Differentiation Of Human Mesenchymal Stromal Cells Research Articles

Human Platelet Lysate as Alternative of Fetal Bovine Serum for Enhanced Human In Vitro Bone Resorption and Remodeling.

IntroductionTo study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells.MethodsWe compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs.Results and DiscussionSupplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations.ConclusionWe conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.

Inhibition of hypoxia-induced Mucin 1 alters the proteomic composition of human osteoblast-produced extracellular matrix, leading to reduced osteogenic and angiogenic potential.

The bone microenvironment is one of the most hypoxic regions of the human body and in experimental models; hypoxia inhibits osteogenic differentiation of mesenchymal stromal cells (MSCs). Our previous work revealed that Mucin 1 (MUC1) was dynamically expressed during osteogenic differentiation of human MSCs and upregulated by hypoxia. Upon stimulation, its C‐terminus (MUC1‐CT) is proteolytically cleaved, translocases to the nucleus, and binds to promoters of target genes. Therefore, we assessed the MUC1‐mediated effect of hypoxia on the proteomic composition of human osteoblast‐derived extracellular matrices (ECMs) and characterized their osteogenic and angiogenic potentials in the produced ECMs. We generated ECMs from osteogenically differentiated human MSC cultured in vitro under 20% or 2% oxygen with or without GO‐201, a MUC1‐CT inhibitor. Hypoxia upregulated MUC1, vascular endothelial growth factor, and connective tissue growth factor independent of MUC1 inhibition, whereas GO‐201 stabilized hypoxia‐inducible factor 1‐alpha. Hypoxia and/or MUC1‐CT inhibition reduced osteogenic differentiation of human MSC by AMP‐activated protein kinase/mTORC1/S6K pathway and dampened their matrix mineralization. Hypoxia modulated ECMs by transforming growth factor‐beta/Smad and phosphorylation of NFκB and upregulated COL1A1, COL5A1, and COL5A3. The ECMs of hypoxic osteoblasts reduced MSC proliferation and accelerated their osteogenic differentiation, whereas MUC1‐CT‐inhibited ECMs counteracted these effects.In addition, ECMs generated under MUC1‐CT inhibition reduced the angiogenic potential independent of oxygen concentration. We claim here that MUC1 is critical for hypoxia‐mediated changes during osteoblastogenesis, which not only alters the proteomic landscape of the ECM but thereby also modulates its osteogenic and angiogenic potentials.

Screening for Growth-Factor Combinations Enabling Synergistic Differentiation of Human MSC to Nucleus Pulposus Cell-Like Cells

Background: Multiple studies have examined the potential of growth factors (GF) to enable mesenchymal stromal cells (MSC) to nucleus pulposus (NP) cell-like cell differentiation. Here we screened a wide range of GF and GF combinations for supporting NP cell-like cell differentiation. Methods: Human MSC were stimulated using 86 different GF combinations of TGF-β1, -2, -3, GDF5, -6, Wnt3a, -5a, -11, and Shh. Differentiation potency was assessed by alcian blue assay and NP cell marker expression (e.g., COL2A1, CD24, etc.). The top four combinations and GDF5/TGF-β1 were further analyzed in 3D pellet cultures, on their ability to similarly induce NP cell differentiation. Results: Almost all 86 GF combinations showed their ability to enhance proteoglycan production in alcian blue assay. Subsequent qPCR analysis revealed TGF-β2/Wnt3a, TGF-β1/Wnt3a, TGF-β1/Wnt3a/GDF6, and Wnt3a/GDF6 as the most potent combinations. Although in pellet cultures, these combinations supported NP marker expression, none showed the ability to significantly induce chondrogenic NP matrix production. Only GDF5/TGF-β1 resulted in chondrogenic pellets with significantly enhanced glycosaminoglycan content. Conclusion: GDF5/TGF-β1 was suggested as an optimal GF combination for MSC to NP cell induction, although further assessment using a 3D and in vivo environment is required. Wnt3a proved promising for monolayer-based NP cell differentiation, although further validation is required.

Effects of Fiber Alignment and Coculture with Endothelial Cells on Osteogenic Differentiation of Mesenchymal Stromal Cells.

Vascularization is a critical process during bone regeneration. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of regenerated bone. One strategy for improving the survival and osteogenesis of tissue-engineered bone grafts involves the coculture of endothelial cells (ECs) with mesenchymal stromal cells (MSCs). Moreover, bone regeneration is especially challenging due to its unique structural properties with aligned topographical cues, with which stem cells can interact. Inspired by the aligned fibrillar nanostructures in human cancellous bone, we fabricated polycaprolactone (PCL) electrospun fibers with aligned and random morphology, cocultured human MSCs with human umbilical vein ECs (HUVECs), and finally investigated how these two factors modulate osteogenic differentiation of human MSCs (hMSCs). After optimizing cell ratio, a hMSCs/HUVECs ratio (90:10) was considered to be the best combination for osteogenic differentiation. Coculture results showed that hMSCs and HUVECs adhered to and proliferated well on both scaffolds. The aligned structure of PCL fibers strongly influenced the morphology and orientation of hMSCs and HUVECs; however, fiber alignment was observed to not affect alkaline phosphate (ALP) activity or mineralization of hMSCs compared with random scaffolds. More importantly, cocultured cells on both random and aligned scaffolds had significantly higher ALP activities than monoculture groups, which indicated that coculture with HUVECs provided a larger relative contribution to the osteogenesis of hMSCs compared with fiber alignment. Taken together, we conclude that coculture of hMSCs with ECs is an effective strategy to promote osteogenesis on electrospun scaffolds, and aligned fibers could be introduced to regenerate bone tissues with oriented topography without significant deleterious effects on hMSCs differentiation. This study shows the ability to grow oriented tissue-engineered cocultures with significant increases in osteogenesis over monoculture conditions. Impact statement This work demonstrates an effective method of enhancing osteogenesis of mesenchymal stromal cells on electrospun scaffolds through coculturing with endothelial cells. Furthermore, we provide the optimized conditions for cocultures on electrospun fibrous scaffolds and engineered bone tissues with oriented topography on aligned fibers. This study demonstrates promising findings for growing oriented tissue-engineered cocultures with significant increase in osteogenesis over monoculture conditions.

Platelet-Rich Plasma Accelerates Bone Differentiation in Human Adipose-Derived Mesenchymal Stromal Cells: An Experimental Study

Background: Mesenchymal stromal cells (MSCs) have high differentiation potential into different cell lines like osteoblasts. Osteogenic differentiation can be regulated through various and complex mechanisms, such as changing the microRNAs expression level. Although platelet-rich plasma (PRP) has been used in MSCs’ osteogenic differentiation process, the molecular mechanisms underlying the effect of PRP induction of osteogenesis by microRNAs is not well understood. Objectives: We evaluated the effect of PRP on the expression of miR-29a, miR-29b, and miR-155 in the PRP-based osteogenic differentiation of human MSCs. Methods: This experimental study was conducted on healthy individuals referred to Taleghani Hospital in Ahvaz, Iran, for abdominoplasty from September 2017 to April 2018. Stromal cells were isolated from human adipose tissue and differentiated into osteoblasts. Effect of 10% PRP on osteoblasts differentiation was monitored by the measurement of alkaline phosphatase activity and calcium deposition. We also evaluated gene expression of the Runx2 and the OPN along with the expression of miRNAs. All tests were performed in triplicate. Results: Treatment of MSCs with 10% PRP resulted in induction of osteogenic differentiation by a significant upregulation of the miR-29a/b (miR-29a: 5.27 (0.77), P < 0.01 (day 3) and 3.76 (0.124), P < 0.01 (day 14); miR-29b: 3.11 (0.35), P < 0.001 (day 3) and 4.25 (0.304), P < 0.01 (day 14)) and a significant downregulation in the miR-155 expression (0.62 (0.006), P < 0.001 (day 3) and 0.55 (0.114), P < 0.05 (day 14)). Conclusions: The remarkable rise in the expression of important osteoblast genes, alkaline phosphatase activity, and calcium deposition verified accelerated differentiation. The present study showed that microRNAs such as miR-29a/b and miR-155 play an active role in the process of bone differentiation during PRP treatment, which in turn, affects mesenchymal cells signaling pathways.

Guanylate Binding Protein 1 Inhibits Osteogenic Differentiation of Human Mesenchymal Stromal Cells Derived from Bone Marrow

Guanylate Binding Proteins (GBPs) are a group of cytokine-inducible large guanosine triphosphatase. Previous studies have shown high expression of GBP1 in circulating monocytes of premenopausal subjects was correlated to extremely low peak bone mass, which is considered as an important determinant of osteoporosis. However, whether GBPs play a role in regulation of osteogenesis of mesenchymal stromal cells (MSCs) remains largely unknown. In the present study, we found that mRNA expression of GBP1 was highest among all the GBPs, and it was dramatically downregulated during osteogenic differentiation of human MSCs derived from bone marrow (hBM-MSCs). While siRNA-mediated knockdown of GBP1 promoted osteogenesis, overexpression of GBP1 suppressed osteogenesis of hBM-MSCs. Furthermore, we found GBP1 is required for expression of indoleamine 2,3 dioxygenase (IDO), Interleukin 6 (IL-6) and IL-8 induced by treatment with Interferon-γ (IFN-γ). Depletion of GBP1 rescued the inhibited osteogenesis induced by IFN-γ treatment, at least in part. Collectively, our findings indicate GBP1 inhibits osteogenic differentiation of MSCs, and inhibition of GBP1 expression may prevent development of osteoporosis and facilitate MSC-based bone regeneration.

Distinctive expression pattern of cystathionine-β-synthase and cystathionine-γ-lyase identifies mesenchymal stromal cells transition to mineralizing osteoblasts.

Mesenchymal stromal cells (MSCs) are key players in the repair or regeneration of the damaged bone tissue. However, heterogeneity exists between MSCs derived from different donors in their bone formation ability both in vitro and in vivo. The identification of markers defining MSCs with different functional phenotypes is fundamental to maximize their clinical potential. In our previous in vivo study, impaired expression in MSCs of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), the two key enzymes in the catabolic pathway of homocysteine, was associated to decreased bone formation and to the onset of osteoporosis in mice. Here, we investigated whether osteogenic differentiation of human MSCs (hMSCs) modulates the expression of CBS and CSE. The expression of CBS and CSE was also assessed during chondrogenesis to confirm the specificity of their expression during osteogenesis. hMSCs displayed a heterogeneous mineralizing capacity between donors (70% of the samples mineralized, while 30% did not mineralize). Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs. In particular, up-regulation of CSE was restricted to hMSCs undergoing mineralization. During chondrogenesis, CBS was significantly up-regulated while CSE expression was not affected. Ex-vivo findings confirmed that mature h-osteoblasts (hOBs) show consistently higher expression of CBS and CSE than hMSCs. Our data provide the first evidence that the expression of CBS and CSE in hMSCs closely correlates with the transition of hMSCs toward the osteoblastic phenotype and that CSE may constitute a novel marker of osteogenic differentiation.

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.

Corrigendum: Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes.

Scientific Reports 6: Article number: 26014; published online: 20 May 2016; updated: 08 November 2016.

Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes

Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.

Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential.Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3–6: 100 ng/ml; days 7–10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure.Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization.The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cellsin vitro

T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset.

Monitoring human mesenchymal stromal cell differentiation by electrochemical impedance sensing

For their wide mesodermal differentiation potential, mesenchymal stromal/stem cells (MSC) are attractive candidates for tissue engineering. However, standardized quality control assays monitoring differentiation that are non-invasive and continuous over time are lacking. We employed a non-invasive assay, using two different systems, to discriminate osteogenic and adipogenic differentiation of MSC by monitoring impedance. Fibroblasts and keratinocytes served as non-specific controls. Impedance profiles were recorded comparing MSC from bone marrow and adipose tissue, either non-induced or induced for osteogenesis or adipogenesis, for 5-14 days, and correlated with differentiation markers assessed by reverse transcription-quantitative polymerase chain reaction and Western blot. Additionally, differentiation modulating effects of extracellular matrix components were analyzed. Adhesion and growth-related impedance profiles of non-induced MSC roughly resembled those of fibroblasts, whereas keratinocytes differed significantly. Distinct from that, osteogenic induction of MSC revealed initially rapid and continuously rising impedance, corresponding to mineralized calcium matrix formation. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte-like cells with numerous lipid vacuoles. Pre-coating with either collagen type I or IV apparently favored osteogenesis and fibronectin adipogenesis. Impedance recordings correlated well with the extent of differentiation evaluated by histochemical staining and protein and gene expression. Overall, our data demonstrate that impedance profiling offers a basis for standardized real-time, non-invasive high-throughput screening of MSC properties. It enables further testing of the influence of diffusible factors or extracellular matrix composites on MSC differentiation or maintenance of stemness, thus substantiating therapeutic application.

Identification of early lineage specific marker genes of human mesenchymal stromal cell differentiation into osteoblast and adipocytes

Identification of early lineage specific marker genes of human mesenchymal stromal cell differentiation into osteoblast and adipocytes

Gelatin/PLLA Sponge‐Like Scaffolds Allow Proliferation and Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].

Comparative Study of Ceramics Structure for Culturing Human Mesenchymal Stromal Cells

Hydroxyapatite (HA) ceramics together with various kinds of osteogenic cells have been used in bone tissue engineering. It is well known that the ceramics structure and composition affect cell proliferation / differentiation. In this study, three different types of HA ceramics were used to investigate initial cell attachment followed by osteoblastic differentiation of human mesenchymal stromal cells (MSCs). The results indicated that micro-pore affected the cell attachment and porosity (pore diameter and inter-pore connection) was the key to allow spacious distribution of the viable cells in the ceramics. This study also confirmed that surface pore areas of HA ceramics support the differentiation of human MSCs and thus the ceramics have the capability to regenerate damaged bone tissue.

T‐cell cytokine induction of BMP‐2 regulates human mesenchymal stromal cell differentiation and mineralization

How T-cells, attracted to local sites of inflammation in arthritides, affect heterotopic ossification is presently unknown. Here, we tested the hypothesis that T-cell cytokines play a role in the differentiation of human mesenchymal stromal cells (HMSC) into the osteoblast phenotype by inducing autologous BMP-2, providing a possible mechanism for heterotopic ossification. HMSC from multiple donor bones were treated with either activated T-cell conditioned medium (ACTTCM) or physiological concentrations of the major inflammatory cytokines, TNF-alpha, TGF-beta, IFN-gamma, and IL-17 (TTII), individually or in combinations. ACTTCM induced BMP-2 protein in a time-dependent manner over a 48 h period and alkaline phosphatase (AlkP) within 7 days. In combination, TTII, like ACTTCM, induced AlkP and synergistically induced BMP-2 protein. Either individually, or in combinations of up to three, the T-cell cytokines failed to induce BMP-2 above control levels while a combination of all four cytokines synergistically induced BMP-2 10-fold as assessed by ELISA. TTII induced mineralized matrix as effectively as dexamethasone. Inhibition of p38 MAPK completely inhibited TTII-induced BMP-2 production and matrix mineralization. Real time RT-PCR analysis demonstrated a striking early (within 4 h) increase in BMP-2 gene expression by TTII, which was suppressed by p38 MAP kinase inhibition. In localized chronic inflammatory diseases, T-cell cytokines released at localized sites of inflammation may be the driving force for differentiation of local mesenchymal stromal cells into the osteoblast phenotype thereby playing a significant role in the heterotopic ossification observed in these diseases.