The doublesex-related Dmrta2 safeguards neural progenitor maintenance involving transcriptional regulation of Hes1.

The mechanisms that determine whether a neural progenitor cell (NPC) reenters the cell cycle or exits and differentiates are pivotal for generating cells in the correct numbers and diverse types, and thus dictate proper brain development. Combining gain-of-function and loss-of-function approaches in an embryonic stem cell-derived cortical differentiation model, we report that doublesex- and mab-3-related transcription factor a2 (Dmrta2, also known as Dmrt5) plays an important role in maintaining NPCs in the cell cycle. Temporally controlled expression of transgenic Dmrta2 in NPCs suppresses differentiation without affecting their neurogenic competence. In contrast, Dmrta2 knockout accelerates the cell cycle exit and differentiation into postmitotic neurons of NPCs derived from embryonic stem cells and in Emx1-cre conditional mutant mice. Dmrta2 function is linked to the regulation of Hes1 and other proneural genes, as demonstrated by genome-wide RNA-seq and direct binding of Dmrta2 to the Hes1 genomic locus. Moreover, transient Hes1 expression rescues precocious neurogenesis in Dmrta2 knockout NPCs. Our study thus establishes a link between Dmrta2 modulation of Hes1 expression and the maintenance of NPCs during cortical development.

A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells.

Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the ß-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format).

Controlled release of dextrin-conjugated growth factors to support growth and differentiation of neural stem cells.

An essential aspect of stem cell in vitro culture and in vivo therapy is achieving sustained levels of growth factors to support stem cell survival and expansion, while maintaining their multipotency and differentiation potential. This study investigated the ability of dextrin (~74,000 g/mol; 27.8 mol% succinoylation) conjugated to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF; or FGF-2) (3.9 and 6.7% w/w protein loading, respectively) to support the expansion and differentiation of stem cells in vitro via sustained, controllable growth factor release. Supplementation of mouse neural stem cells (mNSCs) with dextrin-growth factor conjugates led to greater and prolonged proliferation compared to unbound EGF/bFGF controls, with no detectable apoptosis after 7 days of treatment. Immunocytochemical detection of neural precursor (nestin) and differentiation (Olig2, MAP2, GFAP) markers verified that controlled release of dextrin-conjugated growth factors preserves stem cell properties of mNSCs for up to 7 days. These results show the potential of dextrin-growth factor conjugates for localized delivery of bioactive therapeutic agents to support stem cell expansion and differentiation, and as an adjunct to direct neuronal repair.

Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells.

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K(+) but not outward Na(+) currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

Functionalized α-Helical Peptide Hydrogels for Neural Tissue Engineering.

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair.

Isolation and long-term expansion of functional, myelinating oligodendrocyte progenitor cells from neonatal rat brain.

Oligodendrocytes are the myelinating cells of the central nervous system (CNS). The isolation of purified oligodendrocyte progenitor cells (OPCs) in large numbers has been sought after as a source of cells for repair following CNS-demyelinating diseases and injuries, such as multiple sclerosis (MS) and spinal cord injury (SCI). Methods for isolation of OPCs from rodent neonatal brains are well established and have formed the basis for research in myelin repair within the CNS for many years. However, long-term maintenance of OPCs has been a challenge owing to small cellular yields per animal and spontaneous differentiation within a short period of time. Much effort has been devoted to achieving long-term culture and maintenance of OPCs, but little progress has been made. Here, protocols are presented for preparation of highly enriched rat OPC populations and for their long-term maintenance as oligospheres using mixed-glial-conditioned medium. Functional myelinating oligodendrocytes can be achieved from such protocols, when co-cultured with primary neurons. This approach is an extension of our normal shaking method for isolating OPCs, and incorporates some adaptations from previous OPC culture methods.