J. Doorn

cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

Tissue engineering of large bone defects is approached through implantation of autologous osteogenic cells, generally referred to as multipotent stromal cells or mesenchymal stem cells (MSCs). Animal-derived MSCs successfully bridge large bone defects, but models for ectopic bone formation as well as recent clinical trials demonstrate that bone formation by human MSCs (hMSCs) is inadequate. The expansion phase presents an attractive window to direct hMSCs by pharmacological manipulation, even though no profound effect on bone formation in vivo has been described so far using this approach. We report that activation of protein kinase A elicits an immediate response through induction of genes such as ID2 and FosB, followed by sustained secretion of bone-related cytokines such as BMP-2, IGF-1, and IL-11. As a consequence, PKA activation results in robust in vivo bone formation by hMSCs derived from orthopedic patients.

ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation

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.

A small molecule approach to engineering vascularized tissue

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.

Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on surrounding tissues. In bone tissue engineering applications, the general assumption is that direct differentiation of hMSCs into osteoblasts accounts for newly observed bone formation in vivo. However, the secretion of bone-specific growth factors, but also pro-angiogenic factors, could also contribute to this process. We recently demonstrated that secretion of bone specific growth factors can be enhanced by treatment of hMSCs with the small molecule db-cAMP (cAMP) and here we investigate the biological activity of these secreted factors. We demonstrate that conditioned medium contains a variety of secreted growth factors, with differences between medium from basic-treated and cAMP-treated hMSCs. We show that conditioned medium from cAMP-treated hMSCs increases proliferation of various cell types and also induces osteogenic differentiation, whereas it has differential effects on migration. Microarray analysis on hMSCs exposed to conditioned medium confirmed upregulation of pathways involved in proliferation as well as osteogenic differentiation. Our data suggests that trophic factors secreted by hMSCs can be tuned for specific applications and that a good balance between differentiation on the one hand and secretion of bone trophic factors on the other, could potentially enhance bone formation for bone tissue engineering applications.

Timing, rather than the concentration of cyclic AMP, correlates to osteogenic differentiation of human mesenchymal stem cells

Previously, we demonstrated that protein kinase A (PKA) activation using dibutyryl‐cAMP in human mesenchymal stem cells (hMSCs) induces in vitro osteogenesis and bone formation in vivo. 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. Copyright

High content imaging in the screening of biomaterial-induced MSC behavior

Upon contact with a biomaterial, cells and surrounding tissues respond in a manner dictated by the physicochemical and mechanical properties of the material. Traditionally, cellular responses are monitored using invasive analytical methods that report the expression of genes or proteins. These analytical methods involve assessing commonly used markers for a predefined readout, masking the actual situation induced in the cells. Hence, a broader expression profile of the cellular response should be envisioned, which technically limits up scaling to higher throughput systems. However, it is increasingly recognized that morphometric readouts, obtained non-invasively, are related to gene expression patterns. Here, we introduced distinct surface roughness to three PLA surfaces, by exposure to oxygen plasma of different duration times. The response of mesenchymal stromal cells was compared to smooth untreated PLA surfaces without the addition of differentiation agents. Morphological and genome wide expression profiles revealed underlying cellular changes which was hidden for the commonly used gene markers for osteo-, chondro- and adipogenesis. Using 3 morphometric parameters, obtained by high content imaging, we were able to build a classifier and discriminate between oxygen plasma-induced modified sheets and non-modified PLA sheets where evaluating classical candidates missed this effect. This approach shows the feasibility to use noninvasive morphometric data in high-throughput systems to screen biomaterial surfaces indicating the underlying genetic biomaterial-induced changes.