Jan de Boer | Maastricht University (original) (raw)

Papers by Jan de Boer

Trends in Cell Biology, 2002

Nature Reviews Rheumatology, 2011

Bone, 2004

Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripo... more Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripotent cells for autologous bone tissue engineering. We previously discovered that over activation of the Wnt signal transduction pathway by either lithium or Wnt3A stimulates hMSC proliferation while retaining pluripotency. Release of Wnt3A or lithium from porous calcium phosphate scaffolds, which we use for bone tissue engineering, could provide a mitogenic stimulus to implanted hMSCs. To define the proper release profile, we first assessed the effect of Wnt over activation on osteogenic differentiation of hMSCs. Here, we report that both lithium and Wnt3A strongly inhibit dexamethasone-induced expression of the osteogenic marker alkaline phosphatase (ALP). Moreover, lithium partly inhibited mineralization of hMSCs whereas Wnt3A completely blocked it. Time course analysis during osteogenic differentiation revealed that 4 days of Wnt3A exposure before the onset of mineralization is suffic...

Trends in Cell Biology, 2002

Trends in Cell Biology, 2001

Trends in Cell Biology, 2002

Tissue Engineering Part C: Methods, 2010

Tissue Engineering Part C-methods, 2010

Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxyg... more Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxygen (O(2)) and nutrient diffusion. As such, monitoring the levels of nutrients and O(2) available to the cells is essential to assess the physiology of the cells and to evaluate strategies aiming at improving nutrient availability. In this article, a reporter system containing the luciferase gene driven by a hypoxia responsive promoter was used to monitor cellular hypoxia in a TE context. We report that luciferase activity correlates with the O(2) tension in the cell culture medium. When transgenic cells were seeded onto scaffolds and implanted in immune-deficient mice subcutaneously, luciferase activity was detected. To validate the response to O(2) levels of this reporter system, we cultured transgenic cells on biomaterials in a flow perfusion bioreactor and observed that cells in the bioreactor displayed a drastically lower luciferase activity than conventional static culture, and that higher luciferase activity is observed in the interior of a tissue-engineered construct, illustrating the uneven O(2) distribution in three-dimensional constructs under conventional static culture. We conclude that this reporter system is a versatile tool to investigate cellular O(2) availability in TE both in vitro and in vivo.

Tissue Engineering, 2008

Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they pr... more Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they provide an unlimited supply of cells that can be differentiated into chondrocytes. So far, chondrogenic differentiation of both mouse and human ES cells has only been demonstrated in two-dimensional cultures, in pellet cultures, in a hydrogel, or on thin biomaterials. The next challenge will be to form cartilage on a load-bearing, clinically relevant-sized scaffold in vitro and in vivo, to regenerate defects in patients suffering from articular cartilage disorders. For a successful implant, cells have to be seeded efficiently and homogenously throughout the scaffold. Parameters investigated were the scaffold architecture, seeding method, and cellular condition. Seeding in a three-dimensional fiber-deposited (3DF) scaffold was more homogenous than in a compression-molded scaffold. The seeding efficiency on bare scaffolds was compromised by the absence of serum in the chondrogenic medium, but could be improved by combining the cells with a gel and subsequent injection into the 3DF scaffolds. However, the viability of the cells was unsatisfactory in the interior of the graft. Cell aggregates, the so-called embryoid bodies (EBs), were seeded with increased survival rate. Mouse ES cells readily underwent chondrogenic differentiation in vitro in pellets, on bare scaffolds, in Matrigel, and in agarose, both as single cells and in EBs. The differentiation protocol requires further improvement to achieve homogenous differentiation and abolish teratoma formation in vivo. We conclude that ES cells can be used as a cell source for cartilage tissue engineering, pending further optimization of the strategy.

Tissue Engineering, 2007

During the past decade, there has been much interest in the use of human mesenchymal stem cells (... more During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow-derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm 2 had a significantly higher growth rate than at 5000 cell/cm 2 , which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm 2 in an a-minimal essential medium-based medium containing dexamethasone.

Journal of Tissue Engineering and Regenerative Medicine, 2009

To further investigate the physiological role of PKA in hMSC osteogenesis, we tested a selection ... more To further investigate the physiological role of PKA in hMSC osteogenesis, we tested a selection of G-protein-coupled receptor ligands which signal via intracellular cAMP production and PKA activation. Treatment of hMSCs with parathyroid hormone, parathyroid hormone-related peptide, melatonin, epinephrine, calcitonin or calcitonin gene-related peptide did not result in accumulation of cAMP or induction of alkaline phosphatase (ALP) expression. The only ligand that did induce cAMP, prostaglandin E2, even inhibited ALP expression and mineralization, suggesting that physiological levels of cAMP may inhibit osteogenesis. Furthermore, intermittent exposure of hMSCs to dibutyryl-cAMP inhibited ALP expression, whereas we did not observe an inhibitive effect at low dibutyryl-cAMP concentrations. Taken together, our results demonstrate that cAMP can either stimulate or inhibit osteogenesis in hMSCs, depending on the duration, rather than the strength, of the signal provided.

Journal of Tissue Engineering and Regenerative Medicine, 2009

Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, ... more 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. * The experiments were performed in both basic medium with and without dex. 1 Proliferation was assessed by Alamar blue assay; osteogenic differentiation was assessed by ALP activity induction compared to cells grown in medium without treatment. 2 ALP activity was determined by flow cytometry (FACS), expressing the average ALP signal/cell. 3 Relative ALP activity was determined by ALP biochemical assay normalized to cell number and then the ratio was normalized to the basic control. Figure 2. Effect of PKC activation on hMSC proliferation and osteogenic differentiation. (A) Western blot analysis of hMSCs cultured in the presence of 0, 10 and 1000 nM PMA for 4 h. The results were confirmed with cells from a second donor. (B) Light microscopy pictures with ×100 magnification demonstrated hMSC proliferation in basic medium plus different concentrations of PMA after 4 days. (C) The effect of PMA on the proliferation of hMSCs was expressed as Alamar blue values after 7 days of culture. (D) ALP expression was analysed using FACS after 7 days of culture in the presence of PMA. (E) Gene expression of osteogenic markers COL1A1 and ALP by Q-PCR after 5 days of culture. Expression is indicated as fold-induction compared to cells grown in basic medium and normalized to 18s rRNA. (F) hMSCs were cultured with PMA for 4 days (open bar) or 28 days (black bar) in mineralization medium. At the end of the 28 day culture period, total calcium deposition was determined. 1000× diluted DMSO was taken as a solvent control for 1000 nM PMA. Error bars indicate standard deviation (SD). Basic, basic hMSC medium; dex, osteogenic medium. Statistical significance is calculated relative to cells grown in medium without test compounds Figure 3. Gö6976 stimulates hMSC osteogenic differentiation. (A) hMSC proliferation in the presence of different concentrations of Gö6976 after 5 days. (B) Gö6976 stimulates ALP expression after 5 days. (C) Light microscopy picture with ×100 magnification shows the morphology of hMSCs in basic medium with 1000 nM Gö6976. (D) CoL1A1 and ALP expression in Gö6976-treated cells expressed as fold-induction compared to untreated control cells. (E) hMSCs were cultured in mineralization medium with 100 nM Gö6976 for 20 days. Phase-contrast light microscopy images with ×40 magnification show enhanced hMSCs mineralization in the presence of Gö6976 by von Kossa staining. Error bars indicate SD. Basic, basic hMSC medium;

Trends in Cell Biology - TR CELL BIOL, 2002

Journal of Bone and Mineral Research, 2009

Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heteroto... more Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heterotopic ossification for which there is currently no treatment available. FOP has been linked recently to a heterozygous R206H mutation in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2). Expression of the mutant ALK2-R206H receptor (FOP-ALK2) results in increased phosphorylation of the downstream Smad1 effector proteins and elevated basal BMP-dependent transcriptional reporter activity, indicating that FOP-ALK2 is constitutively active. FOP-ALK2-induced transcriptional activity could be blocked by overexpressing either of the inhibitory Smads, Smad6 or -7, or by treatment with the pharmacological BMP type I receptor inhibitor dorsomorphin. However, in contrast to wild-type ALK2, FOP-ALK2 is not inhibited by the negative regulator FKBP12. Mesenchymal cells expressing the FOP-ALK2 receptor are more sensitive to undergoing BMP-induced osteoblast differentiation and mineralization. In vivo bone formation was assessed by loading human mesenchymal stem cells (hMSCs) expressing the ALK2-R206H receptor onto calcium phosphate scaffolds and implantation in nude mice. Compared with control cells FOP-ALK2-expressing cells induced increased bone formation. Taken together, the R206H mutation in ALK2 confers constitutive activity to the mutant receptor, sensitizes mesenchymal cells to BMP-induced osteoblast differentiation, and stimulates new bone formation. We have generated an animal model that can be used as a stepping stone for preclinical studies aimed at inhibiting the heterotopic ossification characteristic of FOP. ß 2010 American Society for Bone and Mineral Research. KEY WORDS: BONE MORPHOGENETIC PROTEIN; ECTOPIC BONE FORMATION; FIBRODYSPLASIA OSSIFICANS PROGRESSIVA; OSTEOBLAST DIFFERENTIATION; SMAD pulmonary complications of thoracic insufficiency syndrome. (1) At present, no treatment for FOP is available.

PloS one, 2011

Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. The... more 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 ...

Bone, 2008

Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but... more Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but due to their limited number and donor variation, other cell types are used to answer relevant questions in bone tissue engineering. Since the extracellular matrix (ECM) is a complex entity with instructive properties, it is of key importance to analyze its role in osteogenic differentiation

Biomaterials, 2012

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, no... more The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.

Biomaterials, 2013

The repertoire of growth factors determines the biological engagement of human mesenchymal stroma... more The repertoire of growth factors determines the biological engagement of human mesenchymal stromal cells (hMSCs) in processes such as immunomodulation and tissue repair. Hypoxia is a strong modulator of the secretome and well known stimuli to increase the secretion of pro-angiogenic molecules. In this manuscript, we employed a high throughput screening assay on an hMSCs cell line in order to identify small molecules that mimic hypoxia. Importantly, we show that the effect of these small molecules was cell type/species dependent, but we identified phenanthroline as a robust hit in several cell types. We show that phenanthroline induces high expression of hypoxia-target genes in hMSCs when compared with desferoxamine (DFO) (a known hypoxia mimic) and hypoxia incubator (2% O 2 ). Interestingly, our microarray and proteomics analysis show that only phenanthroline induced high expression and secretion of another angiogenic cytokine, interleukin-8, suggesting that the mechanism of phenanthroline-induced hypoxia is distinct from DFO and hypoxia and involves the activation of other signaling pathways. We showed that phenanthroline alone was sufficient to induce blood vessel formation in a Matrigel plug assay in vivo paving the way to its application in ischeamic-related diseases.

Biomaterials, 2012

The response of osteoprogenitors to calcium (Ca 2þ ) is of primary interest for both normal bone ... more The response of osteoprogenitors to calcium (Ca 2þ ) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca 2þ released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca 2þ concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca 2þ enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6 h and pharmaceutical interference with a number of proteins involved in Ca 2þ sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca 2þ and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca 2þ and using microarray analysis, we have identified c-Fos as an early Ca 2þ response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca 2þ , a simple and economic way of priming hMSCs for bone tissue engineering applications.

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2007

Colorectal cancer still represents a paradigm for the elucidation of the cellular, genetic and mo... more Colorectal cancer still represents a paradigm for the elucidation of the cellular, genetic and molecular mechanisms that underly solid tumor initiation, progression to malignancy, and metastasis to distal organ sites. The relative ease with which pathological specimens can be obtained by either surgery or endoscopy from different stages of tumor progression has facilitated the application of omics technologies to allow the genomewide analysis both at the RNA (gene expression) and DNA (aneuploidy) levels. Here, we have reviewed the multiplicity of studies appeared to date in the scientific literature on the expression and genomic analysis of colorectal cancer, and attempted an integration of the profiling data generated and made available in the public domain. This approach is likely to pinpoint specific chromosomal loci and the corresponding genes which (i) play rate-limiting roles in colorectal cancer, (ii) represent putative diagnostic and prognostic markers for the accurate prediction of clinical outcome and response to treatment, and (iii) encompass potential therapeutic targets. Moreover, cross-species data mining and integration of the human colorectal cancer profiles with those obtained from mouse models of intestinal tumorigenesis will even more contribute to the elucidation of highly conserved pathways and cellular functions underlying malignancy in the GI tract. Notwithstanding the above promises, tumor heterogeneity, limited cohort sizes, and methodological differences among experimental and bioinformatic approaches still poses main obstacles towards the optimal utilization and integration of omics profiles.

Trends in Cell Biology, 2002

Nature Reviews Rheumatology, 2011

Bone, 2004

Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripo... more Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripotent cells for autologous bone tissue engineering. We previously discovered that over activation of the Wnt signal transduction pathway by either lithium or Wnt3A stimulates hMSC proliferation while retaining pluripotency. Release of Wnt3A or lithium from porous calcium phosphate scaffolds, which we use for bone tissue engineering, could provide a mitogenic stimulus to implanted hMSCs. To define the proper release profile, we first assessed the effect of Wnt over activation on osteogenic differentiation of hMSCs. Here, we report that both lithium and Wnt3A strongly inhibit dexamethasone-induced expression of the osteogenic marker alkaline phosphatase (ALP). Moreover, lithium partly inhibited mineralization of hMSCs whereas Wnt3A completely blocked it. Time course analysis during osteogenic differentiation revealed that 4 days of Wnt3A exposure before the onset of mineralization is suffic...

Trends in Cell Biology, 2002

Trends in Cell Biology, 2001

Trends in Cell Biology, 2002

Tissue Engineering Part C: Methods, 2010

Tissue Engineering Part C-methods, 2010

Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxyg... more Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxygen (O(2)) and nutrient diffusion. As such, monitoring the levels of nutrients and O(2) available to the cells is essential to assess the physiology of the cells and to evaluate strategies aiming at improving nutrient availability. In this article, a reporter system containing the luciferase gene driven by a hypoxia responsive promoter was used to monitor cellular hypoxia in a TE context. We report that luciferase activity correlates with the O(2) tension in the cell culture medium. When transgenic cells were seeded onto scaffolds and implanted in immune-deficient mice subcutaneously, luciferase activity was detected. To validate the response to O(2) levels of this reporter system, we cultured transgenic cells on biomaterials in a flow perfusion bioreactor and observed that cells in the bioreactor displayed a drastically lower luciferase activity than conventional static culture, and that higher luciferase activity is observed in the interior of a tissue-engineered construct, illustrating the uneven O(2) distribution in three-dimensional constructs under conventional static culture. We conclude that this reporter system is a versatile tool to investigate cellular O(2) availability in TE both in vitro and in vivo.

Tissue Engineering, 2008

Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they pr... more Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they provide an unlimited supply of cells that can be differentiated into chondrocytes. So far, chondrogenic differentiation of both mouse and human ES cells has only been demonstrated in two-dimensional cultures, in pellet cultures, in a hydrogel, or on thin biomaterials. The next challenge will be to form cartilage on a load-bearing, clinically relevant-sized scaffold in vitro and in vivo, to regenerate defects in patients suffering from articular cartilage disorders. For a successful implant, cells have to be seeded efficiently and homogenously throughout the scaffold. Parameters investigated were the scaffold architecture, seeding method, and cellular condition. Seeding in a three-dimensional fiber-deposited (3DF) scaffold was more homogenous than in a compression-molded scaffold. The seeding efficiency on bare scaffolds was compromised by the absence of serum in the chondrogenic medium, but could be improved by combining the cells with a gel and subsequent injection into the 3DF scaffolds. However, the viability of the cells was unsatisfactory in the interior of the graft. Cell aggregates, the so-called embryoid bodies (EBs), were seeded with increased survival rate. Mouse ES cells readily underwent chondrogenic differentiation in vitro in pellets, on bare scaffolds, in Matrigel, and in agarose, both as single cells and in EBs. The differentiation protocol requires further improvement to achieve homogenous differentiation and abolish teratoma formation in vivo. We conclude that ES cells can be used as a cell source for cartilage tissue engineering, pending further optimization of the strategy.

Tissue Engineering, 2007

During the past decade, there has been much interest in the use of human mesenchymal stem cells (... more During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow-derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm 2 had a significantly higher growth rate than at 5000 cell/cm 2 , which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm 2 in an a-minimal essential medium-based medium containing dexamethasone.

Journal of Tissue Engineering and Regenerative Medicine, 2009

To further investigate the physiological role of PKA in hMSC osteogenesis, we tested a selection ... more To further investigate the physiological role of PKA in hMSC osteogenesis, we tested a selection of G-protein-coupled receptor ligands which signal via intracellular cAMP production and PKA activation. Treatment of hMSCs with parathyroid hormone, parathyroid hormone-related peptide, melatonin, epinephrine, calcitonin or calcitonin gene-related peptide did not result in accumulation of cAMP or induction of alkaline phosphatase (ALP) expression. The only ligand that did induce cAMP, prostaglandin E2, even inhibited ALP expression and mineralization, suggesting that physiological levels of cAMP may inhibit osteogenesis. Furthermore, intermittent exposure of hMSCs to dibutyryl-cAMP inhibited ALP expression, whereas we did not observe an inhibitive effect at low dibutyryl-cAMP concentrations. Taken together, our results demonstrate that cAMP can either stimulate or inhibit osteogenesis in hMSCs, depending on the duration, rather than the strength, of the signal provided.

Journal of Tissue Engineering and Regenerative Medicine, 2009

Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, ... more 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. * The experiments were performed in both basic medium with and without dex. 1 Proliferation was assessed by Alamar blue assay; osteogenic differentiation was assessed by ALP activity induction compared to cells grown in medium without treatment. 2 ALP activity was determined by flow cytometry (FACS), expressing the average ALP signal/cell. 3 Relative ALP activity was determined by ALP biochemical assay normalized to cell number and then the ratio was normalized to the basic control. Figure 2. Effect of PKC activation on hMSC proliferation and osteogenic differentiation. (A) Western blot analysis of hMSCs cultured in the presence of 0, 10 and 1000 nM PMA for 4 h. The results were confirmed with cells from a second donor. (B) Light microscopy pictures with ×100 magnification demonstrated hMSC proliferation in basic medium plus different concentrations of PMA after 4 days. (C) The effect of PMA on the proliferation of hMSCs was expressed as Alamar blue values after 7 days of culture. (D) ALP expression was analysed using FACS after 7 days of culture in the presence of PMA. (E) Gene expression of osteogenic markers COL1A1 and ALP by Q-PCR after 5 days of culture. Expression is indicated as fold-induction compared to cells grown in basic medium and normalized to 18s rRNA. (F) hMSCs were cultured with PMA for 4 days (open bar) or 28 days (black bar) in mineralization medium. At the end of the 28 day culture period, total calcium deposition was determined. 1000× diluted DMSO was taken as a solvent control for 1000 nM PMA. Error bars indicate standard deviation (SD). Basic, basic hMSC medium; dex, osteogenic medium. Statistical significance is calculated relative to cells grown in medium without test compounds Figure 3. Gö6976 stimulates hMSC osteogenic differentiation. (A) hMSC proliferation in the presence of different concentrations of Gö6976 after 5 days. (B) Gö6976 stimulates ALP expression after 5 days. (C) Light microscopy picture with ×100 magnification shows the morphology of hMSCs in basic medium with 1000 nM Gö6976. (D) CoL1A1 and ALP expression in Gö6976-treated cells expressed as fold-induction compared to untreated control cells. (E) hMSCs were cultured in mineralization medium with 100 nM Gö6976 for 20 days. Phase-contrast light microscopy images with ×40 magnification show enhanced hMSCs mineralization in the presence of Gö6976 by von Kossa staining. Error bars indicate SD. Basic, basic hMSC medium;

Trends in Cell Biology - TR CELL BIOL, 2002

Journal of Bone and Mineral Research, 2009

Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heteroto... more Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heterotopic ossification for which there is currently no treatment available. FOP has been linked recently to a heterozygous R206H mutation in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2). Expression of the mutant ALK2-R206H receptor (FOP-ALK2) results in increased phosphorylation of the downstream Smad1 effector proteins and elevated basal BMP-dependent transcriptional reporter activity, indicating that FOP-ALK2 is constitutively active. FOP-ALK2-induced transcriptional activity could be blocked by overexpressing either of the inhibitory Smads, Smad6 or -7, or by treatment with the pharmacological BMP type I receptor inhibitor dorsomorphin. However, in contrast to wild-type ALK2, FOP-ALK2 is not inhibited by the negative regulator FKBP12. Mesenchymal cells expressing the FOP-ALK2 receptor are more sensitive to undergoing BMP-induced osteoblast differentiation and mineralization. In vivo bone formation was assessed by loading human mesenchymal stem cells (hMSCs) expressing the ALK2-R206H receptor onto calcium phosphate scaffolds and implantation in nude mice. Compared with control cells FOP-ALK2-expressing cells induced increased bone formation. Taken together, the R206H mutation in ALK2 confers constitutive activity to the mutant receptor, sensitizes mesenchymal cells to BMP-induced osteoblast differentiation, and stimulates new bone formation. We have generated an animal model that can be used as a stepping stone for preclinical studies aimed at inhibiting the heterotopic ossification characteristic of FOP. ß 2010 American Society for Bone and Mineral Research. KEY WORDS: BONE MORPHOGENETIC PROTEIN; ECTOPIC BONE FORMATION; FIBRODYSPLASIA OSSIFICANS PROGRESSIVA; OSTEOBLAST DIFFERENTIATION; SMAD pulmonary complications of thoracic insufficiency syndrome. (1) At present, no treatment for FOP is available.

PloS one, 2011

Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. The... more 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 ...

Bone, 2008

Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but... more Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but due to their limited number and donor variation, other cell types are used to answer relevant questions in bone tissue engineering. Since the extracellular matrix (ECM) is a complex entity with instructive properties, it is of key importance to analyze its role in osteogenic differentiation

Biomaterials, 2012

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, no... more The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.

Biomaterials, 2013

The repertoire of growth factors determines the biological engagement of human mesenchymal stroma... more The repertoire of growth factors determines the biological engagement of human mesenchymal stromal cells (hMSCs) in processes such as immunomodulation and tissue repair. Hypoxia is a strong modulator of the secretome and well known stimuli to increase the secretion of pro-angiogenic molecules. In this manuscript, we employed a high throughput screening assay on an hMSCs cell line in order to identify small molecules that mimic hypoxia. Importantly, we show that the effect of these small molecules was cell type/species dependent, but we identified phenanthroline as a robust hit in several cell types. We show that phenanthroline induces high expression of hypoxia-target genes in hMSCs when compared with desferoxamine (DFO) (a known hypoxia mimic) and hypoxia incubator (2% O 2 ). Interestingly, our microarray and proteomics analysis show that only phenanthroline induced high expression and secretion of another angiogenic cytokine, interleukin-8, suggesting that the mechanism of phenanthroline-induced hypoxia is distinct from DFO and hypoxia and involves the activation of other signaling pathways. We showed that phenanthroline alone was sufficient to induce blood vessel formation in a Matrigel plug assay in vivo paving the way to its application in ischeamic-related diseases.

Biomaterials, 2012

The response of osteoprogenitors to calcium (Ca 2þ ) is of primary interest for both normal bone ... more The response of osteoprogenitors to calcium (Ca 2þ ) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca 2þ released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca 2þ concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca 2þ enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6 h and pharmaceutical interference with a number of proteins involved in Ca 2þ sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca 2þ and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca 2þ and using microarray analysis, we have identified c-Fos as an early Ca 2þ response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca 2þ , a simple and economic way of priming hMSCs for bone tissue engineering applications.

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2007

Colorectal cancer still represents a paradigm for the elucidation of the cellular, genetic and mo... more Colorectal cancer still represents a paradigm for the elucidation of the cellular, genetic and molecular mechanisms that underly solid tumor initiation, progression to malignancy, and metastasis to distal organ sites. The relative ease with which pathological specimens can be obtained by either surgery or endoscopy from different stages of tumor progression has facilitated the application of omics technologies to allow the genomewide analysis both at the RNA (gene expression) and DNA (aneuploidy) levels. Here, we have reviewed the multiplicity of studies appeared to date in the scientific literature on the expression and genomic analysis of colorectal cancer, and attempted an integration of the profiling data generated and made available in the public domain. This approach is likely to pinpoint specific chromosomal loci and the corresponding genes which (i) play rate-limiting roles in colorectal cancer, (ii) represent putative diagnostic and prognostic markers for the accurate prediction of clinical outcome and response to treatment, and (iii) encompass potential therapeutic targets. Moreover, cross-species data mining and integration of the human colorectal cancer profiles with those obtained from mouse models of intestinal tumorigenesis will even more contribute to the elucidation of highly conserved pathways and cellular functions underlying malignancy in the GI tract. Notwithstanding the above promises, tumor heterogeneity, limited cohort sizes, and methodological differences among experimental and bioinformatic approaches still poses main obstacles towards the optimal utilization and integration of omics profiles.