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Macrophage derived-inflammation for confitioning of mesenchymal stromal cells

ABSTRACT: The study: Bone marrow-derived mesenchymal stromal cells (MSC) can be triggered to enhance the expression of repair associated factors by certain stimuli. The purpose of this study was to perform a transcriptome-wide analysis of the effect of macrophage-derived inflammation on MSC and evaluate the repair potential of the conditioned cells. To validate the results, we compared the condition of interest to three control conditions, described bellow. Methods: MSC were cultured in baseline conditions (D10 medium) until reaching passage 4. In parallel, monocytes were differentiated to macrophages and polarized to pro-inflammatory (M1) or non-polarized (M0). After 24h of polarization, the supernatants were collected to produce M1CM and M0CM, respectively. The medium used for macrophage polarization (Pol1) was included in the conditions for MSC culture as a control. The medium of all MSC cultures was changed into fresh D10, M1CM, M0CM or Pol1, and they were cultured for 24h (conditioning). Then, MSC were trypsinized and their RNA was extracted. We worked with Novogene Inc. USA for the RNAsequencing, which they performed based on the Illumina platform. Novogene performed the sequencing, data processing and preliminary analysis. We then did the downstream analysis and data interpretation. Results: Dowstream analysis, after data processing was completed for all samples and groups, was focused on the comparison M1CM vs D10. This is because the effect of M0CM and Pol1 on MSC was minor compared to the effect of M1CM, based on correlation and Venn diagram analysis. M1CM induced 1721 upregulated DEG and 1487 downregulated DEG in MSC compared to D10. Among those genes, the GO and KEGG analyses showed enrichment in GO terms and pathways associated to the enhancement of angiogenesis, immunomodulation, paracrine cell survival, tissue development and regeneration, indicating increased therapeutic potential. Additionally, genes associated with programmed cell death were upregulated, while cell cycle and motility associated genes were downregulated, indicating compromised cell viability and motility. Conclusions: We found that macrophage-derived inflammation induces a transcriptomic makeover on MSC, enhancing their reparative potential. These results reveal the possibility to use inflammation as a priming method for MSC before transplantation, which could potentiate the transplant's efficacy. Further pre-clinical studies are necessary to determine the effects of the beneficial outcomes in tissue, as well as the implications of compromised viability and motility.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE161798 | GEO | 2021/01/21

REPOSITORIES: GEO

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Project description:Background: Macrophage polarization programs, commonly referred to as âclassicalâ and âalternativeâ activation, are widely considered as distinct states that are exclusive of one another, and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages, or if they have adopted a unique state characterized by components of both classical and alternative activation. Results: We analyzed the polarization of individual macrophages responding to TBI using single-cell RNA sequencing. Analysis of signature polarization genes revealed diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNÎ³). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states, and in several distinct and seemingly incompatible combinations. Conclusions: Single-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets, but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarizationâa key consideration for therapeutic modulation. Monocyte derived macrophages were isolated from the ipsilateral hemisphere of mouse brains one day following traumatic brain injury elicited by control cortical impact in C57BL/6 adult male mice. Single-cells were isolated and processed for RNA sequencing using a Fluidigm C1 integrated fluidic circuit chip. 45 biological replicates were analyzed.

Project description:Background: Macrophage polarization programs, commonly referred to as “classical” and “alternative” activation, are widely considered as distinct states that are exclusive of one another, and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages, or if they have adopted a unique state characterized by components of both classical and alternative activation. Results: We analyzed the polarization of individual macrophages responding to TBI using single-cell RNA sequencing. Analysis of signature polarization genes revealed diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNγ). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states, and in several distinct and seemingly incompatible combinations. Conclusions: Single-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets, but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarization—a key consideration for therapeutic modulation.

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Macrophage derived-inflammation for confitioning of mesenchymal stromal cells

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