Mesenchymal Stem Cell functionalization for enhanced therapeutic applications

To date, the therapeutic efficacy of human mesenchymal stem cells (hMSCs) has been investigated in various clinical trials with moderate or in some cases inconsistent results. The still elusive reproducibility relates in part with constitutive differences in the cell preparation, translated into variable “cell potencies”. Other factors include poor cell homing and survival, and age/disease-associated host tissue impairment. It is well accepted that within in vivo niches MSCs exist as heterogeneous cell populations with different stemness propensities and supportive functions. Phenotype-based MSC purification of homogeneous subsets can result in cell populations with distinct biological functions. In addition, preclinical studies have shown that MSC functionalization in vitro, via cell priming, can boost their immunomodulatory, trophic and reparative capacities in vivo. Therefore, in the present review we discuss how phenotype-based MSC purification and MSC priming technologies can contribute to an improved MSC-based product for safer and more effective therapeutic applications

Human infrapatellar fat pad mesenchymal stem cells show immunomodulatory exosomal signatures

Within the human knee infrapatellar fat pad (IFP) and synovium, resident synoviocytes and macrophages contribute to the onset and progression of inflammatory joint diseases. Our hypothesis is that IFP-derived mesenchymal stem cells (IFP-MSC) robust immunomodulatory therapeutic effects are largely exerted via their exosomal (IFP-MSC EXOs) secretome by attenuating synoviocytes and macrophages pro-inflammatory activation. IFP-MSC EXOs showed distinct miRNA and protein immunomodulatory profiles. Reactome analysis of 24 miRNAs highly present in exosomes showed their involvement in the regulation of six gene groups, including immune system. Exosomes were enriched for immunomodulatory and reparative proteins that are involved in positive regulation of cell proliferation, response to stimulus, signal transduction, signal receptor activity, and protein phosphorylation. Stimulated synoviocytes or macrophages exposed to IFP-MSC EXOs demonstrated significantly reduced proliferation, altered inflammation-related molecular profiles, and reduced secretion of pro-inflammatory molecules compared to stimulated alone. In an acute synovial/IFP inflammation rat model, IFP-MSC EXOs therapeutic treatment resulted in robust macrophage polarization towards an anti-inflammatory therapeutic M2 phenotype within the synovium/IFP tissues. Based on these findings, we propose a viable cell-free alternative to MSC-based therapeutics as an alternative approach to treating synovitis and IFP fibrosis

Enhanced immunomodulatory profile of infrapatellar fat pad (IFP)-derived MSC after inflammatory priming, 3D spheroid culture and CD146 selection: a cellular alternative for bone marrow (BM) in orthopedics

Mesenchymal Stem Cells (MSC) exhibit immunomodulatory properties, making them an alternative to treat inflammation-related musculoskeletal conditions. We have identified a CD146-based subset of BM-MSC with enhanced immunomodulatory effects. IFP-MSC have been proposed as an alternative to BM-MSC. Here, we assessed unfractionated and CD146-selected IFP-MSC's secretory response to inflammation (priming) in 2D and 3D spheroids and functional immunomodulatory effects, all contrasted to BM-MSC. Human BM- and IFP-MSC chondrogenic potential and comparative immunophenotype, growth kinetics and key transcripts before and after inflammatory priming (IFNγ/TNFα) were assessed in 2D. Inflammation-related multiplex secretome was interrogated in 2D and 3D spheroids. Fractionated MSC (CD146Pos &CD146Neg) were generated and naïve and primed cells immunomodulatory transcripts and functional immunopotency assay (IPA) with human PBMCs interrogated in vitro. IFP-MSC showed greater chondrogesis compared with BM-MSC. Both cells showed a similar basal phenotypic profile (CD146 higher in BM-MSC). Inflammatory priming arrested growth only in IFP-MSC, sharply increased CD146, CXCR4, CD10, and CD200 markers (more in IFP-MSC) and significantly up-regulated IL-6, IL-8, IDO, CD10, G-CSF, HLA-G,and ICAM-1genes in both cell types.Secretome analysis revealed a comparative up-regulation of the inflammation-related molecules IL-6sR, IP-10, MCP-2, RANTES and VEGF in both cells; members of IGF family increased only in IFP-MSC; AR and NT3/NT4 up in IFP and down in BM-MSC. 3D spheroids resulted in a sustained increase in CD146 expression and protein presence (IHC), with enhanced inflammation-related secretome in primed IFP-MSC compared with BM-MSC, especially the immunosuppressor IDO. Fractionation both cells types (CD146) further increased IDO expression while reduced pro-inflammatory IL-6 and IL-8 in primed CD146Poscells, more pronounced in IFP-MSC. Finally, primed CD146Poscells strongly abrogated proliferation of activated human PBMCs in a dose-dependent manner, outperforming unfractionated cells at large PBMCs:MSC ratios and CD146Negcells at all doses, suggesting CD146Pos as the actual immunomodulatory subset. Inflammatory priming, spheroid cultures and CD146-enrichment result in a significantly enhanced immunomodulatory properties of human IFP-MSC, thus evolving in a viable alternative for cell-based therapy protocols in Orthopedics