Dataset Information

Extracellular vesicles derived from induced pluripotent stem cells mediate anti-inflammatory effects in human primary macrophages. iPSC EV Dataset

ABSTRACT: Extracellular vesicles derived from induced pluripotent stem cells (iPSC EVs) have immunoregulatory potential with the ability to alter monocyte-derived macrophages. Macrophages function in the propagation and resolution of inflammation which is mediated by their phenotype. Macrophages are an ideal therapeutic target as modulating their phenotype towards an anti-inflammatory pro-resolving state may be beneficial in chronic inflammatory diseases such as atherosclerosis. Macrophages are naturally phagocytotic cells and readily take up iPSC EVs however the contents of iPSC EVs and their effects on macrophages are poorly understood. Here iPSC EVs were characterized and analysed by mass-spectrometry based proteomics and a targeted microRNA (miR) panel. Their immunomodulatory effects on macrophages were assessed and a monocyte transmigration assay was used to assess the chemotactic potency of the secretome from iPSC EV treated macrophages. Proteomic analysis on iPSC EVs identified Podocalyxin-like protein 1 (PODXL1), Insulin (INS) and Solute Carrier Family 2 (Facilitated Glucose Transporter), Member 3 (SLC2A3) as the most abundant proteins unique to the iPSC EVs when compared to control NT-2 EVs. Notably, thioredoxin and peroxiredoxin related proteins were detected. miR-302d-3p was the most abundant miR in these iPSC EVs. miR-25-3p, previously reported to alter the macrophage phenotype, was significantly increased in comparison to the control NT-2 EVs. iPSC EVs increased expression of the anti-inflammatory associated MRC1 and miR-21 in human primary macrophages and decreased monocyte chemoattractant protein1 (MCP-1). Mass spectrometry based proteomics revealed that treated macrophages had decreased levels of secretory proteins, some of which have chemotactic properties, these included Azurocidin 1 (AZU1), Growth Differentiation Factor 15 (GDF15), and Ribosomal Protein S19 (RPS19). There was a decrease in monocyte transmigration towards conditioned media from macrophages treated with iPSC EVs. Collectively this study provides insights into the protein content and miR cargo of iPSC EVs and highlights their capacity to inhibit chemotactic proteins in macrophages and upregulate MRC1.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Eugene Dillon

LAB HEAD: Stephen Fitzsimons

PROVIDER: PXD067567 | Pride | 2025-09-08

REPOSITORIES: Pride

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			Action	DRS
	2022_01_21_StephenFitz_01.raw	Raw
	2022_01_21_StephenFitz_02.raw	Raw
	2022_01_21_StephenFitz_03.raw	Raw
	2022_01_21_StephenFitz_04.raw	Raw
	2022_01_21_StephenFitz_05.raw	Raw

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Project description:Extracellular vesicles derived from induced pluripotent stem cells (iPSC EVs) have immunoregulatory potential with the ability to alter monocyte-derived macrophages. Macrophages function in the propagation and resolution of inflammation which is mediated by their phenotype. Macrophages are an ideal therapeutic target as modulating their phenotype towards an anti-inflammatory pro-resolving state may be beneficial in chronic inflammatory diseases such as atherosclerosis. Macrophages are naturally phagocytotic cells and readily take up iPSC EVs however the contents of iPSC EVs and their effects on macrophages are poorly understood. Here iPSC EVs were characterized and analysed by mass-spectrometry based proteomics and a targeted microRNA (miR) panel. Their immunomodulatory effects on macrophages were assessed and a monocyte transmigration assay was used to assess the chemotactic potency of the secretome from iPSC EV treated macrophages. Proteomic analysis on iPSC EVs identified Podocalyxin-like protein 1 (PODXL1), Insulin (INS) and Solute Carrier Family 2 (Facilitated Glucose Transporter), Member 3 (SLC2A3) as the most abundant proteins unique to the iPSC EVs when compared to control NT-2 EVs. Notably, thioredoxin and peroxiredoxin related proteins were detected. miR-302d-3p was the most abundant miR in these iPSC EVs. miR-25-3p, previously reported to alter the macrophage phenotype, was significantly increased in comparison to the control NT-2 EVs. iPSC EVs increased expression of the anti-inflammatory associated MRC1 and miR-21 in human primary macrophages and decreased monocyte chemoattractant protein1 (MCP-1). Mass spectrometry based proteomics revealed that treated macrophages had decreased levels of secretory proteins, some of which have chemotactic properties, these included Azurocidin 1 (AZU1), Growth Differentiation Factor 15 (GDF15), and Ribosomal Protein S19 (RPS19). There was a decrease in monocyte transmigration towards conditioned media from macrophages treated with iPSC EVs. Collectively this study provides insights into the protein content and miR cargo of iPSC EVs and highlights their capacity to inhibit chemotactic proteins in macrophages and upregulate MRC1.

Project description:We investigated the hypothesis that transmigration drives monocyte transcriptional changes. Using Agilent Whole Human Genome Microarrays, we identified over 600 differentially expressed genes (2x, p<0.05) in freshly isolated human monocytes following 1.5 h of transmigration across IL1beta-stimulated ECs compared to untreated monocytes. Genes up-regulated by monocyte transmigration belong to a number of overrepresented functional groups including immune response, and inhibition of apoptosis. qRT-PCR confirmed increased expression of monocyte chemotactic proteins 1 and 3 and of NLR family apoptosis inhibitory protein (NAIP) following monocyte transmigration. In addition, quantification of annexin V binding revealed a reduction in apoptosis following monocyte transmigration. Comparison of gene expression in transmigrated monocytes to additional controls (monocytes which failed to transmigrate and monocytes incubated beneath stimulated ECs) revealed 89 differentially expressed genes which were controlled by the physical process of diapedesis. Functional annotation of these genes showed down-regulation of antimicrobial genes (e.g. alpha-defensin down 50x, cathelicidin down 9x, and cathepsin G down 3x). qRT-PCR confirmed down-regulation of these genes. Immunoblots confirmed that monocyte transmigration down-regulates alpha-defensin protein expression. Overall, these data indicate that exposure of monocytes to stimulated ECs promotes further monocyte recruitment and inhibits monocyte apoptosis. Unexpectedly, following transmigration monocytes displayed reduced anti-microbial expression. Monocytes were allowed to transmigrate across IL1b stimulated HUVEC. Monocytes which transmigrated and monocytes which failed to transmigrate were collected after 1.5 hours from 3 experimental replicates of different human donors. Untreated monocytes were collected as a control from 4 human donors. Monocytes incubated beneath stimulated HUVEC for 1.5 hours were also collected from four human donors as a control.

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Extracellular vesicles derived from induced pluripotent stem cells mediate anti-inflammatory effects in human primary macrophages. iPSC EV Dataset

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