2D and 3D cultured human umbilical cord-derived mesenchymal stem cell-conditioned medium has a dual effect in type 1 diabetes model in rats: immunomodulation and beta-cell regeneration

Abstract

Background Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disease characterized by the irreversible destruction of insulin-producing β-cells in pancreatic islets. Helper and cytotoxic T-cells and cytokine production, which is impaired by this process, take a synergetic role in β-cell destruction, and hyperglycemia develops due to insulin deficiency in the body. Mesenchymal stem cells (MSCs) appear like an excellent therapeutic tool for autoimmune diseases with pluripotent, regenerative, and immunosuppressive properties. Paracrine factors released from MSCs play a role in immunomodulation by increasing angiogenesis and proliferation and suppressing apoptosis. In this context, the study aims to investigate the therapeutic effects of MSC’s secretomes by conditioned medium (CM) obtained from human umbilical cord-derived MSCs cultured in 2-dimensional (2D) and 3-dimensional (3D) environments in the T1D model. Methods First, MSCs were isolated from the human umbilical cord, and the cells were characterized. Then, two different CMs were prepared by culturing MSCs in 2D and 3D environments. The CM contents were analyzed in terms of total protein, IL-4, IL-10, IL-17, and IFN-λ. In vivo studies were performed in Sprague-Dawley-type rats with an autoimmune T1D model, and twelve doses of CM were administered intraperitoneally for 4 weeks within the framework of a particular treatment model. In order to evaluate immunomodulation, the Treg population was determined in lymphocytes isolated from the spleen after sacrification, and IL-4, IL-10, IL-17, and IFN-λ cytokines were analyzed in serum. Finally, β-cell regeneration was evaluated immunohistochemically by labeling Pdx1, Nkx6.1, and insulin markers, which are critical for the formation of β-cells. Results Total protein and IL-4 levels were higher in 3D-CM compared to 2D-CM. In vivo results showed that CMs induce the Treg population and regulate cytokine release. When the immunohistochemical results were evaluated together, it was determined that CM application significantly increased the rate of β-cells in the islets. This increase was at the highest level in the 3D-CM applied group. Conclusion The dual therapeutic effect of MSC-CM on immunomodulation and homeostasis/regeneration of β-cells in the T1D model has been demonstrated. Furthermore, this effect could be improved by using 3D scaffolds for culturing MSCs while preparing CM.

Investigation of conditioned medium properties obtained from human umbilical cord mesenchymal stem/stromal cells preconditioned with dimethyloxalylglycine in a correlation with ultrastructural changes

“…Preconditioning MSCs through hypoxia, chemical agents, trophic factors, cytokines, and physical factors is crucial to improving MSC function in vitro and in vivo (Ferreira et al, 2018;Isildar et al, 2022).…”

Therapeutic Potential of Mesenchymal Stem Cell‐Derived Conditioned Medium for Diabetes Mellitus and Related Complications

“…[42] These proteins are predominantly produced by cells within the human innate and adaptive immune systems and are categorized into pro-inflammatory and antiinflammatory classes. [43] Studies have shown that MSCs secrete anti-inflammatory cytokines, including transforming growth factor 𝛽1 (TGF𝛽1), [44][45][46][47] IL4, [48] IL10, [49][50][51] IL27, [51] IL1Ra, [52,53] as well as pro-inflammatory cytokines, including IL6 [47,54,55] and IL8. [21] Nevertheless, considering the available studies, the question of which of these cytokines are secreted by MSCs isolated from different sources and how effectively they are secreted without external stimuli remains a matter of debate and needs further clarification.…”

“…[ 56 ] This is because there are studies indicating that cytokines are absent or observed at deficient levels in MSC secretome under normal conditions. [ 48,57 ] Indeed, it is widely acknowledged that the secretion of cytokines from MSCs notably rises in response to inflammatory circumstances. [ 58 ] Furthermore, research has revealed that the production of cytokines improves in the presence of external stimuli such as hypoxia and 3D culture conditions.…”

“…These results suggest that UC‐MSC‐CM is effective in the context of immunomodulation and β‐cell regeneration in the T1D model, and this effect could be enhanced by using 3D scaffolds. [ 48 ] Although studies are still in their infancy, the MSC‐CM application appears to be a promising option for T1D. In addition, it is predicted that MSC‐CMs may delay the onset of diabetes‐related complications by showing protective effects on other tissues and organs, as well as their positive effects on immunomodulation and β‐cell regeneration.…”

Pretreated Mesenchymal Stem Cells and Their Secretome: Enhanced Immunotherapeutic Strategies

“…MSCs mainly exhibit their effects due to the expression of various regulatory factors participating in intercellular communication, including cytokines, immune-regulating factors, chemokines, growth factors, nucleic acids, lipids and extracellular vesicles (EVs) in the conditioned media (CM), collectively known as the secretome of MSCs, which is crucial to the regulation of key biological processes [ 14 , 17 , 18 ]. Using the MSC-derived secretome has the following advantages: (1) It avoids potential safety risks associated with cells transplantation, and so far, no safety issues have been reported in secretome; (2) it offers evaluation of dose, potency and safety which is similar to that of traditional medicine; (3) it allows long-term storage under frozen or freeze-dried conditions without losing biological activity; (4) customized cells lines can be used to produce a large quantity of secretome in controlled laboratory conditions without the need to consider which tissue or donor MSCs should be used; (5) pretreatment of MSCs can significantly increase the content of secretome, with relevant therapeutic effects and disease-specific concerns; (6) the regulation of complex immune response requires the synergistic action of various factors, and the use of a single factor cannot reproduce the immunoregulatory effects of MSCs, so the direct use of secretome avoids component screening [ 19 , 20 , 21 , 22 , 23 ].…”