Isolation of human bone marrow stromal cells from bone marrow biopsies for single-cell RNA sequencing.
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ABSTRACT:
SUBMITTER: Gleitz HFE
PROVIDER: S-EPMC8134068 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundNative bone marrow (BM) mesenchymal stem/stromal cells (BM-MSCs) participate in generating and shaping the skeleton and BM throughout the lifespan. Moreover, BM-MSCs regulate hematopoiesis by contributing to the hematopoietic stem cell niche in providing critical cytokines, chemokines and extracellular matrix components. However, BM-MSCs contain a heterogeneous cell population that remains ill-defined. Although studies on the taxonomy of native BM-MSCs in mice have just started to emerge, the taxonomy of native human BM-MSCs remains unelucidated.MethodsBy using single-cell RNA sequencing (scRNA-seq), we aimed to define a proper taxonomy for native human BM non-hematopoietic subsets including endothelial cells (ECs) and mural cells (MCs) but with a focal point on MSCs. To this end, transcriptomic scRNA-seq data were generated from 5 distinct BM donors and were analyzed together with other transcriptomic data and with computational biology analyses at different levels to identify, characterize and classify distinct native cell subsets with relevant biomarkers.ResultsWe could ascribe novel specific biomarkers to ECs, MCs and MSCs. Unlike ECs and MCs, MSCs exhibited an adipogenic transcriptomic pattern while co-expressing genes related to hematopoiesis support and multilineage commitment potential. Furthermore, by a comparative analysis of scRNA-seq of BM cells from humans and mice, we identified core genes conserved in both species. Notably, we identified MARCKS, CXCL12, PDGFRA, and LEPR together with adipogenic factors as archetypal biomarkers of native MSCs within BM. In addition, our data suggest some complex gene nodes regulating critical biological functions of native BM-MSCs together with a preferential commitment toward an adipocyte lineage.ConclusionsOverall, our taxonomy for native BM non-hematopoietic compartment provides an explicit depiction of gene expression in human ECs, MCs and MSCs at single-cell resolution. This analysis helps enhance our understanding of the phenotype and the complexity of biological functions of native human BM-MSCs.
| S-EPMC10469496 | biostudies-literature
Project description:Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing (scRNAseq), we systematically characterized the human non-hematopoietic BM stromal compartment and we investigated stromal cell regulation principles based on the RNA velocity analysis using scVelo and studied the interactions between the human BM stromal cells and hematopoietic cells based on ligand-receptor (LR) expression using CellPhoneDB. scRNAseq led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis and in vitro proliferation capacities and differentiation potentials. Potential key factors that might govern the transition from stem and progenitor cells to fate-committed cells were identified. In situ localization analysis demonstrated that different stromal cells were localized in different niches in the bone marrow. In silico cell-cell communication analysis further predicted that different stromal cell types might regulate hematopoiesis through distinct mechanisms. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on human hematopoietic niche organization.
| S-EPMC10097421 | biostudies-literature
Project description:New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from 20 healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated a strong correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference data set and highlights opportunities for further refinement.
| S-EPMC6328018 | biostudies-literature
Project description:BACKGROUND: Bone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN-? and TNF-? change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs. METHODS: BMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1? and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs. RESULTS: Co-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF-? signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells. CONCLUSIONS: BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells.
| S-EPMC3882878 | biostudies-literature
Project description:Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytometry. We then provide an overview of downstream computational techniques to analyze sequencing data. For complete details on the use and execution of this protocol, please refer to Cook et al. (2022)1 and Liu et al. (2022).2.
| S-EPMC10173011 | biostudies-literature
Project description:Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.
| S-EPMC8579438 | biostudies-literature
Project description:BackgroundMultipotent stromal cells (MSCs) are being studied in the field of regenerative medicine for their multi-lineage differentiation and immunoregulatory capacity. MicroRNAs (miRNAs) are short non-coding RNAs that are responsible for regulating gene expression by targeting transcripts, which can impact MSC functions such as cellular proliferation, differentiation, migration and cell death. miRNAs are expressed in MSCs; however, the impact of miRNAs on cellular functions and donor variability is not well understood. Eight MSC lines were expanded to passages 3, 5 and 7, and their miRNA expression was evaluated using microarray technology.ResultsStatistical analyses of our data revealed that 71 miRNAs out of 939 examined were expressed by this set of MSC lines at all passages and the expression of 11 miRNAs were significantly different between passages 3 and 7, while the expression of 7 miRNAs was significantly different between passages 3 and 5. The expression of these identified miRNAs was evaluated using RT-qPCR for both the first set of MSC lines (n = 6) and a second set of MSC lines (n = 7) expanded from passages 4 to 8. By RT-qPCR only 2 miRNAs, miR-638 and miR-572 were upregulated at passage 7 compared to passage 3 in the first set of MSC lines by 1.71 and 1.54 fold, respectively; and upregulated at passage 8 compared to passage 4 in the second set of MSC lines, 1.35 and 1.59 fold, respectively.ConclusionsThe expression of miR-638 and miR-572 can distinguish MSCs from two different passages of cell culture. These results may be useful in establishing critical quality attributes of MSCs and determining whether changes in these two miRNAs impact cellular functions.
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Project description:Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.
| S-EPMC3175557 | biostudies-literature
Project description:There is a wealth of data indicating human bone marrow contains skeletal stem cells (SSC) with the capacity for osteogenic, chondrogenic and adipogenic differentiation. However, current methods to isolate SSCs are restricted by the lack of a defined marker, limiting understanding of SSC fate, immunophenotype, function and clinical application. The current study applied single-cell RNA-sequencing to profile human adult bone marrow populations from 11 donors and identified novel targets for SSC enrichment. Spherical nucleic acids were used to detect these mRNA targets in SSCs. This methodology was able to rapidly isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with the capacity for tri-lineage differentiation in vitro and ectopic bone formation in vivo. The current studies detail the development of a platform to advance SSC enrichment from human bone marrow, offering an invaluable resource for further SSC characterisation, with significant therapeutic impact therein.
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Project description:Cultured human bone marrow stromal (mesenchymal) stem cells (hBM-MSCs) are heterogenous cell populations exhibiting variable biological properties. Quantitative high-content imaging technology allows identification of morphological markers at a single cell resolution that are determinant for cellular functions. We determined the morphological characteristics of cultured primary hBM-MSCs and examined their predictive value for hBM-MSC functionality. BM-MSCs were isolated from 56 donors and characterized for their proliferative and differentiation potential. We correlated these data with cellular and nuclear morphological features determined by Operetta; a high-content imaging system. Cell area, cell geometry, and nucleus geometry of cultured hBM-MSCs exhibited significant correlation with expression of hBM-MSC membrane markers: ALP, CD146, and CD271. Proliferation capacity correlated negatively with cell and nucleus area and positively with cytoskeleton texture features. In addition, in vitro differentiation to osteoblasts as well as in vivo heterotopic bone formation was associated with decreased ratio of nucleus width to length. Multivariable analysis applying a stability selection procedure identified nuclear geometry and texture as predictors for hBM-MSCs differentiation potential to osteoblasts or adipocytes. Our data demonstrate that by employing a limited number of cell morphological characteristics, it is possible to predict the functional phenotype of cultured hBM-MSCs and thus can be used as a screening test for "quality" of hBM-MSCs prior their use in clinical protocols.
| S-EPMC6988772 | biostudies-literature