiation of NS to NPCs; NPCs to neurons; NPCs to oligodendrocyte progenitor cells (OPCs); and OPCs to oligodendrocytes (OLG). Fibrin plays a crucial role in the conversion of hADMSC to NS and NPCs to OPCs; but, not essential for OPC to OLG maturation. Co-survival and cell-cell interaction of NPC derived neurons and OPCs promoting OLG maturation is illustrated. The designed biomimetic niche shows the potential for directing autologous ADMSCs to neural cells for applications in regenerative medicine.

Isolation, culture, and induced multiple differentiation of Mongolian sheep adipose-derived mesenchymal stem cells

ABSTRACT Adipose-derived mesenchymal stem cells (ADSC) are adult pluripotent cells and important resources for cell-based therapies of animals. There are presently different kinds of somatic cells used as donor cells for clone successfully. However, studies on somatic cell nuclear transplantation (SCNT) using ADSC as donor cells from Mongolian sheep have not been reported up to now. This study tested optimal methods of isolating, purifying, and proliferating Mongolian sheep ADSC, and determine their multiple differentiation potentiality. Adipose tissue was removed from approximately 2-year-old sheep and ADSC were harvested by pancreatic enzyme decomposition and adherent culture method. The growth curves of the Passages 1, 5, and 10 cultures were plotted and the exponential growth was determined as a population doubling time of 34.1 h. The expression of OCT4, SOX2, and NANOG genes were increased at Passage 3 (P3) as seen by reverse transcription polymerase chain reaction (RT-PCR) analysis. ADSC from Passage 3 were induced to undergo neurogenesis and form cardiomyocytes and pancreatic islet-like cells under inductive environments in vitro. The differentiation properties of cardiomyocytes and islet-like cells were confirmed by histological staining with toluidine blue, periodic acid-Schiff, and dithizone. The expression of specific genes in these cells were also detected by RT-PCR. Our study results confirm that isolated cells were indeed ADSC and may provide valuable materials for somatic cell clone and transgenic research.

In Vitro Anti-cancer Activity of Adipose-Derived Mesenchymal Stem Cells Increased after Infection with Oncolytic Reovirus.

Purpose: Reovirus type 3 Dearing (ReoT3D), a wild type oncolytic virus (OV) from the Reoviridae family, kills KRAS mutant cancer cells. However, the use of OVs has faced with some limitations such as immune responses, and delivery of OVs to the tumor sites in systemic therapy. To solve this, and also to increase the anti-cancer effects of these OVs, mesenchymal stem cells (MSCs) might be used as an effective vehicle for OVs delivery. In this study, we examined the anti-cancer effects of human adipose derived-MSCs (AD-MSCs) as a vehicle of ReoT3D against human glioblastoma cells. Methods: Here, AD-MSCs were characterized and toxicity of ReoT3D on them was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Then, capability of AD-MSCs for virus production was assessed by real-time polymerase chain reaction (PCR), and different in vitro anti-cancer experiments were applied for our anti-cancer purposes.Results: Our results from toxicity assay revealed that the isolated and provoked AD-MSCs were resistant to nontoxic concentration multiplicity of infection (MOI) >1 pfu/cells of ReoT3D. In addition, the results indicated that AD-MSCs were susceptible for virus life cycle complementation and were capable for production of virus progenies. Furthermore, our results showed that AD-MSCs had oncolysis effects and increased the anti-cancer effects of ReoT3D. Conclusion: AD-MSCs as a susceptible host for oncolytic reovirus could increase the anti-cancer activity of this OV against glioblastoma multiforme (GBM) cell line.

Regenerative Potential of Simvastatin on Dental Pulp and adipose-derived stem in immature dog’s teeth: A histologic and radiographic study

Regenerative Potential of Simvastatin on Dental Pulp and adipose-derived stem in immature dog’s teeth: A histologic and radiographic study

PuraMatrix hydrogel enhances the expression of motor neuron progenitor marker and improves adhesion and proliferation of motor neuron-like cells.

Objective(s):Cell therapy has provided clinical applications to the treatment of motor neuron diseases. The current obstacle in stem cell therapy is to direct differentiation of stem cells into neurons in the neurodegenerative disorders. Biomaterial scaffolds can improve cell differentiation and are widely used in translational medicine and tissue engineering. The aim of this study was to compare the efficiency of two-dimensional with a three-dimensional culture system in their ability to generate functional motor neuron-like cells from adipose-derived stem cells. Materials and Methods:We compared motor neuron-like cells derived from rat adipose tissue in differentiation, adhesion, proliferation, and functional properties on two-dimensional with three-dimensional culture systems. Neural differentiation was analyzed by immunocytochemistry for immature (Islet1) and mature (HB9, ChAT, and synaptophysin) motor neuron markers. Results:Our results indicated that the three-dimensional environment exhibited an increase in the number of Islet1. In contrast, two-dimensional culture system resulted in more homeobox gene (HB9), Choline Acetyltransferase (ChAT), and synaptophysin positive cells. The results of this investigation showed that proliferation and adhesion of motor neuron-like cells significantly increased in three-dimensional compared with two-dimensional environments. Conclusion:The findings of this study suggested that three-dimension may create a proliferative niche for motor neuron-like cells. Overall, this study strengthens the idea that three-dimensional culture may mimic neural stem cell environment for neural tissue regeneration.

Differentiation of Adipose-Derived Mesenchymal Stem Cells into Neuron-Like Cells induced by using β-mercaptoethanol

Background: Adipose derived-mesenchymal stem cells have been used as an alternative to bone marrow cells in this study. Objective: We investigated the in vitro isolation, identification, and differentiation of stem cells into neuron cells, in order to produce neuron cells via cell culture, which would be useful in nerve injury treatment. Method: Mouse adipose mesenchymal stem cells were dissected from the abdominal subcutaneous region. Neural differentiation was induced using β-mercaptoethanol. This study included two different neural stage markers, i.e. nestin and neurofilament light-chain, to detect immature and mature neurons, respectively. Results: The immunocytochemistry results showed that the use of β-mercaptoethanol resulted in the successful production of neuron cells. This was attributable to the increase and significant overexpression of the nestin protein during the early exposure period, which resulted in the expression of the highest levels of nestin. In comparison, the expression level of neurofilament light-chain protein also increased with time but less than nestin. Non-treated mesenchymal stem cells, considered as control showed very low expression for both markers. Conclusion: The results of this study indicate that adipose mesenchymal cells represent a good, easily obtainable source of bone marrow cells used to developing the differentiation process.

Bisphenol A enhances adipogenic signaling pathways in human mesenchymal stem cells

BackgroundThe endocrine disruptor Bisphenol-A (BPA), has been involved in dysregulating adipose tissue development and increasing the risk of obesity. The objective of this experiment was to investigate whether treatment of human mesenchymal stem cells with BPA could modulate adipogenesis and adipocyte differentiation.MethodsIn this experimental study, the human adipose-derived mesenchymal stem cells (hASCs) were cultured for 2 weeks with continuous exposure to 10− 10 M or 10− 8 M concentrations of BPA. The extent of triglyceride accumulation was visualized by Oil Red O staining. To evaluate BPA effect on the expression levels of key adipogenic trascripotion factors and proteins, we used Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and ELISA.ResultsThe results presented a dose-dependent triglyceride accumulation in treated cells with BPA. Additionally, we observed that BPA induced transcription of the Peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT-enhancer-binding protein-alpha (C/EBPα), CCAAT-enhancer-binding protein-beta (C/EBPβ), sterol regulatory element-binding protein-1c (SREBP1c), Fatty acid synthase (FASN), and lipoprotein lipase (LPL); BPA suppressed the expression of Fatty acid binding protein-4 (FABP4) and Estrogen receptor-beta (ERβ).ConclusionsOur findings supported the hypothesis that BPA enhances adipogenic differentiation thereby may play a role in development of obesity and dysregulation of metabolic homoeostasis.

Co-culture of mesenchymal stem cells and human umbilical vein endothelial cells on heparinized polycaprolactone/gelatin co-spun nanofibers for improved endothelium remodeling

Endothelization of a tissue-engineered substrate is important for its application as an artificial vascular graft. Despite recent advancements in artificial graft fabrication, a graft of <5 mm is difficult to fabricate owing to insufficient endothelization that results in thrombosis after transplantation. We aimed to perform a co-culture of adipose-derived mesenchymal stem cells (MSCs) with human umbilical vein endothelial cells (HUVECs) on antithrombogenic polycaprolactone (PCL)/heparin-gelatin co-spun nanofibers to evaluate the role of co-culturing in promoting quick endothelization of vascular substrates without surface modification by growth factors or other ECM proteins that trigger the endothelization process. Using a co-axial electrospinning technique, we attempted to fabricate our scaffold balancing between mechanical properties and biocompatibility. Antithrombogenic characteristics were then imparted to the fabricated nanofiber substrate by grafting of heparin. Finally, we performed a co-culture of MSCs and HUVECs on the fabricated co-spun nanofiber substrate to obtain proper endothelization of our material under the in-vitro culture. Staining for CD-31 at seven days of culture revealed enhanced CD-31 expression under the co-culture condition; actin staining revealed healthy cobblestone HUVEC morphology, suggesting that MSCs can aid in proper endothelization. Hence, we conclude that co-culture is effective for quick endothelization of vascular substrates.

Dynamic of miRNA-101a-3p and miRNA-200a during Induction of Osteoblast Differentiation in Adipose-derived Mesenchymal Stem Cells.

miRNAs are known as the cellular phenomena regulators that exert their effects in post-transcriptional level. Recent studies highlight the role of miRNAs in mesenchymal stem cells differentiation into osteoblasts. The purpose of this study was to recognize the pattern of miRNA-101a-3p and miRNA-200a expression during osteoblastic differentiation of human adipose tissue-derived mesenchymal stem cells. The cells were incubated in osteoblastic differentiation medium for a period of 21 days. Alizarin red S staining was performed to confirm the successful differentiation of adipose-derived mesenchymal stem cells into osteoblast cells. The expression levels of miRNA-101a-3p and miRNA-200a were analyzed by real-time PCR during 0, 7, 14, and 21 days after differentiation induction. Data exhibited the increase of extracellular red color deposition which was evident at the end of the incubation period. The expression of miRNA-101a-3p and miRNA-200a was up regulated during adipose-derived mesenchymal stem cells trans-differentiation into osteoblast-like cells. These miRNAs could be potential novel biomarkers for monitoring successful differentiation of mesenchymal stem cells toward osteoblasts.

Fibrin Hydrogel Aided Cardiac Progenitor Cell Delivery Enhances Regenerative Tendency in Myocardial Infarct Model

Attempts to regenerate myocardial infarct (MI) using stem cells have been achieving random success rates. This study considered delivering cardiac progenitors (rCPs), developed from rat adipose-derived mesenchymal stem cells (rADMSCs), into rat MI tissue. The study aimed to employ fibrin-based insoluble matrix as a niche for in vitro differentiation of rADMSCs to rCPs and specific composition of fibrin hydrogel to deliver the cells into MI tissue. PKH26 tracker dye labelled-rCPs/rADMSCs suspended in fibrinogen/thrombin was injected to peri-infarct zone in rat heart created by coronary artery ligation. The MI tissues were harvested after 28d, imaged to locate transplanted cells, and analyzed to detect markers of differentiation, angiogenesis, and immune response. Co-localization of Connexin 43/TroponinT with PKH26 indicated differentiation of transplanted progenitors into cardiomyocyte like cells. Additionally, the presence of CD31+ve cells, fewer CD68+ve cells, and reduced collagen deposition are appreciable in tissues transplanted with rCP in fibrin, as compared to those that received rADMSC. The results obtained indicate that fibrin acts as a favorable niche for in vitro priming of rADMSCs into transplantable lineagecommitted rCPs. The in situ clottable fibrin aided the retention and differentiation of transplanted rCPs into cardiomyocytes. Also by 28d, the tissues showed better angiogenic/ immunomodulatory effects and reduced the calcification tendency.

Daphne mucronata enhances cell proliferation and protects human adipose stem cells against monosodium iodoacetate induced oxidative stress in vitro

ABSTRACT Mesenchymal stem cells (MSCs) are being used to treat many diseases as they exhibit great regenerative potential. However, MSC’s transplantation sometimes does not yield the maximum regenerative outcome as they are unable to survive in inflammatory conditions. Several approaches including preconditioning are used to improve the survival rate of mesenchymal stem cells. One such recently reported approach is preconditioning MSCs with plant extracts. The present study was designed to evaluate the effect of Daphne mucronata extract on stressed human adipose-derived mesenchymal stem cells (hADMSCs). Isolated hADMSCs were preconditioned with different concentrations of Daphne muconata extract and the protective, proliferative, antioxidant and anti-inflammatory effect was assessed through various assays and expression analysis of inflammatory markers regulated through NF-κB pathway. Results suggest that preconditioning hADMSCs with Daphne mucronata increased the cell viability, proliferative and protective potential of hADMSCs with a concomitant reduction in LDH, ROS and elevation in SOD activity. Moreover, both the ELISA and gene expression analysis demonstrated down regulations of inflammatory markers (IL1-β, TNF-α, p65, p50, MMP13) in Daphne mucronata preconditioned hADMSCs as compared to stress. This is the first study to report the use of MIA induced oxidative stress against hADMSC’s and effect of Daphne mucronata on stressed hADMSCs. Results of these studies provided evidence that Daphne mucronata protects the hADMSCs during stress conditions by down regulating the inflammatory markers and hence increase the viability and proliferative potential of hADMSCs that is crucial for transplantation purposes.

Abstract A32: Adipose-derived mesenchymal stem cell has the differentiation/reprogramming capacity towards two distinct cancer-associated fibroblasts

Abstract Cancer-associated fibroblasts (CAFs) are well known of its heterogeneity with distinct phenotypes and functions in tumor progression. CAFs are believed to be composed mainly of resident tissue fibroblast, such as pancreas stellate cells, but bone marrow-derived mesenchymal stem cells (MSC) are also known to be their source (Ishii G et al., Adv Drug Deliv Rev 2016). Herein, we focus on adipose-derived MSC (AD-MSC) as an alternative source of CAF, as one has high reactivity of differentiation and reprogramming to the many cell types (Ronnov-Jessen L et al., J Clin Invest 1995; Sugii/Kida et al., PNAS 2010). It was reported that CAFs include myofibroblastic CAFs (myCAFs), which are found adjacent to tumor cells, and inflammatory CAFs (iCAFs), which localize farther away within the dense stroma (Ohlund, Tuveson et al., J Exp Med 2017). We investigated whether AD-MSC can be a CAF progenitor and differentiated to the different subpopulations of CAFs by employing different culture condition. To evaluate the differentiation capacity, a GFP labeled AD-MSC cell line and the human pancreatic-cancer cell line (Capan-1) were co-cultured in two condition, including direct (DR) co-culture or indirect transwell (TW) co-culture. We then performed qRT-PCR analysis and immunocytochemistry to examine their myCAF marker (ACTA-2, CTGF) or iCAF markers (IL-6, CXCL1, LIF) and compared with those in monocultured AD-MSC. We also analyzed in vitro tumor formation in DR co-culture using time-lapse imaging. In DR co-culture, the morphology of Capan-1 and AD-MSC recapitulated the clinical pancreatic cancer, with those CAF markers expressions. In the course of co-culture, sequential morphologic alterations were observed in AD-MSCs. The AD-MSC with stellate shape changed to large and small spindle-like cells through crosstalk with cancer cells. In sharp contrast, only iCAF marker expression was elevated in TW co-culture. These results collectively suggest that the AD-MSC have a potential to be progenitors of both myCAF and iCAF, explaining the heterogeneity of CAFs under the direct crosstalk with cancer cells. In the presentation, we discuss the differentiation/reprogramming capacity of AD-MSC towards two distinct CAFs (i.e., myCAF and iCAF), focusing on the cell morphology and RNA-seq analysis. Citation Format: Yoshihiro Miyazaki, Yutato Kumagai, Hiroko Kushige, Osamu Shimomura, Yasuyuki Kida, Tatsuya Oda. Adipose-derived mesenchymal stem cell has the differentiation/reprogramming capacity towards two distinct cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A32.

MiR-150 Affects Wnt/β-Catenin Pathway Activity and Regulates MSC Osteogenic Differentiation

The Wnt/β-catenin pathway promotes osteoblast differentiation. Bioinformatics analysis showed a targeting relationship between miR-150 and β-catenin 3′-UTR. This study investigated miR-150's role in β-catenin expression and osteoblast differentiation of Adipose-Derived Mesenchymal Stem Cells (ADMSCs). Differentiation of ADMSCs into osteoblasts was induced and the expression of miR-150, β-catenin, RUNX2 and OPN on the 0th, 9th and 18th day during differentiation was detected. The dual luciferase gene reporter assay validated the relationship between miR-150 and β-catenin. ADMSCs were divided into angomir-NC group and angomir-150 group, and then induced for differentiation for 18 days followed by measuring the level of miR-150, β-catenin, RUNX2 and OPN and the degree of osteoblast differentiation by alizarin red staining. Compared with Day 0, miR-150 expression was gradually decreased on 9th and 18th day of osteoblast differentiation, and the expression of β-catenin, RUNX2 and OPN was gradually increased. There was a targeted regulation relationship between miR-150 and β-catenin. Compared with angomir-NC group, miR-150 expression was significantly increased in angomir-150 transfection group with decreased expression of β-catenin, RUNX2 and OPN protein and reduced degree of alizarin red staining. miR-150 expression is decreased and β-catenin expression is increased during osteoblast differentiation of ADMSCs. Up-regulation of miR-150 expression attenuates the ability of ADMSCs to differentiate into osteoblasts by inhibiting β-catenin expression and Wnt/β-catenin pathway.