Extracellular matrices of stromal cell subtypes regulate phenotype and contribute to the stromal microenvironment in vivo.

BACKGROUND: Bone marrow stromal cells (BMSCs) are highly heterogeneous, which may reflect their diverse biological functions, including tissue maintenance, haematopoietic support and immune control. The current understanding of the mechanisms that drive the onset and resolution of heterogeneity, and how BMSCs influence other cells in their environment is limited. Here, we determined how the secretome and importantly the extracellular matrix of BMSCs can influence cellular phenotype. METHODS: We used two immortalised clonal BMSC lines isolated from the same heterogeneous culture as model stromal subtypes with distinct phenotypic traits; a multipotent stem-cell-like stromal line (Y201) and a nullipotent non-stem cell stromal line (Y202), isolated from the same donor BMSC pool. Label-free quantitative phase imaging was used to track cell morphology and migration of the BMSC lines over 96 h in colony-forming assays. We quantified the secreted factors of each cell line by mass spectrometry and confirmed presence of proteins in human bone marrow by immunofluorescence. RESULTS: Transfer of secreted signals from a stem cell to a non-stem cell resulted in a change in morphology and enhanced migration to more closely match stem cell-like features. Mass spectrometry analysis revealed a significant enrichment of extracellular matrix (ECM) proteins in the Y201 stem cell secretome compared to Y202 stromal cells. We confirmed that Y201 produced a more robust ECM in culture compared to Y202. Growth of Y202 on ECM produced by Y201 or Y202 restored migration and fibroblastic morphology, suggesting that it is the deficiency of ECM production that contributes to its phenotype. The proteins periostin and aggrecan, were detected at 71- and 104-fold higher levels in the Y201 versus Y202 secretome and were subsequently identified by immunofluorescence at rare sites on the endosteal surfaces of mouse and human bone, underlying CD271-positive stromal cells. These proteins may represent key non-cellular components of the microenvironment for bona-fide stem cells important for cell maintenance and phenotype in vivo. CONCLUSIONS: We identified plasticity in BMSC morphology and migratory characteristics that can be modified through secreted proteins, particularly from multipotent stem cells. Overall, we demonstrate the importance of specific ECM proteins in co-ordination of cellular phenotype and highlight how non-cellular components of the BMSC microenvironment may provide insights into cell population heterogeneity and the role of BMSCs in health and disease.

Corrigendum: CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

[This corrects the article DOI: 10.3389/fimmu.2022.903796.].

CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Self-assembled gel tubes, filaments and 3D-printing with in situ metal nanoparticle formation and enhanced stem cell growth.

This paper reports simple strategies to fabricate self-assembled artificial tubular and filamentous systems from a low molecular weight gelator (LMWG). In the first strategy, tubular 'core-shell' gel structures based on the dibenzylidenesorbitol-based LMWG DBS-CONHNH2 were made in combination with the polymer gelator (PG) calcium alginate. In the second approach, gel filaments based on DBS-CONHNH2 alone were prepared by wet spinning at elevated concentrations using a 'solvent-switch' approach. The higher concentrations used in wet-spinning prevent the need for a supporting PG. Furthermore, this can be extended into a 3D-printing method, with the printed LMWG objects showing excellent stability for at least a week in water. The LMWG retains its unique ability for in situ precious metal reduction, yielding Au nanoparticles (AuNPs) within the tubes and filaments when they are exposed to AuCl3 solutions. Since the gel filaments have a higher loading of DBS-CONHNH2, they can be loaded with significantly more AuNPs. Cytotoxicity and viability studies on human mesenchymal stem cells show that the DBS-CONHNH2 and DBS-CONHNH2/alginate hybrid gels loaded with AuNPs are biocompatible, with the presence of AuNPs enhancing stem cell metabolism. Taken together, these results indicate that DBS-CONHNH2 can be shaped and 3D-printed, and has considerable potential for use in tissue engineering applications.

Self-assembled low-molecular-weight gelator injectable microgel beads for delivery of bioactive agents.

We report the preparation of hybrid self-assembled microgel beads by combining the low molecular weight gelator (LMWG) DBS-CONHNH2 and the natural polysaccharide calcium alginate polymer gelator (PG). Microgel formulations based on LMWGs are extremely rare due to the fragility of the self-assembled networks and the difficulty of retaining any imposed shape. Our hybrid beads contain interpenetrated LMWG and PG networks, and are obtained by an emulsion method, allowing the preparation of spherical gel particles of controllable sizes with diameters in the mm or µm range. Microgels based on LMWG/alginate can be easily prepared with reproducible diameters <1 µm (ca. 800 nm). They are stable in water at room temperature for many months, and survive injection through a syringe. The rapid assembly of the LMWG on cooling plays an active role in helping control the diameter of the microgel beads. These LMWG microbeads retained the ability of the parent gel to deliver the bioactive molecule heparin, and in cell culture medium this enhanced the growth of human mesenchymal stem cells. Such microgels may therefore have future applications in tissue repair. This approach to fabricating LMWG microgels is a platform technology, which could potentially be applied to a variety of different functional LMWGs, and hence has wide-ranging potential.

Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis.

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

Syndecan-3 regulates MSC adhesion, ERK and AKT signalling in vitro and its deletion enhances MSC efficacy in a model of inflammatory arthritis in vivo.

Rheumatoid arthritis (RA) is a debilitating and painful inflammatory autoimmune disease characterised by the accumulation of leukocytes in the synovium, cartilage destruction and bone erosion. The immunomodulatory effects of bone marrow derived mesenchymal stem cells (MSCs) has been widely studied and the recent observations that syndecan-3 (SDC3) is selectively pro-inflammatory in the joint led us to hypothesise that SDC3 might play an important role in MSC biology. MSCs isolated from bone marrow of wild type and Sdc3-/- mice were used to assess immunophenotype, differentiation, adhesion and migration properties and cell signalling pathways. While both cell types show similar differentiation potential and forward scatter values, the cell complexity in wild type MSCs was significantly higher than in Sdc3-/- cells and was accompanied by lower spread surface area. Moreover, Sdc3-/- MSCs adhered more rapidly to collagen type I and showed a dramatic increase in AKT phosphorylation, accompanied by a decrease in ERK1/2 phosphorylation compared with control cells. In a mouse model of antigen-induced inflammatory arthritis, intraarticular injection of Sdc3-/- MSCs yielded enhanced efficacy compared to injection of wild type MSCs. In conclusion, our data suggest that syndecan-3 regulates MSC adhesion and efficacy in inflammatory arthritis, likely via induction of the AKT pathway.

Mesenchymal Stem Cell-Conditioned Medium Reduces Disease Severity and Immune Responses in Inflammatory Arthritis.

We evaluated the therapeutic potential of mesenchymal stem cell-conditioned medium (CM-MSC) as an alternative to cell therapy in an antigen-induced model of arthritis (AIA). Disease severity and cartilage loss were evaluated by histopathological analysis of arthritic knee joints and immunostaining of aggrecan neoepitopes. Cell proliferation was assessed for activated and naïve CD4+ T cells from healthy mice following culture with CM-MSC or co-culture with MSCs. T cell polarization was analysed in CD4+ T cells isolated from spleens and lymph nodes of arthritic mice treated with CM-MSC or MSCs. CM-MSC treatment significantly reduced knee-joint swelling, histopathological signs of AIA, cartilage loss and suppressed TNFα induction. Proliferation of CD4+ cells from spleens of healthy mice was not affected by CM-MSC but reduced when cells were co-cultured with MSCs. In the presence of CM-MSC or MSCs, increases in IL-10 concentration were observed in culture medium. Finally, CD4+ T cells from arthritic mice treated with CM-MSC showed increases in FOXP3 and IL-4 expression and positively affected the Treg:Th17 balance in the tissue. CM-MSC treatment reduces cartilage damage and suppresses immune responses by reducing aggrecan cleavage, enhancing Treg function and adjusting the Treg:Th17 ratio. CM-MSC may provide an effective cell-free therapy for inflammatory arthritis.