Hanna Taipaleenmäki

Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations.

The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC(Bone)) or adipogenic (mMSC(Adipo)) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC(Bone) versus mMSC(Adipo) using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC(Bone) while genes related to lipid metabolism and immune response were highly expressed in mMSC(Adipo). In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC(Bone) compared to mMSC(Adipo) (p<0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC(Bone) compared to mMSC(Adipo). Reduced Wnt activity in mMSC(Adipo) was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC(Adipo). Interestingly, conditioned medium (CM) collected from mMSC(Adipo) (mMSC-CM(Adipo)) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM(Adipo) abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.

MicroRNAs Regulate Osteogenesis and Chondrogenesis of Mouse Bone Marrow Stromal Cells

MicroRNAs (miRNAs) are non-coding RNAs that bind to target mRNA leading to translational arrest or mRNA degradation. To study miRNA-mediated regulation of osteogenesis and chondrogenesis, we compared the expression of 35 miRNAs in osteoblasts and chondroblasts derived from mouse marrow stromal cells (MSCs). Differentiation of MSCs resulted in up- or downregulation of several miRNAs, with miR-199a expression being over 10-fold higher in chondroblasts than in undifferentiated MSCs. In addition, miR-124a was strongly upregulated during chondrogenesis while the expression of miR-96 was substantially suppressed. A systems biological analysis of the potential miRNA target genes and their interaction networks was combined with promoter analysis. These studies link the differentially expressed miRNAs to collagen synthesis and hypoxia, key pathways related to bone and cartilage physiology. The global regulatory networks described here suggest for the first time how miRNAs and transcription factors are capable of fine-tuning the osteogenic and chondrogenic differentiation of mouse MSCs.

Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis.

Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker.

Few surface markers are available to monitor lineage differentiation during chondrogenesis. Recently, delta-like1/fetal antigen1 (dlk1/FA1), a transmembrane protein of the Notch/Delta/Serrata family, was shown to be essential for inducing early chondrogenesis. Thus, we investigated the possible use of dlk1/FA1 as a novel surface marker for chondroprogenitor cells during hESC differentiation. We found that, Dlk1/FA1 is expressed specifically in cells undergoing transition from proliferating to prehypertrophic chondrocytes during endochondral ossification of the mouse limb. In hESC cells, dlk1/FA1 was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B, a member of TGF-ß family, markedly increased Dlk1 expression in association with up-regulation of the mesoderm-specific markers (e.g. FOXF1, KDR and VE-cadherin) and SOX9. dlk1/FA1+ cells isolated by fluorescence activated cell sorting (FACS) were capable of differentiating into chondrocytic cells when cultured as micromass pellets in a xeno-free system containing TGFβ1. In conclusion, we identified dlk1/FA1 as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage. Tracking dlk1/FA1 expression as a mesoderm/chondroprogenitor surface marker provides a novel strategy for designing clinically relevant protocols to direct the differentiation of hESC into chondrocytes.

Impact of stromal cell composition on BMP-induced chondrogenic differentiation of mouse bone marrow derived mesenchymal cells

Chondrogenic differentiation in mesenchymal stromal cells (MSCs) has been actively studied due to their potential use in mesenchymal tissue repair. Our goal was to develop a simple isolation protocol for adherent mouse MSCs to simultaneously clear off hematopoietic cells and expand to obtain enough starting material for differentiation studies. CD34 and CD45 expressing cells were rapidly removed by inhibiting growth of hematopoietic cells to yield short-term selected (STS) cells. Further passaging enriched more primitive, uniformly Sca-1 expressing, long-term selected (LTS) cells. The efficacy of several BMPs to induce chondrogenesis in pellet culture was compared in STS and LTS cells. In STS cells, chondrogenesis progressed rapidly to terminal differentiation while LTS cells differentiated at a slower rate with no hypertrophy. In LTS cells, rhBMP homodimers -2, -4, -6 and rhBMP2/7 heterodimer were effective enhancers of chondrogenesis over that of rhBMP-5 and -7. In STS cells, rhBMP-2 and rhBMP-7 supported rapid chondrogenesis and terminal differentiation over that of rhBMP-6. These data indicate the impact of stromal cell composition on the chondrogenic differentiation profile, which is an important aspect to be considered when standardizing differentiation assay conditions as well as developing MSC based cartilage repair technologies.

Reduced expression of Sfrp1 during chondrogenesis and in articular chondrocytes correlates with osteoarthritis in STR/ort mice.

To circumvent the problems of genetic and environmental diversity hampering the analysis in humans, we turned to a murine model for human knee osteoarthritis (OA) and fine mapped a previously defined OA-quantitative trait locus (QTL). We here focused on one of the candidate genes within the OA-QTL encoding the Wnt antagonist secreted frizzled related protein 1 (Sfrp1). Sequence analysis of the Sfrp1 gene in the OA strain STR/ort revealed 23 polymorphic changes with a potential to alter the gene expression. Indeed, a reduced expression in STR/ort mice was demonstrated for articular chondrocytes and hypertrophic chondrocytes of the femoral growth plate as shown by immunohistochemistry. RT-PCR of in vitro generated mesenchymal stem cells (MSC) and chondrogenically differentiated MSC (cMSC) confirmed the reduced Sfrp1 expression in STR/ort mice. This reduced Sfrp1 expression in MSC correlated with an increased amount of cytoplasmic β-catenin, a downregulation of the Wnt target gene PPARγ and an upregulation of Runx2 as well as a preferential differentiation of the MSC along the osteoblasts lineage. Given the role of Wnt signalling during chondrogenesis and in maintaining the integrity of the long lived articular chondrocytes, we conclude from our results that the reduced Sfrp1 expression in STR/ort mice not only leads to an increased activation of the Wnt/β-catenin signalling early in life but also renders the articular cartilage prone to premature ageing and to the development of OA.

Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases

The Synechocystis sp. PCC 6803 genome harbours a deg gene family consisting of three members, degP (htrA, slr1204), degQ (hhoA, sll1679) and degS (hhoB, sll1427). We studied the environmental regulation of the Synechocystis sp. PCC 6803 deg genes at the level of transcription and protein structures of the gene products to evaluate their hypothetical role in D1 protein turnover. Northern blotting showed that transcription of the deg genes is differentially regulated, supporting a view of distinct roles of Degs in cellular processes. The oligomerization state as well as the three dimensional structures of the Synechocystis sp. PCC 6803 Deg proteases were predicted based on an amino acid sequence alignment and comparison of the Deg crystal structures from human, Escherichia coli and Thermotoga maritima. The structures of the Synechocystis sp. PCC 6803 Degs resemble more the Thermotoga maritima Deg enzyme structure than the Escherichia coli one. Moreover, the structures of the LA-loops hint towards a homotrimeric form of the Synechocystis sp. PCC 6803 Deg proteases.

The Crosstalk Between Transforming Growth Factor-β1 and Delta Like-1 Mediates Early Chondrogenesis During Embryonic Endochondral Ossification †‡§

Delta like‐1 (Dlk1)/preadipocyte factor‐1 (Pref‐1)/fetal antigen‐1 (FA1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis are not known. Thus, we examined the effect of a number of signaling molecules and their inhibitors on Dlk1 expression during in vitro chondrogenic differentiation in mouse embryonic limb bud mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) pellet cultures. Dlk1/Pref‐1 was initially expressed during mesenchymal condensation and chondrocyte proliferation, in parallel with expression of Sox9 and Col2a1, and was downregulated upon the expression of Col10a1 by hypertrophic chondrocytes. Among a number of molecules that affected chondrogenesis, transforming growth factor‐β1 (TGF‐β1)‐induced proliferation of chondroprogenitors was associated with decreased Dlk1 expression. This effect was abolished by TGF‐β signaling inhibitor SB431542, suggesting regulation of Dlk1/FA1 by TGF‐β1 signaling in chondrogenesis. TGF‐β1‐induced Smad phosphorylation and chondrogenesis were significantly increased in Dlk1−/− MEF, while they were blocked in Dlk1 overexpressing MEF, in comparison with wild‐type MEF. Furthermore, overexpression of Dlk1 or addition of its secreted form FA1 dramatically inhibited TGF‐β1‐induced Smad reporter activity. In conclusion, our data identified Dlk1/FA1 as a downstream target of TGF‐β1 signaling molecule that mediates its function in embryonic chondrogenesis. The crosstalk between TGF‐β1 and Dlk1/FA1 was shown to promote early chondrogenesis during the embryonic endochondral ossification process. STEM CELLS 2012; 30:304–313.

Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes.

OBJECTIVE Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. METHODS Open access bioinformatics tools and databases were used to characterize the gene, predicted protein and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA-tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Yin-Yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). CONCLUSION A novel cartilage specific molecule was identified which marks the differentiating chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype.