Koen J. Dechering

Osteo-transcriptomics of human mesenchymal stem cells: accelerated gene expression and osteoblast differentiation induced by vitamin D reveals c-MYC as an enhancer of BMP2-induced osteogenesis.

Bone marrow-derived human mesenchymal stem cells (hMSCs) have the in vitro capacity to differentiate into osteoblasts, chondrocytes or adipocytes, depending on the applied stimulus. In order to identify novel regulators of osteogenesis in hMSCs, osteo-transcriptomics was performed whereby differentiation induced by dexamethasone (DEX), DEX+ bone morphogenetic protein 2 (BMP2), and DEX+ Vitamin D(3) (1,25(OH)(2)D(3)) was studied over a course of 12 days. Microarray analysis revealed that 2095 genes were significantly regulated by DEX+ 1,25(OH)(2)D(3), of which 961 showed accelerated expression kinetics compared to treatment by DEX alone. The majority of these genes were accelerated 24-48 h after onset of osteogenic treatment. Gene ontology (GO) analysis of these 1,25(OH)(2)D(3)-accelerated genes indicated their involvement in biological processes related to cellular differentiation and cell cycle regulation. When compared to cells treated with DEX or DEX+BMP2, treatment with DEX+ 1,25(OH)(2)D(3) clearly accelerated osteoprogenitor commitment and osteoblast maturation, as measured by alkaline phosphatase (ALP) activity and calcification of the matrix. Cell cycle progression, as observed after initial growth arrest, was not significantly accelerated by 1,25(OH)(2)D(3) and was not required for onset and progression of osteogenesis. However, expression of c-Myc was accelerated by 1,25(OH)(2)D(3), and binding sites for c-MYC were enriched in promoters of genes accelerated by 1,25(OH)(2)D(3). Lentiviral overexpression of c-MYC strongly promoted DEX+ BMP2-induced osteoblast differentiation and matrix maturation. In conclusion, our studies show for the first time that 1,25(OH)(2)D(3) strongly accelerates expression of genes involved in differentiation of hMSCs and, moreover, identify c-MYC as a novel regulator of osteogenesis.

Epigenetics: DNA demethylation promotes skeletal myotube maturation

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation‐contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 μM of the DNA methylation inhibitor 5‐azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up‐regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC‐treated myotubes displayed a mature, well‐defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 μM ryanodine, and 100 μM nifedipine, suggesting that action potential‐induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.—Hupkes, M., Jonsson, M. K. B., Scheenen, W. J., van Rotterdam, W., Sotoca, A. M., van Someren, E. P., van der Heyden, M. A. G., van Veen, T. A., van Ravestein‐van Os, R. I., Bauerschmidt, S., Piek, E., Ypey, D. L., van Zoelen, E. J., Dechering, K. J. Epigenetics: DNA demethylation promotes skeletal myotube maturation. FASEB J. 25, 3861–3872 (2011). www.fasebj.org

Predicting the therapeutic efficacy of MSC in bone tissue engineering using the molecular marker CADM1

Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.

The Role of Collagen Crosslinking in Differentiation of Human Mesenchymal Stem Cells and MC3T3-E1 Cells

Collagen is the main protein component of the extracellular matrix of bone, and it has structural and instructive properties. Collagen undergoes many post-translational modifications, including extensive crosslinking. Although defective crosslinking has been implicated in human syndromes (e.g., osteogenesis imperfecta or Ehlers-Danlos syndrome), it is not clear to what extent crosslinking is necessary for collagens instructive properties during bone formation. Here we report that inhibition of collagen crosslinking in the mouse pre-osteoblast cell line MC3T3-E1 impairs the osteogenic program. Genome-wide expression profiling of beta-aminopropionitrile-treated and control cells revealed that matrix deposition by MC3T3-E1 cells provides a feed back signal, driving cells through the differentiation process, that is strongly impaired when crosslinking is inhibited. Inhibition of crosslinking did not affect osteogenic differentiation of human mesenchymal stem cells (hMSCs), shown by the expression of alkaline phosphatase and genome-wide gene expression analysis, although it enhances matrix mineralization. In conclusion, collagen crosslinking harbors instructive properties in MC3T3-E1 differentiation but plays a more-passive role in differentiation of bone marrow-derived hMSCs.

DNA methylation restricts spontaneous multi-lineage differentiation of mesenchymal progenitor cells, but is stable during growth factor-induced terminal differentiation

The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

High-throughput assay for the identification of compounds regulating osteogenic differentiation of human mesenchymal stromal cells

Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS) not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.

MicroRNA miR-378 promotes BMP2-induced osteogenic differentiation of mesenchymal progenitor cells

BackgroundMicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression.ResultsUsing a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2.ConclusionsOur results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.

TSLP is differentially regulated by vitamin D3 and cytokines in human skin

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic diseases and is highly expressed in keratinocytes in human lesional atopic dermatitis (AD) skin. In nonlesional AD skin TSLP expression can be induced by applying house dust mite allergen onto the skin in the atopy patch test. Several studies have demonstrated that the induction of TSLP expression in mouse skin does not only lead to AD‐like inflammation of the skin, but also predisposes to severe inflammation of the airways. In mice, TSLP expression can be induced by application of the 1,25‐dihydroxyvitamin D3 (VD3) analogue calcipotriol and results in the development of eczema‐like lesions. The objective is to investigate the effect of VD3 (calcitriol) or calcipotriol on TSLP expression in normal human skin and skin from AD patients. Using multiple ex vivo experimental setups, the effects of calci(po)triol on TSLP expression by normal human skin, and skin from AD patients were investigated and compared to effects of calcipotriol on mouse and non‐human primates (NHP) skin. No induction of TSLP expression (mRNA or protein) was observed in human keratinocytes, normal human skin, nonlesional AD skin, or NHP skin samples after stimulation with calcipotriol or topical application of calcitriol. The biological activity of calci(po)triol in human skin samples was demonstrated by the increased expression of the VD3‐responsive Cyp24a1 gene. TSLP expression was induced by cytokines (IL‐4, IL‐13, and TNF‐α) in skin samples from all three species. In contrast to the findings in human and NHP, a consistent increase in TSLP expression was confirmed in mouse skin biopsies after stimulation with calcipotriol. VD3 failed to induce expression of TSLP in human or monkey skin in contrast to mouse, implicating careful extrapolation of this often‐used mouse model to AD patients.

The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically applied. Protein kinase C (PKC) family members have been implicated in bone metabolism, which prompted us to use a pharmaceutical approach to manipulate PKC signalling in hMSCs. Inhibition of PKC resulted in a dose‐dependent inhibition of dexamethasone‐induced osteogenic differentiation. Surprisingly, PKC activation using phorbol 12‐myristate 13‐acetate (PMA) also resulted in inhibition of osteogenesis, although we observed that inhibition was more pronounced at low than at high concentrations of PMA. Furthermore, we observed that inhibition of PKCδ blocked alkaline phosphatase (ALP, an early marker of osteogenic differentiation) expression, whereas inhibition of the conventional PKC subfamily and PKCµ using Gö6976 resulted in an induction of ALP activity, collagen (I) expression and mineralization. In conclusion, inhibition of the conventional PKCs/PKCµ and activation of PKCδ could further benefit osteogenic differentiation of hMSCs in vitro and in vivo, which is currently under investigation. Copyright

Delineation of amplification, hybridization and location effects in microarray data yields better-quality normalization

BackgroundOligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct.ResultsWe show that by a careful decomposition of probe specific amplification, hybridization and array location effects, a normalization can be performed that allows for a much improved analysis of these data. Identification of the technical sources of variation between arrays has allowed us to build statistical models that are used to estimate how the signal of individual probes is affected, based on their properties. This enables a model-based normalization that is probe-specific, in contrast with the signal intensity distribution normalization performed by many current methods. Next to this, we propose a novel way of handling background correction, enabling the use of background information to weight probes during summarization. Testing of the proposed method shows a much improved detection of differentially expressed genes over earlier proposed methods, even when tested on (experimentally tightly controlled and replicated) spike-in datasets.ConclusionsWhen a limited number of arrays are available, or when arrays are run in different batches, technical effects have a large influence on the measured expression of genes. We show that a detailed modelling and correction of these technical effects allows for an improved analysis in these situations.