Pascal Vaudin

Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin α5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/β-catenin signaling-dependent Runx2 expression

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step in bone formation. However, the mechanisms involved in the early stages of osteogenic differentiation are not well understood. In this study, we identified FHL2, a member of the LIM‐only subclass of the LIM protein superfamily, that is up‐regulated during early osteoblast differentiation induced by dexamethasone in murine and human MSCs. Gain‐of‐function studies showed that FHL2 promotes the expression of the osteoblast transcription factor Runx2, alkaline phosphatase, type I collagen, as well as in vitro extracellular matrix mineralization in murine and human mesenchymal cells. Knocking down FHL2 using sh‐RNA reduces basal and dexamethasone‐induced osteoblast marker gene expression in MSCs. We demonstrate that FHL2 interacts with β‐catenin, a key player involved in bone formation induced by Wnt signaling. FHL2‐β‐catenin interaction potentiates β‐catenin nuclear translocation and TCF/LEF transcription, resulting in increased Runx2 and alkaline phosphatase expression, which was inhibited by the Wnt inhibitor DKK1. Reduction of Runx2 transcriptional activity using a mutant Runx2 results in inhibition of FHL2‐induced alkaline phosphatase expression in MSCs. These findings reveal that FHL2 acts as an endogenous activator of mesenchymal cell differentiation into osteoblasts and mediates osteogenic differentiation induced by dexamethasone in MSCs through activation of Wnt/β‐catenin signaling‐dependent Runx2 expression.— Hamidouche, Z., Haÿ, E., Vaudin, P., Charbord, P., Schüle, R., Marie, P. J., and Fromigue, O. FHL2 mediates dexamethasone‐induced mesenchymal cell differentiation into osteoblasts by activating Wnt/β‐catenin signaling‐dependent Runx2 expression. FASEB J. 22, 3813–3822 (2008)

Influence of hypoxia on the domiciliation of mesenchymal stem cells after infusion into rats: possibilities of targeting pulmonary artery remodeling via cells therapies?

BackgroundBone marrow (BM) cells are promising tools for vascular therapies. Here, we focused on the possibility of targeting the hypoxia-induced pulmonary artery hypertension remodeling with systemic delivery of BM-derived mesenchymal stem cells (MSCs) into non-irradiated rats.MethodsSix-week-old Wistar rats were exposed to 3-week chronic hypoxia leading to pulmonary artery wall remodeling. Domiciliation of adhesive BM-derived CD45- CD73+ CD90+ MSCs was first studied after a single intravenous infusion of Indium-111-labeled MSCs followed by whole body scintigraphies and autoradiographies of different harvested organs. In a second set of experiments, enhanced-GFP labeling allowed to observe distribution at later times using sequential infusions during the 3-week hypoxia exposure.ResultsA 30% pulmonary retention was observed by scintigraphies and no differences were observed in the global repartition between hypoxic and control groups. Intrapulmonary radioactivity repartition was homogenous in both groups, as shown by autoradiographies. BM-derived GFP-labeled MSCs were observed with a global repartition in liver, in spleen, in lung parenchyma and rarely in the adventitial layer of remodeled vessels. Furthermore this global repartition was not modified by hypoxia. Interestingly, these cells displayed in vivo bone marrow homing, proving a preservation of their viability and function. Bone marrow homing of GFP-labeled MSCs was increased in the hypoxic group.ConclusionAdhesive BM-derived CD45- CD73+ CD90+ MSCs are not integrated in the pulmonary arteries remodeled media after repeated intravenous infusions in contrast to previously described in systemic vascular remodeling or with endothelial progenitor cells infusions.

Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies

The hepatitis B virus (HBV) envelope protein (S) self-assembles into subviral particles used as commercial vaccines against hepatitis B. These particles are excellent carriers for foreign epitopes, which can be inserted into the external hydrophilic loop or at the N- or C-terminal end of the HBV S protein. We show here that the N-terminal transmembrane domain (TMD) of HBV S can be replaced by the TMDs of the hepatitis C virus (HCV) envelope proteins E1 and E2, to generate fusion proteins containing the entire HCV E1 or E2 sequence that are efficiently coassembled with the HBV S into particles. This demonstrates the remarkable tolerance of the HBV S protein to sequence substitutions conserving its subviral particle assembly properties. These findings may have implications for the design of new vaccine strategies based on the use of HBV subviral particles as carriers for various transmembrane proteins and produced using the same industrial procedures that are established for the HBV vaccine.

Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells: Implication of FGFR2 signaling

The capacity of mesenchymal stem cells (MSCs) to differentiate into functional osteoblasts is tightly controlled by transcription factors that trigger osteoblast commitment and differentiation. The role of Twist1, a basic helix‐loop‐helix (bHLH) transcription factor, in osteogenic differentiation of MSCs remains unclear. Here we investigated the role of Twist1 in the osteogenic differentiation program of murine C3H10T1/2 mesenchymal cells. We showed that molecular silencing of Twist1 using short hairpin RNA (shRNA) expression moderately increased C3H10T1/2 cell proliferation and had no effect on cell survival. In contrast, Twist1 silencing enhanced osteoblast gene expression and matrix mineralization in vitro. Biochemical analyses revealed that Twist1 silencing increased the expression of FGFR2 protein level, which was reduced by a mutant Runx2. Consistent with this finding, Twist1 silencing increased ERK1/2 and PI3K signaling. Moreover, molecular or pharmacological inhibition of FGFR2 or of ERK1/2 and PI3K signaling partly abolished the increased osteoblast gene expression induced by Twist1 silencing in C3H10T1/2 cells. These results reveal that Twist1 silencing upregulates osteoblast differentiation of murine mesenchymal cells in part via activation of FGFR2 expression and downstream signaling pathways, which provides novel insights into the molecular signals by which this transcription factor regulates the osteogenic differentiation program in MSCs. J. Cell. Biochem. 110: 1147–1154, 2010. Published 2010 Wiley‐Liss, Inc.

Olfactory Ensheathing Cells Form the Microenvironment of Migrating GnRH-1 Neurons During Mouse Development

During development, GnRH‐1 neurons differentiate extracerebraly from the nasal placode and migrate from the vomeronasal organ to the forebrain along vomeronasal and terminal nerves. Numerous studies have described the influence of different molecules on the migration of GnRH‐1 neurons, however, the role of microenvironment cells remains poorly understood. This study used GFAP‐GFP transgenic mice to detect glial cells at early developmental stages. Using nasal explant cultures, the comigration of glial cells with GnRH‐1 neurons was clearly demonstrated. This in vitro approach showed that glial cells began migrating from the explants before GnRH‐1 neurons. They remained ahead of the GnRH‐1 migratory front and stopped migrating after the GnRH‐1 neurons. The association of these glial cells with the axons combined with gene expression analysis of GFAP‐GFP sorted cells enabled them to be identified as olfactory ensheathing cells (OEC). Immunohistochemical analysis revealed the presence of multiple glial cell‐type markers showing several OEC subpopulations surrounding GnRH‐1 neurons. Moreover, these OEC expressed genes whose products are involved in the migration of GnRH‐1 neurons, such as Nelf and Semaphorin 4. In situ data confirmed that the majority of the GnRH‐1 neurons were associated with glial cells along the vomeronasal axons in nasal septum and terminal nerves in the nasal forebrain junction as early as E12.5. Overall, these data demonstrate an OEC microenvironment for migrating GnRH‐1 neurons during mouse development. The fact that this glial cell type precedes GnRH‐1 neurons and encodes for molecules involved in their nasal migration suggests that it participates in the GnRH‐1 system ontogenesis.

Lentiviral-Mediated Integrin α5 Expression in Human Adult Mesenchymal Stromal Cells Promotes Bone Repair in Mouse Cranial and Long-Bone Defects

Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

Rostro‐caudal maturation of glial cells in the accessory olfactory system during development: involvement in outgrowth of GnRH neurites

During mammalian embryonic development, GnRH neurones differentiate from the nasal placode and migrate through the nasal septum towards the forebrain. We previously showed that a category of glial cells, the olfactory ensheathing cells (OEC), forms the microenvironment of migrating GnRH neurones. Here, to characterize the quantitative and qualitative importance of this glial, we investigated the spatiotemporal maturation of glial cells in situ and the role of maturing glia in GnRH neurones development ex vivo. More than 90% of migrating GnRH neurones were found to be associated with glial cells. There was no change in the cellular microenvironment of GnRH neurones in the regions crossed during embryonic development as glial cells formed the main microenvironment of these neurones (53.4%). However, the phenotype of OEC associated with GnRH neurones changed across regions. The OEC progenitors immunoreactive to brain lipid binding protein formed the microenvironment of migrating GnRH neurones from the vomeronasal organ to the telencephalon and were also present in the diencephalon. However, during GnRH neurone migration, maturation of OEC to [GFAP+] state (glial fibrillary acid protein) was only observed in the nasal septum. Inducing depletion of OEC in maturation, using transgenic mice expressing herpes simplex virus thymidine kinase driven by the GFAP promoter, had no impact on neurogenesis or on triggering GnRH neurones migration in nasal explant culture. Nevertheless, depletion of [GFAP+] cells decreased GnRH neurites outgrowth by 57.4%. This study suggests that specific maturation of OEC in the nasal septum plays a role in morphological differentiation of GnRH neurones.

LA PLASTICITÉ NEURO-GLIALE DES RÉSEAUX NEUROENDOCRINES

Neuro-glial plasticity of neuroendocrine networks is a major mechanism involved in key events of physiological functions such as parturition and lactation (oxytocinergic system) and preovulatory surge (GnRH system). This type of plasticity is classically described as rearrangements between glial cells and neuroendocrine neurones. Neuro-glial plasticity can occur within several hours. Cellular and molecular mechanisms involved are complex and imply an active regulation of neuroendocrine networks activity. In the present study we show that GnRH pulsatile secretion studied in vitro is regulated by gap junction communication between glial cells. Glial cells forming the microenvironment of GnRH neuronal network could represent a new system for integrating environmental cues and for regulating GnRH secretion