Characterization and response to inflammatory stimulation of human endometrial-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: The human endometrium has emerged as an attractive source of endometrial-derived mesenchymal stem/stromal cells (eMSCs) that can be easily isolated by non-invasive procedures. The prominent capacity of the endometrium for efficient and scarless regeneration each menstrual cycle indicates the increased eMSC immunomodulatory and pro-angiogenic properties. Herein the authors investigated the molecular responses of eMSCs to an inflammatory environment and whether those intrinsic responses affected their functional attributes. METHODS: Human eMSCs immunophenotypic, transcriptional and secretory profiles were evaluated at passage three (P3) and passage eight (P8) to determine culture effects. Functionally, P3 and P8 non-induced and TNF-α/IFN-γ-induced eMSCs were interrogated for their capacity to suppress stimulated peripheral blood mononuclear cell (PBMC) proliferation, whereas non-induced eMSCs were assessed for their support to vascular network formation in co-cultures with human umbilical vein endothelial cells in vitro. RESULTS: Non-induced P3 and P8 eMSCs exhibited similar spindle-shaped morphology and clonogenic capacity. Nevertheless, P8 eMSCs showed reduced growth rate capacity and telomere length. The eMSCs displayed the typical MSC-related immunophenotypic profile, with P3 and P8 eMSCs expressing high levels (>98%) of CD140ß, intermediate levels (35-60%) of CD146 and SUSD2 and low levels (∼8%) of NG2 pericytic markers. Non-induced P3 and P8 showed similar transcriptional and secretory profiles, though the expression of immunomodulatory HLA-G and IL-8 genes was significantly downregulated in P8 compared with P3 eMSCs. Upon TNF-α/IFN-γ induction, eMSCs showed an immunophenotypic profile similar to that of non-induced eMSCs, except for significant upregulation of HLA-DR protein expression in both induced P3 and P8 eMSCs. However, induced P3 and P8 eMSCs showed significant upregulation of CD10, HLA-G, IDO, IL-6, IL-8, LIF and TSG gene expression compared with non-induced cultures. TNF-α/IFN-γ induction strongly increased the secretion of inflammatory-/angiogenesis-related molecules, whereas growth factor secretion was similar to the non-induced eMSCs. Functionally, P3 and P8 eMSCs showed a strong inhibitory effect on stimulated PBMC proliferation and the capacity to support neovascularization in vitro. CONCLUSIONS: The authors' study suggests that serial expansion does not affect eMSC immunophenotypic, transcriptional and secretory profiles. This is directly reflected by the functional immunomodulatory and pro-angiogenic properties of eMSCs, which remain unaltered until P8 in vitro. However, exposure of eMSCs to inflammatory environments enhances their immunomodulatory transcriptional and inflammatory-/angiogenesis-related secretory profiles. Therefore, the resulting evidence of eMSCs serial expansion and exposure to inflammation could serve as a foundation for improved eMSCs manufacturing and potential clinical translation efforts.

Regulatory-compliant conditions during cell product manufacturing enhance in vitro immunomodulatory properties of infrapatellar fat pad-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cell (MSC)-based therapies have gained attention as potential alternatives for multiple musculoskeletal indications based on their trophic and immunomodulatory properties. The infrapatellar fat pad (IFP) serves as a reservoir of MSCs, which play crucial roles modulating inflammatory and fibrotic events at the IFP and its neighboring tissue, the synovium. In an effort to comply with the existing regulatory framework regarding cell-based product manufacturing, we interrogated the in vitro immunomodulatory capacity of human-derived IFP-MSCs processed under different conditions, including a regulatory-compliant protocol, in addition to their response to the inflammatory and fibrotic environments often present in joint disease. METHODS: Immunophenotype, telomere length, transcriptional and secretory immunomodulatory profiles and functional immunopotency assay were assessed in IFP-MSCs expanded in regular fetal bovine serum (FBS)-supplemented medium and side-by-side compared with same-donor cells processed with two media alternatives (i.e., regulatory-compliant pooled human platelet lysate [hPL] and a chemically reinforced/serum-reduced [Ch-R] formulation). Finally, to assess the effects of such formulations on the ability of the cells to respond to pro-inflammatory and pro-fibrotic conditions, all three groups were stimulated ex vivo (i.e., cell priming) with a cocktail containing TNFα, IFNγ and connective tissue growth factor (tumor-initiating cells) and compared with non-induced cohorts assessing the same outcomes. RESULTS: Non-induced and primed IFP-MSCs expanded in either hPL or Ch-R showed distinct morphology in vitro, similar telomere dynamics and distinct phenotypical and molecular profiles when compared with cohorts grown in FBS. Gene expression of IL-8, CD10 and granulocyte colony-stimulating factor was highly enriched in similarly processed IFP-MSCs. Cell surface markers related to the immunomodulatory capacity, including CD146 and CD10, were highly expressed, and secretion of immunomodulatory and pro-angiogenic factors was significantly enhanced with both hPL and Ch-R formulations. Upon priming, the immunomodulatory phenotype was enhanced, resulting in further increase in CD146 and CD10, significant CXCR4 presence and reduction in TLR3. Similarly, transcriptional and secretory profiles were enriched and more pronounced in IFP-MSCs expanded in either hPL or Ch-R, suggesting a synergistic effect between these formulations and inflammatory/fibrotic priming conditions. Collectively, increased indoleamine-2,3-dioxygenase activity and prostaglandin E2 secretion for hPL- and Ch-R-expanded IFP-MSCs were functionally reflected by their robust T-cell proliferation suppression capacity in vitro compared with IFP-MSCs expanded in FBS, even after priming. CONCLUSIONS: Compared with processing using an FBS-supplemented medium, processing IFP-MSCs with either hPL or Ch-R similarly enhances their immunomodulatory properties, which are further increased after exposure to an inflammatory/fibrotic priming environment. This evidence supports the adoption of regulatory-compliant practices during the manufacturing of a cell-based product based on IFP-MSCs and anticipates a further enhanced response once the cells face the pathological environment after intra-articular administration. Mechanistically, the resulting functionally enhanced cell-based product has potential utilization as a novel, minimally invasive cell therapy for joint disease through modulation of local immune and inflammatory events.

CD146+ Endometrial-Derived Mesenchymal Stem/Stromal Cell Subpopulation Possesses Exosomal Secretomes with Strong Immunomodulatory miRNA Attributes.

The perivascular localization of endometrial mesenchymal stem/stromal cells (eMSC) allows them to sense local and distant tissue damage, promoting tissue repair and healing. Our hypothesis is that eMSC therapeutic effects are largely exerted via their exosomal secretome (eMSC EXOs) by targeting the immune system and angiogenic modulation. For this purpose, EXOs isolated from Crude and CD146+ eMSC populations were compared for their miRNA therapeutic signatures and immunomodulatory functionality under inflammatory conditions. eMSC EXOs profiling revealed 121 in Crude and 88 in CD146+ miRNAs, with 82 commonly present in both populations. Reactome and KEGG analysis of miRNAs highly present in eMSC EXOs indicated their involvement among others in immune system regulation. From the commonly present miRNAs, four miRNAs (hsa-miR-320e, hsa-miR-182-3p, hsa-miR-378g, hsa-let-7e-5p) were more enriched in CD146+ eMSC EXOs. These miRNAs are involved in macrophage polarization, T cell activation, and regulation of inflammatory cytokine transcription (i.e., TNF-α, IL-1ß, and IL-6). Functionally, stimulated macrophages exposed to eMSC EXOs demonstrated a switch towards an alternate M2 status and reduced phagocytic capacity compared to stimulated alone. However, eMSC EXOs did not suppress stimulated human peripheral blood mononuclear cell proliferation, but significantly reduced secretion of 13 pro-inflammatory molecules compared to stimulated alone. In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially upon CD146+ eMSC EXO exposure. Our study suggests that eMSC, and even more, the CD146+ subpopulation, possess exosomal secretomes with strong immunomodulatory miRNA attributes. The resulting evidence could serve as a foundation for eMSC EXO-based therapeutics for the resolution of detrimental aspects of tissue inflammation.

Umbilical cord mesenchymal stem cells for COVID-19 acute respiratory distress syndrome: A double-blind, phase 1/2a, randomized controlled trial.

Acute respiratory distress syndrome (ARDS) in COVID-19 is associated with high mortality. Mesenchymal stem cells are known to exert immunomodulatory and anti-inflammatory effects and could yield beneficial effects in COVID-19 ARDS. The objective of this study was to determine safety and explore efficacy of umbilical cord mesenchymal stem cell (UC-MSC) infusions in subjects with COVID-19 ARDS. A double-blind, phase 1/2a, randomized, controlled trial was performed. Randomization and stratification by ARDS severity was used to foster balance among groups. All subjects were analyzed under intention to treat design. Twenty-four subjects were randomized 1:1 to either UC-MSC treatment (n = 12) or the control group (n = 12). Subjects in the UC-MSC treatment group received two intravenous infusions (at day 0 and 3) of 100 ± 20 × 106 UC-MSCs; controls received two infusions of vehicle solution. Both groups received best standard of care. Primary endpoint was safety (adverse events [AEs]) within 6 hours; cardiac arrest or death within 24 hours postinfusion). Secondary endpoints included patient survival at 31 days after the first infusion and time to recovery. No difference was observed between groups in infusion-associated AEs. No serious adverse events (SAEs) were observed related to UC-MSC infusions. UC-MSC infusions in COVID-19 ARDS were found to be safe. Inflammatory cytokines were significantly decreased in UC-MSC-treated subjects at day 6. Treatment was associated with significantly improved patient survival (91% vs 42%, P = .015), SAE-free survival (P = .008), and time to recovery (P = .03). UC-MSC infusions are safe and could be beneficial in treating subjects with COVID-19 ARDS.