Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells

Dysfunctions in adipose tissue cells are responsible for several obesity-related metabolic diseases. Understanding the process of adipocyte formation is thus fundamental for understanding these diseases. The adipocyte differentiation of adipose-derived stem/stromal cells (ADSCs) showed a reduction in the mRNA level of the interleukin 21 receptor (IL21R) during this process. Although the receptor has been associated with metabolic diseases, few studies have examined its function in stem cells. In this study, we used confocal immunofluorescence assays to determine that IL21R colocalizes with mitochondrial protein ATP5B, ALDH4A1, and the nucleus of human ADSCs. We demonstrated that silencing and overexpression of IL21R did not affect the cell proliferation and mitochondrial activity of ADSCs. However, IL21R silencing did reduce ADSC adipogenic capacity. Further studies are needed to understand the mechanism involved between IL21R and the adipogenic differentiation process.

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.

Preclinical Evaluation of the Safety and Immunological Action of Allogeneic ADSC-Collagen Scaffolds in the Treatment of Chronic Ischemic Cardiomyopathy

The use of allogeneic adipose-derived mesenchymal stromal cells (alloADSCs) represents an attractive approach for treating myocardial infarction (MI). Furthermore, adding a natural support improves alloADSCs engraftment and survival in heart tissues, leading to a greater therapeutic effect. We aimed to examine the safety and immunological reaction induced by epicardial implantation of a clinical-grade collagen scaffold (CS) seeded with alloADSCs for its future application in humans. Thus, cellularized scaffolds were myocardially or subcutaneously implanted in immunosuppressed rodent models. The toxicological parameters were not significantly altered, and tumor formation was not found over the short or long term. Furthermore, biodistribution analyses in the infarcted immunocompetent rats displayed cell engraftment in the myocardium but no migration to other organs. The immunogenicity of alloADSC-CS was also evaluated in a preclinical porcine model of chronic MI; no significant humoral or cellular alloreactive responses were found. Moreover, CS cellularized with human ADSCs cocultured with human allogeneic immune cells produced no alloreactive response. Interestingly, alloADSC-CS significantly inhibited lymphocyte responses, confirming its immunomodulatory action. Thus, alloADSC-CS is likely safe and does not elicit any alloreactive immunological response in the host. Moreover, it exerts an immunomodulatory action, which supports its translation to a clinical setting.

The Identification of HSA-MIR-17-5P Existence in the Exosome of Adipose-Derived Stem Cells and Adipocytes

MicroRNAs (miRNAs) have ability to down-regulate gene expressions. hsa-miR-17-5p, has been confirmed as an oncogene or tumor suppressor. However, the existence on human adipose-derived stem cells (ADSCs) or adipocytes, is still unclear. Many researchers emphasizing the role of hsa-miR-17-5p on cellular senescence, aging and cancer, but not specific on the expression in the exosome of human ADSCs and adipocytes. The primary ADSCs were derived from subcutaneous adipose tissue of pregnant woman during elective cesarean operation, then processed by combining conventional and enzymatic methods. Adipocytes were differentiated by using the StemPro Adipogenesis Differentiation kit® and Oil Red-O staining. Exosomes were isolated using Exosome Purification and RNA Isolation kit® and were characterized by scanning electron microscope. The markers, CD34 and CD44, were identified and analyzed by using FACS analysis. Subsequently, microRNA was extracted and observed for hsa-miR-17-5p expression. This study showed that ADSCs and adipocytes were proved to express CD34+ and CD44+. The hsa-miR-17-5p were also detected in both the exosome of ADSCs and adipocytes. Although the source of the ADSCs was from pregnant woman, the characteristic was similar with the ones from non-pregnant woman. Our study also supports the questionable existence of CD34 in ADSCs. Having confirmed the characteristics, we proved that the exosomes of ADSCs and adipocytes expressed similar hsa-miR-17-5p despite they are from phenotypically different cell types and may have distinct roles. However, further research steps should be done in the future to verify the role of hsa-miR-17-5p towards senescent cell and ADSC differentiation.

O49: EFFECT OF AFIMOXIFENE (Z-4-HYDROXYTAMOXIFEN) ON ADIPOSE-DERIVED STEM CELLS

Background Adipose-derived stem cells (ADSCs) are a promising cell source for adipose tissue engineering. Currently, novel breast reconstruction techniques using ADSCs are actively being explored. Systemic chemo- & hormonal therapy may impede tissue regeneration in breast cancer patients. However, the effect of hormonal therapy on ADSCs and their regenerative capabilities is poorly understood. Aims The current study aims to analyse the effect of Afimoxifene, an active metabolite of Tamoxifen, on proliferation and viability of ADSCs in vitro Method Lipoaspirates or adipose tissue were obtained with informed consent from breast cancer patients and healthy controls. The isolated ADSCs were subjected to single or multiple dose(s) of Z-4-Hydroxytamoxifen (12.5nM, 25nM, 50nM, 100nM and 1μM) and, analyzed on days 1, 3 and 5, using CellTitre 96 ® AQueous Cell proliferation assay. T47D and MDA-MB-231 cell lines were used as positive and negative controls, respectively. Result ADSCs were obtained from a total of 3 patients. ADSCs were isolated from a cancer patient with/without active disease at the time of sample procurement and healthy subjects opting for elective cosmetic procedures. Morphology, CFU assay and adipogenic differentiation assay were used to validate stem cell population. Afimoxifene demonstrated no statistically significant reduction in the viability or proliferation of the ADSCs, irrespective of the dose- or time-dependent exposure or cancer status (p >0.05). Conclusion Afimoxifene has no deleterious effect on viability or proliferation of human ADSCs up to 1μM concentration. Key words Adipose-derived stem cells, Afimoxifene, Breast reconstruction, Hormone therapy, Tamoxifen. Take-home message Afimoxifene has no deleterious effect on viability or proliferation of human ADSCs up to 1μM concentration

Based on a Self-Feeder Layer, a Novel 3D Culture Model of Human ADSCs Facilitates Trans-Differentiation of the Spheroid Cells into Neural Progenitor-Like Cells Using siEID3 with a Laminin/Poly-d-lysine Matrix

Human adipose-derived stromal cells (ADSCs) are receiving unprecedented attention as a potential cellular source for regenerative medicine-based therapies against various diseases and conditions. However, there still have significant issues concerning the translational development of ADSC-based therapies, such as its heterogeneity and being prone to aging. We developed a new simple and economical 3D semi-suspended expansion method in which 3D spheroids reside on an ADSC-derived self-feeder cell layer, producing cells with increased population homogeneity and strong stemness and ensuring that the proliferation and differentiation potency of the cells does not become notably reduced after at least ten passages in culture. To check the potential application of the 3D ADSC spheroids, we discovered that the combination of siEID3, which is a small interfering RNA of EP300 inhibitor of differentiation 3 (EID3), and laminin/poly-d-lysine matrix can rapidly result in trans-differentiation of the 3D spheroid cells to neural progenitor-like cells (NPLCs) in approximately 9 days in vitro. This approach provides a multidisciplinary tool for stem cell research and production in mesenchymal stem cell-related fields.

Effect of Mineral Pitch on the Proliferation of Human Adipose Derived Stem Cells on Acellular Scaffold

Purpose : Acellular scaffold extracted from extracellular matrix (ECM) have been used for constructive and regenerative medicine. Adipose derived stem cells (ADSCs) can enhance the vascularization capacity of scaffolds. High mobility group box 1 (HMGB1) and stromal derived factor1 (SDF1) are considered as two important factors in vascularization and immunologic system. In this study, the effect of mineral pitch on the proliferation of human ADSCs was evaluated. In addition to HMGB1 and SDF1, factors expression in acellular scaffold was also assessed. Methods: To determine acellular scaffold morphology and the degree of decellularization, hematoxylin & eosin (H&E), 6-diamidino-2-phenylindole (DAPI), and Masson’s trichrome staining were applied. The scaffolds were treated with mineral pitch. Also, ADSCs were seeded on the scaffolds, and adhesion of the cells to the scaffolds were assessed using field emission scanning electron microscopy (FE-SEM). In addition, the efficiency of mineral pitch to induce the proliferation of ADSCs on the scaffolds was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. To measure HMGB1 and SDF1 mRNA expression, real-time polymerase chain reactions (RT-PCR) was used. Results: FE-SEM showed that decellularized matrix possesses similar matrix morphology with a randomly oriented fibrillar structure and interconnecting pores. No toxicity was observed in all treatments, and cell proliferation were supported in scaffolds. The important point is that, the proliferation capacity of ADSCs on Mineral pitch loaded scaffolds significantly increased after 48 h incubation time compared to the unloaded scaffold (P<0.001). Conclusion: The results of this study suggest that mineral pitch has potentials to accelerate proliferation of ADSCs on the acellular scaffolds.

Harvest and Culture of Porcine Adipose-Derived Mesenchymal Stem Cells for Autologous Transplantation

ImportanceTransplantation of adipose-derived (mesenchymal) stem cells (ADSCs) are currently under investigation for numerous novel regenerative cell therapies of the head, neck and periphery. Critical to the development of these techniques is the availability of large-animal models that can be used to test the safety and efficacy of these approaches in a manner that provides source material (in this case MSC) analogous to those developed in humans.ObjectiveTo describe the surgical technique and laboratory procedures for harvesting and isolating porcine ADSCs that are functionally equivalent to human ADSCs without sacrificing the donor animal.MethodsThe reagents and methods used in the porcine model described were purposefully focused to be able to be sufficiently analogous to those used in humans such that data developed using these techniques should support the use of porcine models for regulatory submissions.ResultsWe describe a method and confirm the activity of functionally analogous adipose derived porcine MSC. Two conditions were critical to move gain analogous performance: the cells needed to be incubated at porcine body temperature (39°C) and the cells were more sensitive to initial plating densities with plating densities of 20,000 cells/cm2 being optimal.DiscussionThis approach will allow reproducible and predictable use of an autologous large-animal model for testing AMDSC therapies.