GATA3 Mediates a Fast, Irreversible Commitment to BMP4-Driven Differentiation in Human Embryonic Stem Cells.

During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling, we find that hESCs commit to exiting pluripotency unexpectedly early. We show that bone morphogenetic protein 4 (BMP4), an important differentiation trigger, induces a subset of early genes to mirror the sustained, bistable dynamics of upstream signaling. Induction of one of these genes, GATA3, drives differentiation in the absence of BMP4. Conversely, GATA3 knockout delays differentiation and prevents fast commitment to differentiation. We show that positive feedback at the level of the GATA3-BMP4 axis induces fast, irreversible commitment to differentiation. We propose that early commitment may be a feature of BMP-driven fate choices and that interlinked feedback is the molecular basis for an irreversible transition from pluripotency to differentiation.

Site-specific differences in osteoblast phenotype, mechanical loading response and estrogen receptor-related gene expression.

The osteoporosis-resistant nature of skull bones implies inherent differences exist between their cellular responses and those of other osteoporosis-susceptible skeletal sites. Phenotypic differences in calvarial and femoral osteoblastic responses to induction of osteogenesis, mechanical loading, estrogen, growth factor and cytokine stimulation were investigated. Primary rat calvarial and femoral adult male osteoblasts were cultured and osteoblastic mineralisation and maturation determined using Alizarin Red staining and expression of osteogenic marker genes assessed. Expression of known mechanically-responsive genes was compared between sites following loading of scaffold-seeded cells in a bioreactor. Cell proliferation and differentiation following growth factor and estrogen stimulation were also compared. Finally expression of estrogen receptors and associated genes during osteogenic differentiation were investigated. Calvarial osteoblasts exhibited delayed maturation (45d. vs 21d.) and produced less mineralised matrix than femoral osteoblasts when osteogenically induced. PDGF-BB and FGF2 both caused a selective increase in proliferation and decrease in osteoblastic differentiation of femoral osteoblasts. Mechanical stimulation resulted in the induction of the expression of Ccl2 and Anx2a selectively in femoral osteoblasts, but remained unchanged in calvarial cells. Estrogen receptor beta expression was selectively upregulated 2-fold in calvarial osteoblasts. Most interestingly, the estrogen responsive transcriptional repressor RERG was constitutively expressed at 1000-fold greater levels in calvarial compared with femoral osteoblasts. RERG expression in calvarial osteoblasts was down regulated during osteogenic induction whereas upregulation occurred in femoral osteoblasts. Bone cells of the skull are inherently different to those of the femur, and respond differentially to a range of stimuli. These site-specific differences may have important relevance in the development of strategies to tackle metabolic bone disorders.

Cellular differentiation, bioactive and mechanical properties of experimental light-curing pulp protection materials.

OBJECTIVE: Materials for pulp protection should have therapeutic properties in order to stimulate remineralization and pulp reparative processes. The aim of this study was to evaluate the mechanical properties, biocompatibility, cell differentiation and bioactivity of experimental light-curable resin-based materials containing bioactive micro-fillers. METHODS: Four calcium-phosphosilicate micro-fillers were prepared and incorporated into a resin blend: 1) Bioglass 45S5 (BAG); 2) zinc-doped bioglass (BAG-Zn); 3) ßTCP-modified calcium silicate (ß-CS); 4) zinc-doped ß-CS (ß-CS-Zn). These experimental resins were tested for flexural strength (FS) and fracture toughness (FT) after 24h and 30-day storage in simulated body fluid (SBF). Cytotoxicity was evaluated using MTT assay, while bioactivity was evaluated using mineralization and gene expression assays (Runx-2 & ALP). RESULTS: The lowest FS and FT at 24h was attained with ß-CS resin, while all the other tested materials exhibited a decrease in FS after prolonged storage in SBF. ß-CS-Zn maintained a stable FT after 30-day SBF aging. Incorporation of bioactive micro-fillers had no negative effect on the biocompatibility of the experimental materials tested in this study. The inclusion of zinc-doped fillers significantly increased the cellular remineralization potential and expression of the osteogenic genes Runx2 and ALP (p<0.05). SIGNIFICANCE: The innovative materials tested in this study, in particular those containing ß-CS-Zn and BAG-Zn may promote cell differentiation and mineralization. Thus, these materials might represent suitable therapeutic pulp protection materials for minimally invasive and atraumatic restorative treatments.

Sprouty 2, an Early Response Gene Regulator of FosB and Mesenchymal Stem Cell Proliferation During Mechanical Loading and Osteogenic Differentiation.

Sprouty 2 (Spry2), an inhibitor of MAP kinase signaling was previously shown by our group to be induced during mechanical loading of mesenchymal stem cells (MSCs). Here, we studied the implication of Spry2 activation during mechanical loading and chemically induced MSC differentiation. Spry 2 expression showed an immediate early response during mechanical loading and chemical induction of osteogenic differentiation and followed the same pattern as osteogenic associated gene FosB and was necessary for the induction of FosB, as Spry 2 knock down also abrogated the upregulation of FosB expression. Spry 2 knock down was, also associated with an early response of the osteogenic genes Runx-2 and ALP. Neither the knock-down of Spry 2 nor the subsequent reduction in FosB had any effect on mid-late osteogenesis or mineralization but was associated with a significant increase in proliferation of MSC. These effects were possibly governed by negative regulation of MEK/Erk signaling as Spry 2 knock down resulted in an increase in phosphorylation of Erk1/2. In summary, our results shows the involvement of Spry2 in regulation of FosB and Runx2 genes, MAPK signaling and proliferation of MSC. Taken together these results suggest a possible role for Spry2 in regulation of MSC functions in response to mechanical loading and osteogenic differentiation. J. Cell. Biochem. 118: 2606-2614, 2017. © 2017 Wiley Periodicals, Inc.

DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1α and mTOR signalling.

Stem cell fate decisions to remain quiescent, self-renew or differentiate are largely governed by the interplay between extracellular signals from the niche and the cell intrinsic signal cascades and transcriptional programs. Here we demonstrate that DNA Damage Inducible Transcript 4 (DDIT4) acts as a link between HIF1α and mTOR signalling and regulation of adult stem cell fate. Global gene expression analysis of mesenchymal stem cells (MSC) derived from single clones and live RNA cell sorting showed a direct correlation between DDIT4 and differentiation potentials of MSC. Loss and gain of function analysis demonstrated that DDIT4 activity is directly linked to regulation of mTOR signalling, expression of pluripotency genes and differentiation. Further we demonstrated that DDIT4 exert these effects down-stream to HIF1α. Our findings provide an insight in regulation of adult stem cells homeostasis by two major pathways with opposing functions to coordinate between states of self-renewal and differentiation.

Self-assembled monolayers of alendronate on Ti6Al4V alloy surfaces enhance osteogenesis in mesenchymal stem cells.

Phosphonates have emerged as an alternative for functionalization of titanium surfaces by the formation of homogeneous self-assembled monolayers (SAMs) via Ti-O-P linkages. This study presents results from an investigation of the modification of Ti6Al4V alloy by chemisorption of osseoinductive alendronate using a simple, effective and clean methodology. The modified surfaces showed a tailored topography and surface chemistry as determined by SEM microscopy and RAMAN spectroscopy. X-ray photoelectron spectroscopy revealed that an effective mode of bonding is created between the metal oxide surface and the phosphate residue of alendronate, leading to formation of homogenous drug distribution along the surface. In-vitro studies showed that alendronate SAMs induce differentiation of hMSC to a bone cell phenotype and promote bone formation on modified surfaces. Here we show that this novel method for the preparation of functional coatings on titanium-based medical devices provides osseoinductive bioactive molecules to promote enhanced integration at the site of implantation.

Inhibition of Akt/mTOR attenuates age-related changes in mesenchymal stem cells.

The decline in mesenchymal stem cell (MSC) self-renewal and function with aging contributes to diseases associated with impaired osteogenesis. MSC donor age in prolonged culture also limits the therapeutic potential of these cells for tissue engineering and regenerative medicine. Here, we demonstrate an intervention to preserve the immature state MSC and consequently maintain self-renewal and differentiation capacity during in vitro aging. We showed that blocking of phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) prevents the development of an age-related phenotype and maintains MSC morphology of early passage cells with high clonogenic frequency and enhanced proliferative capacity. MSC cultured in the presence of inhibitors of Akt or mTOR also robustly maintain their osteogenic potential, that is otherwise lost during in vitro aging. We further report that these effects may be mediated by induction of expression of pluripotency genes Nanog and Oct-4 and by the reduction in the production of cytoplasmic reactive oxygen species (ROS). Additionally, loss of Akt/mTOR and ROS was accompanied with lower levels of DNA damage. These results provide an insight into mechanisms involved in MSC aging and suggest possible interventions to maintain quiescence and function of MSC prior to in vivo transplantation or as pharmacological agents in diseases associated with loss of MSC function.

Gene expression responses to mechanical stimulation of mesenchymal stem cells seeded on calcium phosphate cement.

INTRODUCTION: The aim of the study reported here was to investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain. METHODS: Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5±4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis. RESULTS: The maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB. There were also changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process. CONCLUSION: The results suggest that mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation. The results provide a valuable insight into molecular events and signal transduction pathways involved in the regulation of MSC osteogenic differentiation in response to a physiological level of mechanical stimulation.

Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess great potential for use in regenerative medicine. However, their clinical application may be limited by the ability to expand their cell numbers in vitro while maintaining their differential potentials and stem cell properties. Thus the aim of this study was to test the effect of a range of medium supplements on MSC self-renewal and differentiation potential. Cells were cultured until confluent and subcultured continuously until reaching senescence. Medium supplementation with fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB, ascorbic acid (AA), and epidermal growth factor (EGF) both increased proliferation rate and markedly increased number of cell doublings before reaching senescence, with a greater than 1,000-fold increase in total cell numbers for AA, FGF-2, and PDGF-BB compared with control cultures. Long-term culture was associated with loss of osteogenic/adipocytic differentiation potential, particularly with FGF-2 supplementation but also with AA, EGF, and PDGF-BB. In addition FGF-2 resulted in reduction in expression of CD146 and alkaline phosphatase, but this was partially reversible on removal of the supplement. Cells expressed surface markers including CD146, CD105, CD44, CD90, and CD71 by flow cytometry throughout, and expression of these putative stem cell markers persisted even after loss of differentiation potentials. Overall, medium supplementation with FGF-2, AA, EGF, and PDGF-BB greatly enhanced the total in vitro expansion capacity of MSC cultures, although differentiation potentials were lost prior to reaching senescence. Loss of differentiation potential was not reflected by changes in stem cell surface marker expression.

Akt- and Erk-mediated regulation of proliferation and differentiation during PDGFRß-induced MSC self-renewal.

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self-renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet-derived growth factor receptor ß (PDGFRß) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF-BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl-2'-deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT-PCR, and by long-term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRß. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR-ß expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E-BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRß signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRß-induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self-renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self-renewal strategies.

Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes.

Osteoblasts and adipocytes differentiate from a common precursor cell, the mesenchymal stem cell (MSC). Adenosine is known to signal via four adenosine receptor subtypes, and significantly, recent findings indicate that these may play a role in MSC differentiation. We therefore investigated adenosine receptor expression and activation during the differentiation of MSCs to osteoblasts and adipocytes. The A(2B) R was dominant in MSCs, and its expression and activity were transiently upregulated at early stages of osteoblastic differentiation. Both activation and overexpression of A(2B) R induced the expression of osteoblast-related genes [Runx2 and alkaline phosphatase (ALP)], as well as ALP activity, and stimulation increased osteoblast mineralization. The expression of A(2A) R was upregulated during later stages of osteoblastic differentiation, when its activation stimulated ALP activity. Differentiation of MSCs to adipocytes was accompanied by significant increases in A(1) R and A(2A) R expression, and their activation was associated with increased adipogenesis. Enhanced A(2A) R expression was sufficient to promote expression of adipocyte-related genes (PPARγ and C/EBPα), and its activation resulted in increased adipocytic differentiation and lipid accumulation. In contrast, the A(1) R was involved mainly in lipogenic activity of adipocytes rather than in their differentiation. These results show that adenosine receptors are differentially expressed and involved in lineage-specific differentiation of MSCs. We conclude, therefore, that fruitful strategies for treating diseases associated with an imbalance in the differentiation and function of these lineages should include targeting adenosine receptor signal pathways. Specifically, these research avenues will be useful in preventing or treating conditions with insufficient bone or excessive adipocyte formation.