Single cell RNA sequencing of sorted bone marrow stromal cells from Osx-Cre, Ai9 mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE200693 | GEO | 2022/04/15
REPOSITORIES: GEO
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Project description:Single cell RNA sequencing of sorted bone marrow stromal cells from Osx-Cre, Ai9 mice
| PRJNA826089 | ENA
Project description:Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as "cell factories" that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies.
| S-EPMC4275011 | biostudies-literature
Project description:Fracture healing is a process that involves many cell populations. In this study we characterized gene expression in a subset of cells involved in fracture healing. αSMACreERT2 mice crossed with Ai9 reporter mice that express tdTomato fluorescent protein after Cre-mediated activation were used as an experimental model. αSMA-expressing cells were labeled by tamoxifen administration, then periosteal cells from the tibia were isolated two days later (controls), or tibial fractures were performed and periosteum/soft callus tissue was collected after 2 and 6 days. The tdTomato positive cell population was isolated by flow cytometry, and subjected to microarray analysis. Histology and cell surface marker analysis indicates that αSMACreERT2 labels a mainly mesenchymal population in the periosteum that expands after fracture, and contributes to both osteogenic and chondrogenic elements of the fracture callus. We were therefore able to examine gene expression in a defined population during the early stages of fracture healing. Total RNA was obtained from the tomato positive cells within the periosteal compartment of fractures from αSMACreERT2/Ai9 mice. Control animals were given 2 doses of tamoxifen, and periosteum was collected and labeled cells sorted (8-9 sex-matched mice per group). Fractures were performed after the second dose of tamoxifen, and tomato positive cells from periosteum/callus tissue were isolated 2 and 6 days after fracture (4-8 animals per sample pooled). 3 replicates for each sample are included.
2013-11-27 | E-GEOD-45156 | biostudies-arrayexpress
Project description:Purpose: To investigate if and how APPswe in OCN-Cre+ OB-lineage cells gives rise to the brain and behavior phenotypes, we purified OCN-Cre+ BMSCs (marked by tdTomato+, believed to be OB progenitors) from both the 6-MO control (OCN-Cre; Ai9) and TgAPPsweOCN; Ai9 mice using fluorescence-activated cell sorting (FACS) and then subjected them to RNA-seq analysis Methods: (1)the whole bone marrow cells of 6-month-old OCn-Cre;Ai9 andTgAPPswe;OCn-Cre;Ai9 flushed out from long bones of mice with DMEM were filtered through a 70-mm filter mesh, washed, re-suspended, and then plated in 100-mm dishes with growth medium (DMEM plus 10% FBS), which were incubated at 37oC with 5% CO2. The non-adherent cells were removed 72 hours after changing the medium. The attached bone marrow cells were cultured with the growth medium for 7 days. These cells were then resuspended and plated at culture dishes and cultured for another 3-6 days with the same growth medium. These cells, so called BMSCs. (2)Then cell media were removed from culture dishes and cells were rinsed with PBS. Trypsin solution was added to incubate at 37°C for 2 min. The detached adherent cells were centrifuged, and the pellet cells were washed with 1 ml cold PBS, and finally resuspended in 0.5 ml PBS with 1% FBS for flow cytometry analysis. Flow cytometric analysis was performed by use of a flow cytometer in CWRU core facility. Acquisition and analysis were performed by using FACSDiva 8.0.1 software (BD).(3)Total RNAs were extracted from purified Td+ OB-progenitor cells from OCN-Cre; Ai9 and TgAPPsweOCN; Ai9 mice by flow cytometer. RNA Integrity Number (RIN) was accessed for every sample, and the samples were considered qualified with RIN> 2. These RNA samples were then subjected to RNA-seq analyses by BGI America (Cambridge, MA) using the DNBseq platform. Firstly, we removed the reads mapped to rRNA and obtained the raw data with 52.47 Mb reads. After filtering low-quality, adaptor-polluted and high content of unknown base reads in the sequencing reads, 51.9 Mb clean reads were obtained per sample on average. Then clean reads were mapped to reference genome using HISAT2. On average 92.91% reads were mapped and the uniformity of the mapping result for each sample suggests that the samples were comparable. Comparisons to RNAseq were normalized to fpkm Results: 917 up- and 1825 down-regulated genes were identified in APPswe+ OB progenitors. Among these genes, 154 up- and 269 down-regulated genes encode secreted proteins. Interestingly, go analysis showed that most up-regulated genes are involved in inflammatory response, cytokine production, and cytokine/chemokine-mediated signaling pathways; and most down-regulated genes are implicated in cell cycle, cell proliferation, and bone mineralization. Conclusions: Increased cytokines and chemokines in APPswe+ OB-progenitors, indicating that APPswe+ OB progenitors may undergo cellular senescence.
2021-11-30 | GSE186827 | GEO
Project description:Fracture healing is a process that involves many cell populations. In this study we characterized gene expression in a subset of cells involved in fracture healing. αSMACreERT2 mice crossed with Ai9 reporter mice that express tdTomato fluorescent protein after Cre-mediated activation were used as an experimental model. αSMA-expressing cells were labeled by tamoxifen administration, then periosteal cells from the tibia were isolated two days later (controls), or tibial fractures were performed and periosteum/soft callus tissue was collected after 2 and 6 days. The tdTomato positive cell population was isolated by flow cytometry, and subjected to microarray analysis. Histology and cell surface marker analysis indicates that αSMACreERT2 labels a mainly mesenchymal population in the periosteum that expands after fracture, and contributes to both osteogenic and chondrogenic elements of the fracture callus. We were therefore able to examine gene expression in a defined population during the early stages of fracture healing.
2013-11-27 | GSE45156 | GEO
Project description:Parental ES reporter line Dlx6a-Cre; Ai9 was differentiated using 2 different transcriptional specifiation strategies: 1) Nkx2-1/Dlx2 or 2) Nkx2-1/Dlx2/St18. Neurons were directeed towards neural, telencephalic and then ventral telencephalic identity prior to neuronal differentiation and reporter gene activation. Cells were differentiated for 12 days. tdTomato+ neurons were isolated by flow cytometry prior to RNA isolationa and library construction.
2022-12-08 | GSE180825 | GEO
Project description:Ocular Sebaceous Carcinoma (OSC) is a rare malignant tumor for which initial clinical and pathological diagnosis is often incorrect. OSC can mimic Squamous Cell Carcinoma of the Conjunctiva (SCCC). Aim of this study was to find microRNA biomarkers to distinguish OSC and SCCC from normal tissue and from each other. Clinical OSC and SCCC case files and corresponding histopathological slides were collected and reviewed. Microdissected formalin-fixed paraffin-embedded tumor and control tissue were sub-jected to semi-high throughput microRNA profiling. MicroRNA expression distinguishes OSC and SCCC from corresponding control tissues. Selected differentially expressed miRNAs were validated using single RT-PCR assays. No prognostic miRNAs could be identified that reliable predict SCCC metastasis or OSC recurrence. A com-parison between OSC (n=14) and SCCC (n=18) revealed 38 differentially expressed microRNAs (p<0.05). Differentially expressed miRNAs were selected for validation in the discovery cohort and an independent validation cohort (OSC, n=11; SCCC, n=12). At least two miRNAs miR-196b-5p (p ≤ 0.05) and miR-107 (p ≤ 0.001) displayed a statistically significant differential expression between OSC and SCCC with miR-196b-5p upregulated in SCCC and miR-107 up-regulated in OSC. In the validation cohort microRNA miR-493-3p also showed significant up-regulation in SCCC when compared to OSC (p ≤ 0.05). ROC analyses indicated that the com-bined miR-196b-5p and miR-107 expression levels predicted OSC with 90.0% sensitivity and 83.3% specificity. In conclusion, combined testing of miR-196b-5p and miR-107, can be of additional use in routine diagnostics to discriminate OSC from SCCC.
2022-04-26 | GSE201490 | GEO
Project description:The single-cell RNA sequencing was focused on the suprafornical LHA and its surrounding areas. For single cell dissociation and collection, we used transgenic animal Agrp-IRES-Cre × Ai9 crosses, where the tdTomato-labeled AGRP neurons extend a prominent set of axonal projections to the suprafornical region of the LHA and provide a useful signal for visually guided dissection of the targeting brain region. Single-cell cDNA libraries were prepared using the Smart-SCRB chemistry.
2022-02-22 | GSE167293 | GEO
Project description:BackgroundSB623 cells are expanded from marrow stromal cells (MSCs) transfected with a Notch intracellular domain (NICD)-expressing plasmid. In stroke-induced animals, these cells reduce infarct size and promote functional recovery. SB623 cells resemble the parental MSCs with respect to morphology and cell surface markers despite having been in extended culture. MSCs are known to have immunosuppressive properties; whether long-term culture of MSCs impact their immunomodulatory activity has not been addressed.MethodsTo assess the possible senescent properties of SB623 cells, we performed cell cycle related assays and beta-galactosidase staining. To assess the immunomodulatory activity of these expanded NICD-transfected MSCs, we performed co-cultures of SB623 cells or MSCs with either enriched human T cells or monocytes and assessed cytokine production by flow cytometry. In addition, we monitored the immunosuppressive activity of SB623 cells in both allogenic and xenogenic mixed lymphocyte reaction (MLR).ResultsCompared to MSCs, we showed that a small number of senescent-like cells appear in each lot of SB623 cells. Nevertheless, we demonstrated that these cells suppress human T cell proliferation in both the allogeneic and xenogeneic mixed lymphocyte reaction (MLR) in a manner comparable to MSCs. IL-10 producing T cells were generated and monocyte-dendritic cell differentiation was dampened by co-culture with SB623 cells. Compared to the parental MSCs, SB623 cells appear to exert a greater inhibitory impact on the maturation of dendritic cells as demonstrated by a greater reduction in the surface expression of the co-stimulatory molecule, CD86.ConclusionThe results demonstrated that the immunosuppressive activity of the expanded NICD-transfected MSCs is comparable to the parental MSCs, in spite of the appearance of a small number of senescent-like cells.
| S-EPMC3228829 | biostudies-literature
Project description:BackgroundAngiogenesis is a critical part of the endogenous repair process in brain injury and disease, and requires at least two sequential steps. First, angiogenic sprouting of endothelial cells occurs, which entails the initial proliferation of endothelial cells and remodeling of the surrounding extracellular matrix. Second, vessel stabilization is necessary to prevent vascular regression, which relies on vascular smooth muscle recruitment to surround the young vessels. Marrow stromal cells (MSCs) have been shown to promote revascularization after hindlimb ischemia, cardiac ischemia, and stroke. SB623 cells are derived from marrow stromal cells by transfection with a Notch1 intracellular domain (NICD)-expressing plasmid and are known to elicit functional improvement in experimental stroke. These cells are currently used in human clinical testing for treatment of chronic stroke. In the current study, the angiogenic property of SB623 cells was investigated using cell-based assays.MethodsAngiogenic paracrine factors secreted by SB623 cells and the parental MSCs were identified using the Qantibody Human Angiogenesis Array. To measure the angiogenic activity of conditioned medium from SB623 cells and MSCs, endothelial tube formation in the human umbilical vein endothelial cell (HUVEC) assay and endothelial cell sprouting and branching in the rodent aortic ring assay were quantified. To validate the angiogenic contribution of VEGF in conditioned medium, endothelial cells and aortic rings were treated with SU5416, which inhibits VEGFR2 at low dose.ResultsConditioned medium from SB623 cells promoted survival and proliferation of endothelial cells under serum-deprived conditions and supports HUVEC vascular tube formation. In a rodent aortic ring assay, there was enhanced endothelial sprouting and branching in response to SB623-derived conditioned medium. SU5416 treatment partially reversed the effect of conditioned medium on endothelial cell survival and proliferation while completely abrogate HUVEC tube formation and endothelial cell sprouting and branching in aortic ring assays.ConclusionsThese data indicate that SB623 cell-secreted angiogenic factors promoted several aspects of angiogenesis, which likely contribute to promoting recovery in the injured brain.
| S-EPMC3615967 | biostudies-literature