Dataset Information

Bone Marrow Microenvironment in Light-Chain Amyloidosis: In Vitro Expansion and Characterization of Mesenchymal Stromal Cells.

ABSTRACT: Immunoglobulin light-chain amyloidosis (AL) is caused by misfolded light chains produced by a small B cell clone. Mesenchymal stromal cells (MSCs) have been reported to affect plasma cell behavior. We aimed to characterize bone marrow (BM)-MSCs from AL patients, considering functional aspects, such as proliferation, differentiation, and immunomodulatory capacities. MSCs were in vitro expanded from the BM of 57 AL patients and 14 healthy donors (HDs). MSC surface markers were analyzed by flow cytometry, osteogenic and adipogenic differentiation capacities were in vitro evaluated, and co-culture experiments were performed in order to investigate MSC immunomodulatory properties towards the ALMC-2 cell line and HD peripheral blood mononuclear cells (PBMCs). AL-MSCs were comparable to HD-MSCs for morphology, immune-phenotype, and differentiation capacities. AL-MSCs showed a reduced proliferation rate, entering senescence at earlier passages than HD-MSCs. The AL-MSC modulatory effect on the plasma-cell line or circulating plasma cells was comparable to that of HD-MSCs. To our knowledge, this is the first study providing a comprehensive characterization of AL-MSCs. It remains to be defined if the observed abnormalities are the consequence of or are involved in the disease pathogenesis. BM microenvironment components in AL may represent the targets for the prevention/treatment of the disease in personalized therapies.

SUBMITTER: Valsecchi C

PROVIDER: S-EPMC8614719 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Bone Marrow Microenvironment in Light-Chain Amyloidosis: In Vitro Expansion and Characterization of Mesenchymal Stromal Cells.

Valsecchi Chiara C Croce Stefania S Maltese Alice A Montagna Lorenza L Lenta Elisa E Nevone Alice A Girelli Maria M Milani Paolo P Bosoni Tiziana T Massa Margherita M Abbà Carlotta C Campanelli Rita R Ripepi Jessica J De Silvestri Annalisa A Carolei Adriana A Palladini Giovanni G Zecca Marco M Nuvolone Mario M Avanzini Maria Antonietta MA

Biomedicines 20211022 11

Immunoglobulin light-chain amyloidosis (AL) is caused by misfolded light chains produced by a small B cell clone. Mesenchymal stromal cells (MSCs) have been reported to affect plasma cell behavior. We aimed to characterize bone marrow (BM)-MSCs from AL patients, considering functional aspects, such as proliferation, differentiation, and immunomodulatory capacities. MSCs were in vitro expanded from the BM of 57 AL patients and 14 healthy donors (HDs). MSC surface markers were analyzed by flow cyt ...[more]

PMID: 34829752

Similar Datasets

Project description:BackgroundMesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.MethodsHuman bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination.ResultsAs compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls.ConclusionOur study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Project description:BackgroundBone marrow stromal cells (BMSCs) are extensively used in regeneration therapy and cytology experiments simulate how BMSCs respond to radiation. Due to the small number and the heterogeneity of primary isolated BMSCs, extensive in vitro expansion is usually required before application, which affects the cellular characteristics and gene expression of BMSCs. However, whether the radiation response of BMSCs changes during in vitro expansion is unclear.MethodsIn this study, BMSCs were passaged in vitro and irradiated at passage 6 (P6) and passage 10 (P10). Then, apoptosis, the cell cycle, senescence, the cytokine secretion and the gene expression profile were analysed for the P6, P10, and non-irradiated (control) BMSCs at different post-irradiation time points.ResultsThe P6 BMSCs had a lower percentage of apoptotic cells than the P10 BMSCs at 24 and 48 h post-irradiation but not compared to that of the controls at 2 and 8 h post-irradiation. The P6 BMSCs had a lower percentage of cells in S phase and a higher percentage in G1 phase than the P10 BMSCs at 2 and 8 h post-irradiation. The radiation had similar effects on the senescent cell level and impaired immunomodulation capacity of the P6 and P10 BMSCs. Regardless of whether they were irradiated, the P6 and P10 BMSCs always expressed a distinctive set of genes. The upregulated genes were enriched in pathways including the cell cycle, DNA replication and oocyte meiosis. Then, a subset of conserved irradiation response genes across the BMSCs was identified, comprising 12 differentially upregulated genes and 5 differentially downregulated genes. These genes were especially associated with the p53 signaling pathway, DNA damage and DNA repair. Furthermore, validation experiments revealed that the mRNA and protein levels of these conserved genes were different between the P6 and P10 BMSCs after irradiation. Weighted gene co-expression network analysis supported these findings and further revealed the effects of cell passage on the irradiation response in BMSCs.ConclusionThe results indicated that cell passage in vitro affected the irradiation response of BMSCs via molecular mechanisms that mediated differences in apoptosis, the cell cycle, senescence and the cytokine secretion. Thus, accurate cell passage information is not only important for transplantation therapy but also for future studies on the radiation response in BMSCs.

Project description:Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded ex vivo. In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in vitro in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells in vitro. Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs). RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.

Project description:BackgroundTestosterone deficiency (TD) occurs most frequently in older men and can cause many health problems. Testosterone replacement therapy (TRT) is widely used to treat TD, but this regimen can lead to a series of side effects. Stem cell therapy has been wildly studied in vitro. However, due to the multidirectional differentiation potential and heterogeneity of stem cells, it is difficult to achieve the good efficiency and reproducibility in basic research and clinical applications. This study aims to identify a new strategy for the treatment of TD.MethodsBone marrow-derived mesenchymal stromal cells (BMSCs) were enucleated by Ficoll density gradient centrifugation. The organelles and cellular functions of enucleated BMSCs were analyzed by immunofluorescence staining and flow cytometry. Extracellular vesicles (EVs) were isolated by ultracentrifugation and characterized. For the animal studies, enucleated BMSCs were labelled with Mitotracker and injected into ethane dimethanesulfone (EDS)-treated rats. Testosterone production and spermatogenesis were detected at different time points through various tests. To determine the mechanism of efferocytosis, we analysed the number of macrophages by immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR).ResultsThe injection of enucleated BMSCs (Cargocytes) into the testes of EDS-treated rats restored the levels of serum testosterone, increased the number of Leydig cells (LCs), and improved spermatogenesis. We found that enucleated BMSCs underwent apoptosis earlier than BMSCs did. Subsequently, testicular interstitial macrophages phagocytosed apoptotic enucleated BMSCs through efferocytosis. Efferocytosis promoted macrophage polarization from the M1 to the M2 phenotype, reduced the expression of proinflammatory cytokines, and decreased the levels of inflammation and oxidative stress.ConclusionsIn summary, this study pioneered the application of stromal cell enucleation technology to repair tissue damage in the reproductive system, explored the potential of cell burial in the treatment of reproductive system diseases and provided a new approach for the clinical treatment of male infertility.

Dataset Information

Bone Marrow Microenvironment in Light-Chain Amyloidosis: In Vitro Expansion and Characterization of Mesenchymal Stromal Cells.

Publications

Bone Marrow Microenvironment in Light-Chain Amyloidosis: In Vitro Expansion and Characterization of Mesenchymal Stromal Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets