Project description:Our previous studies have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is markedly induced in human primary endometrial stromal cells (HESC) as they undergo differentiation in response to steroid hormones and cAMP. WNT4 is a downstream target of BMP2 regulation in these cells. To identify the common downstream targets of BMP2 and WNT4 in human endometrial stromal cells, we performed gene expression profling of human ensometrial stromal cell transduced with BMP2 or WNT4 adenovirus. Gene expression profiling revealed that FOXO1, a forkhead family transcription factor and a known regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. These studies uncovered a linear pathway involving BMP2, WNT4, and FOXO1 that operates in human endometrium to critically control decidualization.
Project description:Our previous studies have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFM-NM-2 superfamily, is markedly induced in human primary endometrial stromal cells (HESC) as they undergo differentiation in response to steroid hormones and cAMP. WNT4 is a downstream target of BMP2 regulation in these cells. To identify the common downstream targets of BMP2 and WNT4 in human endometrial stromal cells, we performed gene expression profling of human ensometrial stromal cell transduced with BMP2 or WNT4 adenovirus. Gene expression profiling revealed that FOXO1, a forkhead family transcription factor and a known regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. These studies uncovered a linear pathway involving BMP2, WNT4, and FOXO1 that operates in human endometrium to critically control decidualization. Human endometrial stromal cells were transduced with recombinant adenovirus expressing BMP2, WNT4, or a negative control GFP at MOI 50:1 in 2 ml of culture medium. After transduction for 24 h, the viral particles were removed and the cells were treated with E+P for 3 days to induce decidualization (n=3 for each treatment), pooled total RNA from these cells was then hybridized to high density affymetrix microarrays according to the Affymetrix protocol (Human Genome HG-U133 A2.0 Array) .
Project description:Human mesenchymal stem cells (MSC) display a high potential for the development of novel treatment strategies for cartilage repair. However, the pathways involved in their differentiation to functional and non hypertrophic chondrocytes remain largely unknown, despite the work on embryologic development and the identification of key growth factors including members of the TGFbeta, Hh, Wnt and FGF families. In this study, we asked if we could identify specific biological networks independently from the growth factor used (TGFbeta-3 or BMP-2). To address this question, we used DNA microarrays and performed large-scale expression profiling of MSC at different time points during their chondral differentiation. By comparing these data with those obtained during their differentiation into osteoblasts and adipocytes, we identified 318 genes specific for chondrogenesis. We distributed the selected genes in 5 classes according to their kinetic of expression and used the Ingenuity software in order to identify new biological networks. We could reconstruct 3 phases for chondral differentiation, characterized by functional pathways. The first phase corresponds to cell attachment and apoptosis prevention with the up-regulation of α5 integrins, BCL6, NFIL3, RGS2 and down-regulation of CTGF and CYR61. The second phase is characterized by a proliferation/differentiation step with the continuous expression of MAF, PGF, HGMA1 or NOTCH3, CHI3L1, WNT5A, LEPR. Finally, the last step of differentiation/hypertrophy is characterized by expression of DKK1, APOD/E, SERPINF1 and TIMP4. These data propose new pathways to understand the complexity of MSC differentiation to chondrocytes and new potential targets for cell therapy applied to cartilage repair. Experiment Overall Design: To identify genes involved in TGFbeta03/BMP2-induced chondrogenesis, MSC were isolated from human bone marrow aspirates by adherence to culture dishes and cultivated to passage 3. After induction of chondrogenic differentiation with BMP2 or TGFbeta-3, gene expression was determined by microarray hybridization after 1, 3, 7 and 21 days and compared to the undifferentiated MSC.
Project description:In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared to healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis.
Project description:Multipotent progenitor cells (iMPCs) created from induced pluripotent stem cells (iPSCs) is a promising cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) were shown to significantly enhance transforming growth factor-b (TGFb)-induced iMPC chondrogenesis. In contrast, TGFb alone is sufficient to induce robust chondrogenesis of human primary mesenchymal stromal cells (MSCs). Currently, the mechanism underlying this difference between iMPCs and MSCs has not been fully understood. In this study, we first generated iMPCs from human iPSCs and examined their differentiation capacity at different passages. We then optimized the conditions for chondrogenesis and determined that medium supplemented with TGFb and BMP6 led to robust cartilage formation from iMPCs with minimal hypertrophy. Moreover, the cartilage generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and led to a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFb alone induced phosphorylation of Smad2/3 in iMPCs but not Smad1/5, which led to poor chondrogenesis. In contrast, TGFb resulted in the phosphorylation of both Smad2/3 and Smad1/5 and robust chondrogenesis in human MSCs. We further discovered that the remarkably low level of Activin receptor-like kinase 1 (ACVRL1/ALK1) in iMPCs, when compared to MSCs, partially accounts for this difference. For instance, overexpression of ALK1 in iMPCs activated both Smad1/5 and Smad2/3 upon TGFb only treatment and resulted in a high level of chondrogenesis, which was not observed in control iMPCs. In summary, this study describes a robust method to generate chondrocytes with low hypertrophy for hyaline cartilage repair and elucidates the difference between MSCs and iMPCs in response to TGFb.
Project description:Multipotent progenitor cells (iMPCs) created from induced pluripotent stem cells (iPSCs) is a promising cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) were shown to significantly enhance transforming growth factor-b (TGFb)-induced iMPC chondrogenesis. In contrast, TGFb alone is sufficient to induce robust chondrogenesis of human primary mesenchymal stromal cells (MSCs). Currently, the mechanism underlying this difference between iMPCs and MSCs has not been fully understood. In this study, we first generated iMPCs from human iPSCs and examined their differentiation capacity at different passages. We then optimized the conditions for chondrogenesis and determined that medium supplemented with TGFb and BMP6 led to robust cartilage formation from iMPCs with minimal hypertrophy. Moreover, the cartilage generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and led to a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFb alone induced phosphorylation of Smad2/3 in iMPCs but not Smad1/5, which led to poor chondrogenesis. In contrast, TGFb resulted in the phosphorylation of both Smad2/3 and Smad1/5 and robust chondrogenesis in human MSCs. We further discovered that the remarkably low level of Activin receptor-like kinase 1 (ACVRL1/ALK1) in iMPCs, when compared to MSCs, partially accounts for this difference. For instance, overexpression of ALK1 in iMPCs activated both Smad1/5 and Smad2/3 upon TGFb only treatment and resulted in a high level of chondrogenesis, which was not observed in control iMPCs. In summary, this study describes a robust method to generate chondrocytes with low hypertrophy for hyaline cartilage repair and elucidates the difference between MSCs and iMPCs in response to TGFb.
Project description:Microarray analysis of bone marrow multipotent mesenchymal stromal cells isolated from type 1 diabetes patients and healthy donors.
Project description:Temporal data on gene expression and context-specific open chromatin states can improve identification of key transcription factors (TFs) and the gene regulatory networks (GRNs) controlling cellular differentiation. However, their integration remains challenging. Here, we delineate a general approach for data-driven and unbiased identification of key TFs and dynamic GRNs, called EPIC-DREM. We generated time-series transcriptomic and epigenomic profiles during differentiation of mouse multipotent bone marrow stromal cells (MSCs) towards adipocytes and osteoblasts. Using our novel approach we constructed time-resolved GRNs for both lineages and identifed the shared TFs involved in both differentiation processes. To take an alternative approach to prioritize the identified shared regulators, we mapped dynamic super-enhancers in both lineages and associated them to target genes with correlated expression profiles. The combination of the two approaches identified aryl hydrocarbon receptor (AHR) and Glis family zinc finger 1 (GLIS1) as mesenchymal key TFs controlled by dynamic MSC-specific super-enhancers that become repressed in both lineages. AHR and GLIS1 control differentiation-induced genes and we propose they function as guardians of mesenchymal multipotency.
Project description:Multipotent Mesenchymal Stromal Cells (MSCs) are multipotent adult cells that can be isolated from different tissues. The current hypothesis supports that the main MSCs action mechanism relates to its paracrine activity, creating a microenvironment with trophic signals. However, preclinical studies with different sources of MSCs and different animal models have shown conflicting results. Therefore, the evaluation of these cells secretome content is of great interest. Here we analyzed the secreted proteins of MSCs, isolated from 3 different sources (adipose tissue, uterine tubes and skeletal muscle), obtained from 5 different donors. Following MSCs characterization, proteins secreted in serum-free conditions (conditioned media, CM) were analyzed by mass-spectrometry-based quantitative proteomics.
Project description:Methods: We used an ex vivo culture model and measured gene expression changes in human lung fibroblasts after stimulation with BMPs and their antagonists using HEEBO microarrays. The in vitro data were correlated with in vivo observations in published expression datasets of human lung adenocarcinomas. Results: We have systematically analyzed the response to BMP2, BMP4, BMP7 and their antagonists, gremlin and noggin, to define common and specific gene expression patterns. A BMP2 induced gene expression signature was defined, which is specific for stromal fibroblasts. Gene expression profiles from lung adenocarcinoma biopsies were analyzed to determine the prognostic significance of the Fibroblast specific BMP2 induced gene list. This gene list successfully segregated patients with different prognostic outcome in 3 datasets. In a small dataset (Garber et al.) there was a strong trend for a worse prognosis of patients with adenocarcinomas of all stages over-expressing the Fibroblast specific BMP2 induced gene list. In two larger datasets with stage I adenocarcinomas we observed a significantly worse disease-free (p=0.002, Lee et al. and p=0.002, Bhattacharjee et al.) and overall survival (p=0.0002). Conclusions: The effects of BMPs and their antagonists are heterogeneous in different cell types. The gene expression pattern induced by BMP2 in primary lung fibroblasts may predict outcomes of patients with lung adenocarcinomas. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment stimulus_or_stress_design