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Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

ABSTRACT: Since the reconstruction of large bone defects remains a challenge, knowledge about the biology of bone healing is desirable to develop novel strategies for improving the treatment of bone defects. In osteoimmunology, macrophages are the central component in the early stage of physiological response after bone injury and bone remodeling in the late stage. During this process, a switch of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) is observed. An appealing option for bone regeneration would be to exploit this regulatory role for the benefit of osteogenic differentiation of osteoprogenitor cells (e.g., mesenchymal stem cells; MSCs) and to eventually utilize this knowledge to improve the therapeutic outcome of bone regenerative treatment. In view of this, we focused on the in vitro interaction of different macrophage subtypes with adipose tissue MSCs to monitor the behavior (i.e. proliferation, differentiation and mineralization) of the latter in dedicated co-culture models. Our data show that co-culture of MSCs with M2 macrophages, but not with M1 macrophages or M0 macrophages, results in significantly increased MSC mineralization caused by soluble factors. Specifically, M2 macrophages promoted the proliferation and osteogenic differentiation of MSCs, while M0 and M1 macrophages solely stimulated the osteogenic differentiation of MSCs in the early and middle stages during co-culture. Secretion of the soluble factors oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) by macrophages showed correlation with MSC gene expression levels for OSM-receptor and BMP-2, suggesting the involvement of both signaling pathways in the osteogenic differentiation of MSCs.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5552848 | biostudies-other | 2017 Mar

REPOSITORIES: biostudies-other

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Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

Zhang Yang Y Böse Thomas T Unger Ronald E RE Jansen John A JA Kirkpatrick Charles James CJ van den Beucken Jeroen J J P JJJP

Cell and tissue research 20170330 2

Since the reconstruction of large bone defects remains a challenge, knowledge about the biology of bone healing is desirable to develop novel strategies for improving the treatment of bone defects. In osteoimmunology, macrophages are the central component in the early stage of physiological response after bone injury and bone remodeling in the late stage. During this process, a switch of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) is observed. An appealing option fo ...[more]

PMID: 28361303

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Project description:Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

Project description:Human adipose tissue has been recently recognized as a potential source of stem cells for regenerative medicine applications, including bone tissue engineering (TE). Despite the gathered knowledge regarding the differentiation potential of human adipose tissue-derived stem cells (hASCs), in what concerns the endothelial lineage many uncertainties are still present. The existence of a cell subpopulation within the human adipose tissue that expresses a SSEA-4 marker, usually associated to pluripotency, raises expectations on the differentiation capacity of these cells (SSEA-4(+)hASCs). In the present study, the endothelial and osteogenic differentiation potential of the SSEA-4(+)hASCs was analyzed, aiming at proposing a single-cell source/subpopulation for the development of vascularized bone TE constructs. SSEA-4(+)hASCs were isolated using immunomagnetic sorting and cultured either in α-MEM, in EGM-2 MV (endothelial growth medium), or in osteogenic medium. SSEA-4(+)hASCs cultured in EGM-2 MV formed endothelial cell-like colonies characterized by a cobblestone morphology and expression of CD31, CD34, CD105, and von Willebrand factor as determined by quantitative reverse transcriptase (RT)-polymerase chain reaction, immunofluorescence, and flow cytometry. The endothelial phenotype was also confirmed by their ability to incorporate acetylated low-density lipoprotein and to form capillary-like structures when seeded on Matrigel. SSEA-4(+)hASCs cultured in α-MEM displayed fibroblastic-like morphology and exhibited a mesenchymal surface marker profile (>90% CD90(+)/CD73(+)/CD105(+)). After culture in osteogenic conditions, an overexpression of osteogenic-related markers (osteopontin and osteocalcin) was observed both at molecular and protein levels. Matrix mineralization detected by Alizarin Red staining confirmed SSEA-4(+)hASCs osteogenic differentiation. Herein, we demonstrate that from a single-cell source, human adipose tissue, and by selecting the appropriate subpopulation it is possible to obtain microvascular-like endothelial cells and osteoblasts, the most relevant cell types for the creation of vascularized bone tissue-engineered constructs.

Dataset Information

Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

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Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

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