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.

Attitudes Towards Standardization of Mesenchymal Stromal Cells-A Qualitative Exploration of Expert Views.

Pharmacopoeial standards ensure quality control of established medicines. It is widely believed that translation of cell therapy medicines will be facilitated by defining and adopting relevant standards. Mesenchymal stromal cells (MSCs) are used extensively for multiple indications in regenerative medicine. They are highly heterogeneous in terms of their biological characteristics and their mechanisms of action, making standardization a challenging undertaking. Furthermore, the use of MSCs in therapy appears to attract diverse views, ranging from concern and caution to enthusiastic positivity. We conducted semi-structured interviews with 20 expert stakeholders from academia, industry, regulatory agencies, non-governmental organizations and clinicians to explore their views, experiences, recommendations, and concerns regarding standardization of MSCs. Qualitative thematic analysis of transcribed records led to development of a consensus framework, which identified 5 key themes to facilitate exploration of the interviews' content. On the basis of our findings, we conclude that (1) there is undoubtedly an appetite for standardization, particularly in development of assays that enable comparison or benchmarking across manufacturers, processes, and cell sources; (2) stakeholder groups are not homogeneous in their concerns and attitudes; (3) careful consideration must be given to the points along the development timeline at which different standardization approaches could be beneficial; and (4) the roles of standards could be promoted further for specific aspects of advanced therapy medicinal product (ATMP) development and regulation such as qualification of decentralized manufacturing sites. A unified cross-stakeholder approach will help to advance MSC therapeutics and other cell therapy medicines.

Selection of tRNA Genes in Human Breast Tumours Varies Substantially between Individuals.

Abnormally elevated expression of tRNA is a common feature of breast tumours. Rather than a uniform increase in all tRNAs, some are deregulated more strongly than others. Elevation of particular tRNAs has been associated with poor prognosis for patients, and experimental models have demonstrated the ability of some tRNAs to promote proliferation or metastasis. Each tRNA isoacceptor is encoded redundantly by multiple genes, which are commonly dispersed across several chromosomes. An unanswered question is whether the consistently high expression of a tRNA in a cancer type reflects the consistent activation of the same members of a gene family, or whether different family members are activated from one patient to the next. To address this question, we interrogated ChIP-seq data to determine which tRNA genes were active in individual breast tumours. This revealed that distinct sets of tRNA genes become activated in individual cancers, whereas there is much less variation in the expression patterns of families. Several pathways have been described that are likely to contribute to increases in tRNA gene transcription in breast tumours, but none of these can adequately explain the observed variation in the choice of genes between tumours. Current models may therefore lack at least one level of regulation.

Characterisation of mesenchymal stromal cells in clinical trial reports: analysis of published descriptors.

BACKGROUND: Mesenchymal stem or stromal cells are the most widely used cell therapy to date. They are heterogeneous, with variations in growth potential, differentiation capacity and protein expression profile depending on tissue source and production process. Nomenclature and defining characteristics have been debated for almost 20 years, yet the generic term 'MSC' is used to cover a wide range of cellular phenotypes. Against a documented lack of definition of cellular populations used in clinical trials, our study evaluated the extent of characterisation of the cellular population or study drug. METHODS: A literature search of clinical trials involving mesenchymal stem/stromal cells was refined to 84 papers upon application of pre-defined inclusion/exclusion criteria. Data were extracted covering background trial information including location, phase, indication, tissue source and details of clinical cell population characterisation (expression of surface markers, viability, differentiation assays and potency/functionality assays). Descriptive statistics were applied, and tests of association between groups were explored using Fisher's exact test for count data with simulated p value. RESULTS: Twenty-eight studies (33.3%) include no characterisation data. Forty-five (53.6%) reported average values per marker for all cell lots used in the trial, and 11 (13.1%) studies included individual values per cell lot. Viability was reported in 57% of studies. Differentiation was discussed: osteogenesis (29% of papers), adipogenesis (27%), and chondrogenesis (20%) and other functional assays arose in 7 papers (8%). The extent of characterisation was not related to the clinical phase of development. Assessment of functionality was very limited and did not always relate to the likely mechanism of action. CONCLUSIONS: The extent of characterisation was poor and variable. Our findings concur with those in other fields including bone marrow aspirate and platelet-rich plasma therapy. We discuss the potential implications of these findings for the use of mesenchymal stem or stromal cells in regenerative medicine, and the importance of characterisation for transparency and comparability of literature.

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses.

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.