Adipogenic Differentiation Of Mesenchymal Stromal Cells Research Articles

Epigenetic regulation by long noncoding RNAs in osteo-/adipogenic differentiation of mesenchymal stromal cells and degenerative bone diseases.

Bone is a complex tissue that undergoes constant remodeling to maintain homeostasis, which requires coordinated multilineage differentiation and proper proliferation of mesenchymal stromal cells (MSCs). Mounting evidence indicates that a disturbance of bone homeostasis can trigger degenerative bone diseases, including osteoporosis and osteoarthritis. In addition to conventional genetic modifications, epigenetic modifications (i.e., DNA methylation, histone modifications, and the expression of noncoding RNAs) are considered to be contributing factors that affect bone homeostasis. Long noncoding RNAs (lncRNAs) were previously regarded as ‘transcriptional noise’ with no biological functions. However, substantial evidence suggests that lncRNAs have roles in the epigenetic regulation of biological processes in MSCs and related diseases. In this review, we summarized the interactions between lncRNAs and epigenetic modifiers associated with osteo-/adipogenic differentiation of MSCs and the pathogenesis of degenerative bone diseases and highlighted promising lncRNA-based diagnostic and therapeutic targets for bone diseases.

Mesenchymal Stromal Cells Regulate Sialylations of N-Glycans, Affecting Cell Migration and Survival.

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.

The oncometabolite R-2-hydroxyglutarate dysregulates the differentiation of human mesenchymal stromal cells via inducing DNA hypermethylation

BackgroundIsocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs.MethodsHuman bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in.ResultsR-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition.ConclusionsThe oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.

Evaluation of 3D Printed Gelatin-Based Scaffolds with Varying Pore Size for MSC-Based Adipose Tissue Engineering.

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion-based 3D printing is used to fabricate photo-crosslinkable gelatin-based scaffolds with pore sizes in the range of 200-600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC-based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.

Potential Marker Genes for Predicting Adipogenic Differentiation of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are a promising source for tissue engineering of soft connective tissues. However, the differentiation capacity of MSCs varies among individual cell lines. Here, we show marker genes to predict the adipogenic potential of MSCs. To clarify the correlation between gene expression patterns before adipogenic induction and the differentiation level of MSCs after differentiation, we compared mRNA levels of 95 genes and glycerol-3-phosphate dehydrogenase (GPDH) activities in 15 MSC lines (five jaw and 10 ilium MSCs) from 15 donors. Expression profiles of 22 genes before differentiation significantly correlated with GPDH activities after differentiation. Expression levels of 11 out of the 22 genes in highly potent ilium MSCs were at least three times higher compared with jaw MSCs, which have limited differentiation potential. Furthermore, three-dimensional scatter plot for mRNA expression of ITGA5, CDKN2D, and CD74 could completely distinguish highly potent MSCs from poorly potent MSCs for adipogenesis. The treatment of MSC cultures with the anti-ITGA5 antibody reduced adipogenic differentiation of MSCs. Collectively, these results suggest that the three genes play a role in adipogenesis before induction and can serve as predictors to select potent MSCs for adipogenic differentiation.

Thy-1: more than a marker for mesenchymal stromal cells.

Balanced differentiation of mesenchymal stromal cells (MSCs) into osteoblasts and adipocytes is essential for healthy bone and fat homeostasis. A disturbed balance of MSC differentiation results in bone loss and increased adipogenesis as observed in several human conditions, such as osteoporosis, obesity, and aging. This reflects the importance of MSC fate decision. Therefore, it is important to identify factors that regulate the balance between osteogenic and adipogenic differentiation of MSCs so as to identify new targets to improve bone formation in osteoporosis and control adipose tissue development. There is accumulating evidence that thymus cell antigen 1 (Thy-1), also known as CD90, expressed on MSCs, plays a critical role in the fate decision of MSCs. Recently, Thy-1 has been shown to promote osteogenic differentiation of MSCs and thus bone formation while inhibiting adipogenic differentiation and restricting adipose tissue accumulation. The clinical importance of these findings was validated by the detection of reduced serum soluble Thy-1 in patients with disturbed bone remodeling. In this review, we aim to summarize data on the impact of Thy-1 on the control of MSC differentiation and its consequences for bone and fat formation.-Saalbach, A., Anderegg, U. Thy-1: more than a marker for mesenchymal stromal cells.

AML-induced osteogenic differentiation in mesenchymal stromal cells supports leukemia growth.

Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

Labeling Mesenchymal Stromal Cells with PKH26 or VybrantDil Significantly Diminishes their Migration, but does not affect their Viability, Attachment, Proliferation and Differentiation Capacities

Fluorescent dyes such as PKH26 and VybrantDil facilitate rapid and simple labeling of cells for later detection in various assays. But covering the cell surface with lipophilic fluorescent dyes may impair cellular functions. We therefore investigated the effects of PKH26 and VybrantDil on viability, proliferation, differentiation, attachment and migration of human mesenchymal stromal cells (MSCs) in vitro. To this end, MSCs were harvested from bone marrow of 12 individuals, expanded employing methods compliant to good manufacturing practice, and stained with PKH26 or VybrantDil. MSCs without label served as controls. The intensity of fluorescent staining as function of label and incubation time was investigated. Viability and proliferation were enumerated by cell counting. The osteogenic and adipogenic differentiations of MSCs were explored by cytochemical staining and transcript analyses, the cell migration and attachment by specific in vitro assays. We report that labeling of human MSCs with PKH26 yielded brighter signals facilitating prolonged detection compared to VybrantDil. No significant effects of PKH26 and VybrantDil were recorded when the viability, proliferation, attachment, osteogenic and adipogenic differentiation of human MSCs were investigated. But loading cells with PKH26 or VybrantDil significantly diminished the migration of the MSCs in vitro. We conclude that analyses depending on cellular migration may be biased when the cells are loaded with these lipophilic dyes.