Sox-positive cell population in the adult cerebellum increases upon tissue degeneration.

Adult neurogenesis is well-described in the subventricular and subgranular zones of the mammalian brain. Recent observations that resident glia express stem cell markers in some areas of the brain not traditionally associated with neurogenesis hint to a possible role in tissue repair. The Bergmann glia (BG) population in the cerebellum displays markers and in vitro features associated with neural stem cells (NSC), however the physiological relevance of this phenotypic overlap remains unclear in the absence of established in vivo evidence of tissue regeneration in the adult cerebellum. Here, this BG population was analysed in the adult cerebellum of different species and showed conservation of NSC-associated marker expression including Sox1, Sox2 and Sox9, in chick, primate and mouse cerebellum tissue. NSC-like cells isolated from adult mouse cerebellum showed slower growth when compared to lateral ventricle NSC, as well as differences upon differentiation. In a mouse model of cerebellar degeneration, progressive Purkinje cell loss was linked to cerebellar cortex disorganisation and a significant increase in Sox-positive cells compared to matching controls. These results show that this Sox-positive population responds to cerebellar tissue disruption, suggesting it may represent a mobilisable cellular resource for targeted strategies to promote tissue repair.

Tailoring Pyro-and Orthophosphate Species to Enhance Stem Cell Adhesion to Phosphate Glasses.

Phosphate-based glasses (PBGs) offer significant therapeutic potential due to their bioactivity, controllable compositions, and degradation rates. Several PBGs have already demonstrated their ability to support direct cell growth and in vivo cytocompatibility for bone repair applications. This study investigated development of PBG formulations with pyro- and orthophosphate species within the glass system (40 - x)P2O5·(16 + x)CaO·20Na2O·24MgO (x = 0, 5, 10 mol%) and their effect on stem cell adhesion properties. Substitution of phosphate for calcium revealed a gradual transition within the glass structure from Q2 to Q0 phosphate species. Human mesenchymal stem cells were cultured directly onto discs made from three PBG compositions. Analysis of cells seeded onto the discs revealed that PBG with higher concentration of pyro- and orthophosphate content (61% Q1 and 39% Q0) supported a 4.3-fold increase in adhered cells compared to glasses with metaphosphate connectivity (49% Q2 and 51% Q1). This study highlights that tuning the composition of PBGs to possess pyro- and orthophosphate species only, enables the possibility to control cell adhesion performance. PBGs with superior cell adhesion profiles represent ideal candidates for biomedical applications, where cell recruitment and support for tissue ingrowth are of critical importance for orthopaedic interventions.

Live Simultaneous Monitoring of Mineral Deposition and Lipid Accumulation in Differentiating Stem Cells.

Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells.

Live Quantitative Monitoring of Mineral Deposition in Stem Cells Using Tetracycline Hydrochloride.

The final stage of in vitro osteogenic differentiation is characterized by the production of mineral deposits containing calcium cations and inorganic phosphates, which populate the extracellular matrix (ECM) surrounding the cell monolayer. Conventional histological techniques for the assessment of mineralization, such as Von Kossa and Alizarin Red S staining, are end point techniques requiring cell fixation. Moreover, in both cases staining quantitation requires dye extraction, which irreversibly alters the ECM conformation and structure, therefore preventing the use of the sample for further analysis. In this study, the use of tetracycline hydrochloride (TC) is proposed for the nondestructive staining, quantitation, and imaging of mineralizing bone-like nodules in live cultures of human bone marrow mesenchymal stem cells cultured under osteogenic conditions. Overnight administration of TC to living cells was shown not to alter the metabolic activity or the progression of cell differentiation. When applied to differentiating cultures, cell exposure to serial doses of TC was found to produce quantifiable fluorescence emission specifically in osteogenic cultures. Incubation with TC enabled fluorescence imaging of mineralized areas in live cultures and the combination with other fluorophores using appropriate filters. These results demonstrate that serial TC administration over the differentiation time course provides a qualitative and quantitative tool for the monitoring and evaluation of the differentiation process in live cells.