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Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

ABSTRACT: Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

SUBMITTER: Rinker TE

PROVIDER: S-EPMC3965183 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

Rinker Torri E TE Hammoudi Taymour M TM Kemp Melissa L ML Lu Hang H Temenoff Johnna S JS

Integrative biology : quantitative biosciences from nano to macro 20140124 3

Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D ...[more]

PMID: 24463781

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Project description:BackgroundThe use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA.ResultsRelative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P < 0.001) and 5-bromo-2'-deoxyuridine (BrdU) incorporation (167 ± 6 and 120 ± 6%, respectively; P < 0.001). Treatment with DEX increased ALP activity compared with control (1,638 ± 38%; P < 0.001). In the absence and presence of Dex, BMP-2 did not alter ALP activity (P > 0.8). Runt-related transcription factor2 expression increased 3-fold (P < 0.001) by d 6 of culture. Osterix expression increased 9-fold (P < 0.05) by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 (P < 0.01); however expression was reduced 4-fold at d 18 (P < 0.01).ConclusionsDexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.

Project description:BackgroundOsteoporosis is a common bone disease in elderly population caused by imbalanced bone formation and bone resorption. Mesenchymal stem cells (MSCs) are responsible for maintaining this bone homeostasis. The phenotype of transmembrane 9 superfamily 4 (TM9SF4) knockout mice suggests a relationship between TM9SF4 proteins and bone homeostasis. But the effect of TM9SF4 in osteology has never been reported. In the present study, we investigated the function of TM9SF4 in MSC differentiation commitment, as well as its role in osteoporosis.MethodsPrimary bone marrow MSCs, isolated from TM9SF4 wildtype (TM9SF4+/+) and knockout (TM9SF4-/-) mice, were induced to differentiate into osteoblasts or adipocytes, respectively. The osteogenesis was examined by qRT-PCR detection of osteogenic markers, ALP staining and Alizarin Red S staining. The adipogenesis was tested by qRT-PCR quantification of adipogenic markers and Oil Red O staining. The cytoskeletal organization of MSCs was observed under confocal microscope. The osteoporotic model was induced by ovariectomy in TM9SF4+/+ and TM9SF4-/- mice, followed by Toluidine blue and H&E staining to assess lipid accumulation in trabecular bones, as well as micro-computed tomography scanning and immunohistochemistry staining for bone mass density assessment. The experiments on signaling pathways were conducted using qRT-PCR, Western blot and Alizarin Red S staining.ResultsWe determined the role of TM9SF4 in MSC differentiation and found that TM9SF4-/- MSCs had higher potential to differentiate into osteoblasts and lower capability into adipocytes, without affecting osteoclastogenesis in vitro. In ovariectomy-induced osteoporotic model, TM9SF4-/- mice retained higher bone mass and less lipid accumulation in trabecular bones, indicating an important role of TM9SF4 in the regulation of osteoporosis. Mechanistically, TM9SF4-depleted cells showed elongated actin fibers, which may act through mTORC2/Akt/β-catenin pathway to promote their commitment into osteoblasts. Furthermore, TM9SF4-depleted cells showed higher activity of canonical Wnt pathway, suggesting the participation of Wnt/β-catenin during TM9SF4-regulated osteogenesis.ConclusionsOur study demonstrates TM9SF4 as a novel regulator for MSC lineage commitment. Depletion of TM9SF4 preferentially drives MSCs into osteoblasts instead of adipocytes. Furthermore, TM9SF4-/- mice show delayed bone loss and reduced lipid accumulation during ovariectomy-induced osteoporosis. Our results indicate TM9SF4 as a promising target for the future clinical osteoporotic treatment.

Project description:BackgroundIn osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model.ResultsWe observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts.ConclusionsHere, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.

Dataset Information

Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

Publications

Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

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