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Abstract Mesenchymal stem cells (MSCs) interact with their surroundings via integrins, which link to the actin cytoskeleton and translate physical cues into biochemical signals through mechanotransduction. N-cadherins enable cell-cell communication and are also linked to the cytoskeleton. This crosstalk between integrins and cadherins modulates MSC mechanotransduction and fate. Here we show the role of this crosstalk in the mechanosensing of viscosity using supported lipid bilayers as substrates of varying viscosity. We functionalize these lipid bilayers with adhesion peptides for integrins (RGD) and N-cadherins (HAVDI), to demonstrate that integrins and cadherins compete for the actin cytoskeleton, leading to an altered MSC mechanosensing response. This response is characterised by a weaker integrin adhesion to the environment when cadherin ligation occurs. We model this competition via a modified molecular clutch model, which drives the integrin/cadherin crosstalk in response to surface viscosity, ultimately controlling MSC lineage commitment.

IntroductionOpening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy.MethodsIntravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonally-derived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods.Results3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines.ConclusionThis novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BM-derived MSC sub-population could achieve this.

Over the last decade, the acceleration in the clinical use of mesenchymal stromal cells (MSCs) has been nothing short of spectacular. Perhaps most surprising is how little we know about the “MSC product.” Although MSCs are being delivered to patients at an alarming rate, the regulatory requirements for MSC therapies (for example in terms of quality assurance and quality control) are nowhere near the expectations of traditional pharmaceuticals. That said, the standards that define a chemical compound or purified recombinant protein cannot be applied with the same stringency to a cell-based therapy. Biological processes are dynamic, adaptive and variable. Heterogeneity will always exist or emerge within even the most rigorously sorted clonal cell populations. With MSCs, perhaps more so than any other therapeutic cell, heterogeneity pervades at multiple levels, from the sample source to the single cell. The research and clinical communities collectively need to recognize and take steps to address this troublesome truth, to ensure that the promise of MSC-based therapies is fulfilled.

Summary: In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1−/− mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7+ stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages. : Balzano et al. show that bone marrow peri-sinusoidal stromal cells, which form the hematopoietic stem cell niche, also express B cell niche genes including IL-7 and Nidogen-1. Loss of Nidogen-1 expression specifically affects access of pro-B cells to IL-7, resulting in impaired expansion and differentiation of early B cells. Keywords: B cell development, hematopoietic stem cell, stromal cell niche, interaction network, bone marrow

Bone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis of functional markers for BMSC subsets. All clones expressed typical BMSC cell-surface antigens; however, clones with trilineage differentiation capacity exhibited enhanced vascular interaction gene sets, whereas non-differentiating clones were uniquely CD317 positive with significantly enriched immunomodulatory transcriptional networks and high IL-7 production. IL-7 lineage tracing and CD317 immunolocalization confirmed the existence of a rare non-differentiating BMSC subtype, distinct from Cxcl12-DsRed+ perivascular stromal cells in vivo. Colony-forming CD317+ IL-7hi cells, identified at ∼1%–3% frequency in heterogeneous human BMSC fractions, were found to have the same biomolecular profile as non-differentiating BMSC clones using Raman spectroscopy. Distinct functional identities can be assigned to BMSC subpopulations, which are likely to have specific roles in immune control, lymphopoiesis, and bone homeostasis.

Wnt signalling has been implicated in the regulation of stem cell self-renewal and differentiation; however, the majority of in vitro studies are carried out using monolayer 2D culture techniques. Here, we used mesenchymal stromal cell (MSC) EGFP reporter lines responsive to Wnt pathway activation in a 3D spheroid culture system to mimic better the in vivo environment. Endogenous Wnt signalling was then investigated under basal conditions and when MSCs were induced to undergo osteogenic and adipogenic differentiation. Interestingly, endogenous Wnt signalling was only active during 3D differentiation whereas 2D cultures showed no EGFP expression throughout an extended differentiation time-course. Furthermore, exogenous Wnt signalling in 3D adipogenic conditions inhibited differentiation compared to unstimulated controls. In addition, suppressing Wnt signalling by Dkk-1 restored and facilitated adipogenic differentiation in MSC spheroids. Our findings indicate that endogenous Wnt signalling is active and can be tracked in 3D MSC cultures where it may act as a molecular switch in adipogenesis. The identification of the signalling pathways that regulate MSCs in a 3D in vivo-like environment will advance our understanding of the molecular mechanisms that control MSC fate.

Mesenchymal stem/stromal cells (MSCs) are typically characterised by their ability to differentiate into skeletal (osteogenic, chondrogenic and adipogenic) lineages. MSCs also appear to have additional non-stem cell functions in coordinating tissue morphogenesis and organising vascular networks through interactions with endothelial cells (ECs). However, suitable experimental models to examine these apparently unique MSC properties are lacking. Following previous work, we have developed our 3D in vitro co-culture models to enable us to track cellular self-organisation events in heterotypic cell spheroids combining ECs, MSCs and their differentiated progeny. In these systems, MSCs, but not related fibroblastic cell types, promote the assembly of ECs into interconnected networks through intrinsic mechanisms, dependent on the relative abundance of MSC and EC numbers. Perturbation of endogenous platelet-derived growth factor (PDGF) signalling significantly increased EC network length, width and branching. When MSCs were pre-differentiated towards an osteogenic or chondrogenic lineage and co-cultured as mixed 3D spheroids, they segregated into polarised osseous and chondral regions. In the presence of ECs, the pre-differentiated MSCs redistributed to form a central mixed cell core with an outer osseous layer. Our findings demonstrate the intrinsic self-organising properties of MSCs, which may broaden their use in regenerative medicine and advance current approaches.