Evaluation of a cell-based osteogenic formulation compliant with good manufacturing practice for use in tissue engineering

Molecular Biology Reports - Proper bony tissue regeneration requires mechanical stabilization, an osteogenic biological activity and appropriate scaffolds. The latter two elements can be combined...     

Loss and rescue of osteocalcin and osteopontin modulate osteogenic and angiogenic features of mesenchymal stem/stromal cells

Noncollagenous proteins in the bone extracellular matrix, such as osteocalcin (OC) and osteopontin (OPN), inherent to evolution of bone as a skeletal tissue, are known to regulate bone formation and mineralization. However, the fundamental basis of this regulatory role remains unknown. Here, for the first time, we use mouse mesenchymal stem/stromal cells (MSC) lacking both OC and OPN to investigate the mechanistic roles of OC and OPN on the proliferation capacity and differentiation ability of MSC. We found that the loss of OC and OPN reduces stem cells self-renewal potential and multipotency, affects their differentiation into an osteogenic lineage, and impairs their angiogenic potential while maintaining chondrogenic and adipogenic lineages. Moreover, loss of OC and OPN compromises the extracellular matrix integrity and maturation, observed by an unexpected enhancement of glycosaminoglycans content that are associated with a more primitive skeletal connective tissue, and by a delay on the maturation of mineral species produced. Interestingly, exogenously supplemented OC and OPN were able to rescue MSC proliferative and osteogenic potential along with matrix integrity and mineral quality. Taken together, these results highlight the key contributions of OC and OPN in enhancing osteogenesis and angiogenesis over primitive connective tissue, and support a potential therapeutic approach based on their exogenous supplementation.     

The crystal structure of human telomeric DNA complexed with berberine: an interesting case of stacked ligand to G-tetrad ratio higher than 1:1

The first crystal structure of human telomeric DNA in complex with the natural alkaloid berberine, produced by different plant families and used in folk medicine for millennia, was solved by X-ray diffraction method. The G-quadruplex unit features all-parallel strands. The overall folding assumed by DNA is the same found in previously reported crystal structures. Similarly to previously reported structures the ligand molecules were found to be stacked onto the external 5′ and 3′-end G-tetrads. However, the present crystal structure highlighted for the first time, the presence of two berberine molecules in the two binding sites, directly interacting with each tetrad. As a consequence, our structural data point out a 2:1 ligand to G-tetrad molar ratio, which has never been reported before in a telomeric intramolecular quadruplex structure.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep

The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P < 0.001). The efficiency of the extraction method was confirmed by the results obtained from qPCR which showed a Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.     

Late Jurassic ichthyosaurs from the Neuquén Basin,Argentina

Late Jurassic ichthyosaurs are well represented in the Tithonian of the Neuquén Basin, in northwestern Patagonia, Argentina. Most of the ichthyosaur material from the Neuquén Basin was originally identified as Ophthalmosaurus. Recently, the new ichthyosaur genus Caypullisaurus was described, based on an almost complete mature specimen from Cerro Lotena. Some material previously referred to Ophlhalmosaurus has been referred to the new genus. However, both genera are present in the Tithonian of the Neuquén Basin. The discovery of an articulated forefin in Cajón de Almanza (near Loncopue, Neuquén) confirms the presence of Ophthalmosaurus in the uppermost Tithonian of the Neuquén Basin.     

Remodelling of skeletal tissues bone and structural specialisations in an elasmosaurid (Sauropterygia: Plesiosauroidea) from the Upper Cretaceous of Patagonia,Argentina

Elasmosauridae were cosmopolitan Late Cretaceous plesiosaurs with conspicuous morphological diversity. Within this group, vertebral morphology is a criterion for estimating relative age in plesiosaur. On the other hand, the microstructure of plesiosaur bone is considered as indicative of ontogenetic stage. However, knowledge about ontogenetic tissue transformation in different elements of the skeleton is poorly known. Resorption and remodelling of skeletal tissues are required for development and growth, mechanical adaptation, repair and mineral homeostasis of the vertebrate skeleton. This contribution analyses different postcranial elements of a Late Cretaceous elasmosaurid from Patagonia. Characterisation of bone microstructure indicates the presence of compact bone inner organisation in an adult derived plesiosaur from the Cretaceous and that the distribution of bone specialisations depicts conspicuous variations within a single skeleton depending on the skeletal element considered. Bone compactness or degree of remodelling in elasmosaurids is not necessarily correlated with the ontogenetic age of the animal or to costal versus pelagic lifestyles. The available data are still scarce, but we propose a topic of discussion: perhaps the degree of remodelling and compactness also may be related to the activity level and increased mechanical load in different skeletal elements.     

CXCL12-Mediated Murine Neural Progenitor Cell Movement Requires PI3Kβ Activation

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.