Osteogenesis Of MSCs Research Articles

Quantifying BMP2 Dynamics during Stem Cell Differentiation using, Real Time Imaging, Combined Confocal AFM and Systems Biology Approaches

Bone morphogenetic proteins (BMP2) are key regulators during mesenchymal stem (MSC) cell differentiation; however how these growth factors achieve lineage specification is not well understood. BMP-2, a frequently studied BMP, is known to induce adipogenesis, chondrogenesis and osteogenesis in MSCs, however the exact dynamics leading to the determination of the fate of the stem cell is not well understood. We hypothesize those regions on the plasma membrane, where BMP-2 binds direct signaling pathway and therefore regulate stem cell fate.In order to determine the dynamics of BMP2 and binding sites on the plasma membrane we covalently link Quantum Dots (QDs) to BMP-2. Creating stable fluorescent BMP2 allowed for the first time measurements of BMP2 dynamics on the cell surface in real time. QD’s, semi-conducting nanocrystals, superior to conventional organic dyes, are highly efficient due to high extinction coefficient and stability (minimizing photo bleaching), which are 20 times brighter and 100 times more stable than traditional fluorescent reporters. Additionally results were verified using combined AFM and Confocal imaging. Using mutated RFP tagged BMP type - I receptors, or gfp tagged caveolin-1 or AP-2 constructs we were able to identify different regions important for osteogenesis, chondrogenesis and adipogenesis. Further using these data combined with a systems biology approach created a signaling model for BMP2 during stem cell differentiation.Our data indicate BMP2 interacts with receptors localized in caveolae and clathrin coated pits and these domains are key regions for signaling. Dependent on BMP receptor localization MSCs are directed toward specific stem cell fate.

Interleukin-17A increases leptin production in human bone marrow mesenchymal stem cells

Lineage commitment of human bone marrow mesenchymal stem cells (hBM-MSCs) to adipocytes or osteoblasts has been suggested as a model system to study the relationship between type II diabetes and abnormal bone metabolism. Leptin and IL-17A inhibit adipogenesis whereas they promote osteogenesis in MSCs. Due to pathophysiologic roles of IL-17A in human metabolic diseases and bone metabolism, it was evaluated whether IL-17A-dependent inverse regulation on adipogenesis and osteogenesis was related to endogenous leptin production in hBM-MSCs. In the analysis of adiponectin and leptin secretion profiles of hBM-MSCs in response to various combinations of differentiation inducing factors, it was found that dexamethasone, a common molecule used for both adipogenesis and osteogenesis, increased leptin production in hBM-MSCs. Importantly, the level of leptin production during osteogenesis in hBM-MSCs was higher than that during adipogenesis, implicating a significant leptin production in extra-adipose tissues. IL-17A increased leptin production in hBM-MSCs and also under the condition of osteogenesis. In spite of direct inhibition on adipogenesis, IL-17A up-regulated leptin production in hBM-MSC-derived adipocytes. Anti-leptin antibody treatment partially antagonized the IL-17A dependent inhibition of adipogenesis in hBM-MSCs, suggesting a role of leptin in mediating the inverse regulation of IL-17A on osteogenesis and adipogenesis in hBM-MSCs. Therefore, the IL-17A-induced leptin production may provide a key clue to understand a molecular mechanism on the lineage commitment of hBM-MSCs into adipocytes or osteoblasts. In addition, leptin production in extra-adipose tissues like MSCs and osteoblasts should be considered in future studies on leptin-associated human diseases.

Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

Mesenchymal stem (stromal) cells (MSCs) are present in a variety of tissues during prenatal and postnatal human development. In adult organism, they are prevalent in bone marrow and supposed to be involved in space-flight induced osteopenia. We studied expression of various genes in human bone marrow MSCs after different terms of simulated microgravity (SMG) provided by Random Positioning Machine. Simulated microgravity induced transient changes in expression level of genes associated with actin cytoskeleton, especially after 48 h of SMG. However, after 120 h exposure in SMG partial restoration of gene expression levels (relative to the control) was found. Similar results were obtained with bmMSCs subjected to 24 h readaptation in static state after 24 h in SMG. Analysis of 84 genes related to identification, growth and differentiation of stem cells revealed that expression of nine genes was changed slightly after 48 h in SMG. More pronounced changes in gene expression of “stem cells markers” were observed after 120 h of simulated microgravity. Among 84 investigated genes, 30 were up-regulated and 24 were down-regulated. Finally, MSCs osteogenesis induced by long-term (10–20 days) simulation of microgravity was accompanied by down-regulation of gene expression of the main osteogenic differentiation markers ( ALPL, OMD) and master transcription osteogenic factor of MSCs ( Runx2). Thus, our study demonstrated that changes in expression level of some genes associated with actin cytoskeleton and stem cell markers are supposed to be one of the mechanisms, which contribute to precursor's cellular adaptation to the microgravity conditions. These results can clarify genomic mechanisms through which SMG reduces osteogenic differentiation of bmMSCs.

Rescuing replication and osteogenesis of aged mesenchymal stem cells by exposure to a young extracellular matrix

This study aimed to determine whether aging negatively affects MSC replication and osteogenesis and whether these features could be altered by exposure to an extracellular matrix (ECM) generated by marrow cells from young or old mice. A cell-free ECM was prepared from cultured femoral marrow cells from either 3- or 18-mo-old C57BL/6 mice (young-ECM or old-ECM, respectively). The replication and osteogenesis of young or old MSCs maintained on young-ECM vs. old-ECM as well as plastic were examined in vitro and in vivo. We found that the frequency of MSCs in marrow from old mice, measured by colony-forming cells, was only marginally lower than that of young mice. In contrast, defects in the self-renewal and bone formation capacity of old MSCs were remarkable. These defects were corrected by provision of a young-ECM but not old-ECM. In parallel cultures maintained on a young-ECM, the intracellular levels of reactive oxygen species from both old and young mice were reduced 30-50% compared to those maintained on old-ECM or plastic. We concluded that aging negatively affects the formation of an ECM that normally preserves MSC function, and aged MSCs can be rejuvenated by culture on a young-ECM.

Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Multiple Myeloma Patients Is Associated with a Failure In the Deactivation of the NOTCH Pathway

AbstractAbstract 1894Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients. Disclosures:No relevant conflicts of interest to declare.

Microsphere-based drug releasing scaffolds for inducing osteogenesis of human mesenchymal stem cells in vitro

In this study, in vitro osteogenesis was successfully achieved in human mesenchymal stem cells (hMSCs) by controlled release of the osteogenesis-inducing drugs dexamethasone, ascorbic acid (AA) and β-glycerophosphate (GP) from poly(lactic-co-glycolic acid) (PLGA) sintered microsphere scaffolds (SMS). We investigated the osteogenesis of human MSCs (hMSCs) on dexamethasone laden PLGA-SMS (PLGA-Dex-SMS), and dexamethasone, AA and GP laden PLGA-SMS (PLGA-Com-SMS). hMSCs cultured on the microsphere systems, which act as drug release vehicles and also promote cell growth/tissue formation—displayed a strong osteogenic commitment locally. The osteogenic commitment of hMSCs on the scaffolds were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real-time PCR and immunohistochemistry analysis. The results indicated hMSCs cultured on PLGA-Com-SMS exhibited superior osteogenic differentiation owing to significantly high phenotypic expression of typical osteogenic genes—osteocalcin (OC), type I collagen, alkaline phosphatase (ALP), and Runx-2/Cbfa-1, and protein secretion of bone-relevant markers such as osteoclast and type I collagen when compared with PLGA-Dex-SMS. In conclusion, by promoting osteogenic development of hMSCs in vitro, this newly designed controlled release system opens a new door to bone reparation and regeneration.

VN and Col I differentially regulate osteogenesis in MSCs

VN and Col I differentially regulate osteogenesis in MSCs

The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells

Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg–Gly–Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 m m) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 m m RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 m m being optimal concentration.