The Secretome of Mesenchymal Stem Cells Prevents Islet Beta Cell Apoptosis via an IL-10-Dependent Mechanism

Background: Type 1 Diabetes Mellitus (T1DM) is partly driven by autoimmune destruction of the pancreatic beta cell, facilitated by the release of inflammatory cytokines, including IFN-γ, TNF-α and IL-1β by cells of the innate immune system. Mesenchymal Stem Cells (MSCs) have been used to counteract autoimmunity in a range of therapeutic settings due to their secretion of trophic and immunomodulatory factors that ameliorate disease independently of the cells themselves. Objective: The aim of this study was to assess the effect of the secretome of human bone-marrow derived MSCs on cytokine-driven beta cell apoptosis. Methods: All experiments were conducted in two insulin-secreting islet cell lines (BRIN-BD11 and βTC1.6) with selected experiments confirmed in primary islets. MSC secretome was generated by conditioning serum-free media (MSC-CM) for 24 hours on sub-confluent MSC populations. The media was then removed and filtered in readiness for use. Results: Exposure to IFN-γ, TNF-α and IL-1β induced apoptosis in cell lines and primary islets. The addition of MSC-CM to cell lines and primary islets partially reversed cytokine-driven apoptosis. MSC-CM also restored glucose-stimulated insulin secretion in cytokine-treated cell lines, which was linked to improved cell viability following from cytokine challenge. Characterization of MSC-CM revealed significant concentrations of IL-4, IL-10, PIGF and VEGF. Of these, IL-10 alone prevented cytokine-driven apoptosis. Furthermore, the inhibition of IL-10 through the addition of a blocking antibody reversed the anti-apoptotic effects of MSC-CM. Conclusion: Overall, the protective effects of MSC-CM on islet beta cell survival appear to be largely IL-10-dependent.

Exploring the Role of Mesenchymal Stem Cells During Normothermic Organ Perfusion: A New Paradigm to Enhance Outcome Following Allograft Transplantation

Background: Normothermic Machine Perfusion (NMP) has been established in the field of solid organ transplantation for both liver and kidney allografts. The ability to perfuse organs at body temperature enables viability assessment as well as optimisation prior to implantation. Discussion: A recent in vitro report of the use of Mesenchymal Stem Cells (MSCs) in the use of a normothermic lung perfusion circuit has raised the possibility of their use in solid organ transplantation. The aim of this short review is to outline the potential uses of bone marrow derived MSCs for their use in renal allograft ex vivo NMP. An overview is provided of current literature of NMP as well as theorised uses for MSCs.

Ocular Stem Cells to Treat Retinal and Corneal Disorders

Background:According to WHO, 285 million people are visually impaired out of which, 39 million are classified as blind and the remaining 246 million people have low vision which comprises of moderate vision impairment and severe vision impairment. Therapies to treat major disorders leading to visual impairment like Age-related Macular Degeneration (AMD), Stargardt’s Disease (STGD), Retinitis Pigmentosa (RP) and corneal scarring are required.In the last decade, many advances have been made to treat these disorders using stem cell therapy. For corneal damage by accidental burns, scarring or limbal stem cell deficiencies which can lead to partial or total blindness, are treated with a risky intervention like keratoplasty. To overcome issues like graft rejection caused by keratoplasty as well as have a better outcome, limbal stem cell therapy has been introduced. Similarly, Retinal Pigment Epithelium (RPE) is a supporting tissue essential in nutrient transport, production of growth factors, phagocytosis of the photoreceptors and retinol cycling.Discussion and Conlusion:Degeneration of this monolayer causes many diseases that have no prevailing treatment; however, research is being carried out to replace this simple epithelial monolayer primarily with an autologous source of cells and currently using stem cells. This review discusses the advances made in the field of ocular stem cell therapy with regards to development, cultivation and novel methods used to deliver these cells to replace the corneal and retinal epithelium as a new standard for treatment.

Upregulation of Adhesion Molecules Sustains Matrix-Free Growth of Human Embryonic Stem Cells

Background: Despite recent advances in culture techniques for undifferentiated human Embryonic Stem Cells (hESCs), further improvements are required to facilitate research and translation of these cells in clinical settings. We have previously derived hESC lines that can be cultured in their undifferentiated state on regular plastic culture dishes, without the need for feeder cells or other coating supports, denoted Matrix-Free Growth hESCs (MFG-hESCs). Objective: In this study, we further characterize and compare MFG-hESCs to hESCs in order to understand the molecular differences responsible for the unique ability of MFG-hESCs. Results: Microarray analysis demonstrated that MFG-hESCs highly resemble feeder-cultured hESCs in global gene expression profile. Two identified groups of genes with differential expression were those encoding for ribosomal proteins and attachment proteins, such as the RGD (Arg-Gly-Asp)-associated proteins. Real-time PCR and flow cytometry corroborated the microarray results. Culture of MFG-hESCs in the presence of RGD peptides resulted in decreased attachment ability compared to cells cultured in the presence of RGES (Arg-Gly-Asp-Ser) peptides. Conclusion: This study demonstrates that MFG-hESC lines overexpress cell attachment proteins but retain the typical characteristics of undifferentiated feeder-cultured hESCs. The ability to culture high-quality pluripotent stem cells in feeder- and matrix-free conditions creates a new opportunities for their large-scale manufacturing for experimental research and translational applications.

Isolation, Culture and Characterization of Cancer Stem Cells from Primary Osteosarcoma

Background:Osteosarcoma cancer stem cells (CSCs) are defined as a subpopulation of osteosarcoma cells, which have the ability of self-renewal, proliferation and differentiation. This study aimed to identify CSCs from human osteosarcomain vitro.Methods:Osteosarcoma CSCs were isolated and cultured with sphere-forming assay technique on an ultra-low well attachment surface plate. After sarcosphere colonies were formed, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the expression of genes of embryonic stem cells such asNANOG, Oct3/4, STAT3 and gene of MSC CD133. Immunofluorescence analysis (IFA) of alkaline phosphatase (ALP), osteocalcin, and CD 133 was also performed to see the expression of osteosarcoma CSC surface protein with immuno-enzymatic staining principle. We also performed alizarin red staining to evaluate calcification in osteosarcoma CSCs.Results:The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies (sarcospheres) in slightly hypoxicand serum free condition which was not attached to the substrate with tight density. RT-PCR demonstrated that sarcospheres expressed genes which encodeNANOG, Oct3/4 STAT 3, but not for CD 133. IFA showed positive protein expression of ALP, osteocalcin and CD 133 which was moderate, strong, and weak positive respectively. Sarcospheres also had a positive reaction toward alizarin red staining.Conclusion:Osteosarcoma CSCs could be isolated from human osteosarcoma by sphere-forming assay technique and characterized by the expression of genes of embryonic stem cells,such asNANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.