From Primary MSC Culture of Adipose Tissue to Immortalized Cell Line Producing Cytokines for Potential Use in Regenerative Medicine Therapy or Immunotherapy.

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors.

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds-In Vitro Study.

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

Can supernatant from immortalized adipose tissue MSC replace cell therapy? An in vitro study in chronic wounds model.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical practice, post-culture MSC supernatant treatment could be a more attractive alternative to autologous stem cell transplantation. In this study, we compared the regenerative properties of supernatants harvested from four newly established human adipose tissue mesenchymal stem cell lines (HATMSCs) derived from chronic wound patients or healthy donors. METHODS: HATMSC supernatants were produced in a serum-free medium under hypoxia and their content was analyzed by a human angiogenesis antibody array. The regenerative effect of HATMSCs supernatants was investigated in an in vitro model of chronic wound, where cells originating from human skin, such as microvascular endothelial cells (HSkMEC.2), keratinocytes (HaCaT), and fibroblasts (MSU-1.1), were cultured in serum-free and oxygen-reduced conditions. The effect of supernatant treatment was evaluated using an MTT assay and light microscopy. In addition, fibroblasts and HATMSCs were labeled with PKH67 and PKH26 dye, respectively, and the effect of supernatant treatment was compared to that obtained when fibroblasts and HATMSCs were co-cultured, using flow cytometry and fluorescent microscopy. RESULTS: A wide panel of angiogenesis-associated cytokines such as angiogenin, growth-regulated oncogene (GRO), interleukin-6 and 8 (IL-6, IL-8), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), and matrix metalloproteinase (MMP) were found in all tested HATMSCs supernatants. Moreover, supernatant treatment significantly enhanced the survival of fibroblasts, endothelial cells, and keratinocytes in our chronic wound model in vitro. Importantly, we have shown that in in vitro settings, HATMSC supernatant treatment results in superior fibroblast proliferation than in the case of co-culture with HATMSCs. CONCLUSIONS: Our results suggest that therapy based on bioactive factors released by the immortalized atMSC into supernatant has important effect on skin-derived cell proliferation and might preclude the need for a more expensive and difficult cell therapy approach to improve chronic wound healing.

Assembly of protein-based hollow spheres encapsulating a therapeutic factor.

Neurotrophins, as important regulators of neural development, function, and survival, have a therapeutic potential to repair damaged neurons. However, a controlled delivery of therapeutic molecules to injured tissue remains one of the greatest challenges facing the translation of novel drug therapeutics field. This study presents the development of an innovative protein-protein delivery technology of nerve growth factor (NGF) by an electrostatically assembled protein-based (collagen) reservoir system that can be directly injected into the injury site and provide long-term release of the therapeutic. A protein-based biomimetic hollow reservoir system was fabricated using a template method. The capability of neurotrophins to localize in these reservoir systems was confirmed by confocal images of fluorescently labeled collagen and NGF. In addition, high loading efficiency of the reservoir system was proven using ELISA. By comparing release profile from microspheres with varying cross-linking, highly cross-linked collagen spheres were chosen as they have the slowest release rate. Finally, biological activity of released NGF was assessed using rat pheochromocytoma (PC12) cell line and primary rat dorsal root ganglion (DRG) cell bioassay where cell treatment with NGF-loaded reservoirs induced significant neuronal outgrowth, similar to that seen in NGF treated controls. Data presented here highlights the potential of a high capacity reservoir-growth factor technology as a promising therapeutic treatment for neuroregenerative applications and other neurodegenerative diseases.

InterfERing with endoplasmic reticulum stress.

Stress to the endoplasmic reticulum (ER) is a recognized factor in Alzheimer's and Parkinson's diseases, diabetes, heart disease, liver disorders and cancer. Thus, drugs that interfere with ER stress have wide therapeutic potential. Here we review the effects of drugs on three arms of ER stress: the protein kinase RNA-activated (PKR)-like ER kinase (PERK) arm, the activated transcription factor 6 (ATF6) arm and the inositol-requiring enzyme 1 (IRE1) arm. Drugs fall into five groups: (i) compounds directly binding to ER stress molecules; (ii) chemical chaperones; (iii) inhibitors of protein degradation; (iv) antioxidants; (v) drugs affecting calcium signaling. Treatments are generally inhibitory and lead to increased viability, except when applied to cancer cells. A focus on interfering with the ATF6 arm is required, and more in vivo testing of these compounds concurrently across all three arms is needed if the full importance of ER stress to human disease is to be realized.