Human platelet lysate enhances proliferation but not chondrogenic differentiation of pediatric mesenchymal progenitors.

BACKGROUND AIMS: Cell therapies have the potential to improve reconstructive procedures for congenital craniofacial cartilage anomalies such as microtia. Adipose-derived stem cells (ADSCs) and auricular cartilage stem/progenitor cells (CSPCs) are promising candidates for cartilage reconstruction, but their successful use in the clinic will require the development of xeno-free expansion and differentiation protocols that can maximize their capacity for chondrogenesis. METHODS: We assessed the behavior of human ADSCs and CSPCs grown either in qualified fetal bovine serum (FBS) or human platelet lysate (hPL), a xeno-free alternative, in conventional monolayer and 3-dimensional spheroid cultures. RESULTS: We show that CSPCs and ADSCs display greater proliferation rate in hPL than FBS and express typical mesenchymal stromal cell surface antigens in both media. When expanded in hPL, both cell types, particularly CSPCs, maintain a spindle-like morphology and lower surface area over more passages than in FBS. Both media supplements support chondrogenic differentiation of CSPCs and ADSCs grown either as monolayers or spheroids. However, chondrogenesis appears less ordered in hPL than FBS, with reduced co-localization of aggrecan and collagen type II in spheroids. CONCLUSIONS: hPL may be beneficial for the expansion of cells with chondrogenic potential and maintaining stemness, but not for their chondrogenic differentiation for tissue engineering or disease modeling.

Dystrophin deficiency affects human astrocyte properties and response to damage.

In addition to progressive muscular degeneration due to dystrophin mutations, 1/3 of Duchenne muscular dystrophy (DMD) patients present cognitive deficits. However, there is currently an incomplete understanding about the function of the multiple dystrophin isoforms in human brains. Here, we tested the hypothesis that dystrophin deficiency affects glial function in DMD and could therefore contribute to neural impairment. We investigated human dystrophin isoform expression with development and differentiation and response to damage in human astrocytes from control and induced pluripotent stem cells from DMD patients. In control cells, short dystrophin isoforms were up-regulated with development and their expression levels changed differently upon neuronal and astrocytic differentiation, as well as in 2-dimensional versus 3-dimensional astrocyte cultures. All DMD-astrocytes tested displayed altered morphology, proliferative activity and AQP4 expression. Furthermore, they did not show any morphological change in response to inflammatory stimuli and their number was significantly lower as compared to stimulated healthy astrocytes. Finally, DMD-astrocytes appeared to be more sensitive than controls to oxidative damage as shown by their increased cell death. Behavioral and metabolic defects in DMD-astrocytes were consistent with gene pathway dysregulation shared by lines with different mutations as demonstrated by bulk RNA-seq analysis. Together, our DMD model provides evidence for altered astrocyte function in DMD suggesting that defective astrocyte responses may contribute to neural impairment and might provide additional potential therapeutic targets.

Adipose-Derived Stem Cells in Aesthetic Surgery.

Adipose-derived stem cells (ADSC) have come to be viewed as a ubiquitous solution for aesthetic and reconstructive problems involving loss of tissue volume and age or radiation-induced loss of tissue pliability and vascularity. As the theoretical potential of "stem cell therapy" has captured the public imagination, so the commercial potential of novel therapies is being exploited beyond scientifically sound, hypothesis-driven paradigms and in the absence of evidence establishing clinical efficacy and safety. Moreover, with variations in methods of isolation, manipulation, and reintroduction described, it is unclear how the practitioner with an interest in ADSC can harness the clinical potential in reproducible and scientifically measurable ways. This Continuing Medical Education (CME) article presents a summary of our understanding of what ADSC are, their utility within the field of aesthetic surgery, and the current and future directions for adipose stem cell research.

Pulling and Pushing Stem Cells to Control Their Differentiation.

Much has already been done to achieve precisely controlled and customised regenerative therapies. Thanks to recent advances made in several areas relevant to regenerative medicine including the use of stimuli-responsive materials, 4-dimensional biofabrication, inducible pluripotent stem cells, control of stem cell fate using chemical and physical factors, minimal access delivery, and information-communication technology. In this short perspective, recent advances are discussed with a focus on a recent report on the use of mechanical stretching of nanoparticle-laden stem cells by using external magnetic field to induce defined cardiac line differentiation. Although more and more tools are becoming available for engineering tissue models tissues and the range of potential applications is expanding, there is still much work to be done before it is proved to work with human cells, form tissues and ultimately achieve application in the clinic.

Objectifying Micrognathia Using Three-Dimensional Photogrammetric Analysis.

BACKGROUND: Micrognathia occurs isolated and as part of entities like Robin sequence (RS). An objective measurement of mandible size and growth is needed to determine the degree of micrognathia and enable a comparison of treatment outcomes. A pilot study was conducted to investigate the usability of 3-dimensional (3D) facial photogrammetry, a fast, noninvasive method, to estimate mandible size and growth in a small cohort of newborns and infants. METHODS: Exterior mandibular volume was estimated using a tetrahedron defined by 4 facial landmarks. Twelve patients with RS with different etiologies were selected and photogrammetric images were obtained prospectively in 3 patients with RS in whom mandibular growth in the first year of life was determined. We used 3 tetrahedra defined by 6 landmarks on mandibular computed tomography (CT) scans to estimate an interior mandibular volume, which we compared to the exterior mandibular volume in 10 patients. RESULTS: The exterior mandibular volume using 3D photography could be determined in all patients. Signature heat maps allowed visualization of facial dysmorphism in 3D; signature graphs demonstrated similarities of facial dysmorphism in patients with the same etiology and differences from those with other diagnoses and from controls. The correlation between interior (3D photogrammetry) and exterior mandibular volumes (CT imaging) was 0.8789. CONCLUSION: The 3D facial photogrammetry delineates the general facial characteristics in patients with different syndromes involving micrognathia, and can objectively estimate mandibular volume and growth, with excellent correlation with bony measurement. It has been concluded that 3D facial photogrammetry could be a clinically effective instrument for delineating and quantifying micrognathia.

An overview of the therapeutic potential of regenerative medicine in cutaneous wound healing.

The global burden of disease associated with wounds is an increasingly significant public health concern. Current treatments are often expensive, time-consuming and limited in their efficacy in chronic wounds. The challenge of overcoming current barriers associated with wound care requires innovative management techniques. Regenerative medicine is an emerging field of research that focuses on the repair, replacement or regeneration of cells, tissues or organs to restore impaired function. This article provides an overview of the pathophysiology of wound healing and reviews the latest evidence on the application of the principal components of regenerative medicine (growth factors, stem cell transplantation, biomaterials and tissue engineering) as therapeutic targets. Improved knowledge and understanding of the pathophysiology of wound healing has pointed to new therapeutic targets. Regenerative medicine has the potential to underpin the design of specific target therapies in acute and chronic wound healing. This personalised approach could eventually reduce the burden of disease associated with wound healing. Further evidence is required in the form of large animal studies and clinical trials to assess long-term efficacy and safety of these new treatments.

Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase-AIF pathway.

PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to citrulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated apoptosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskeleton disassembly may play a role in AIF translocation to the nucleus. This is the first study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium-induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved.

Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells.

Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

Considering the evolution of regeneration in the central nervous system.

For many years the mammalian CNS has been seen as an organ that is unable to regenerate. However, it was also long known that lower vertebrate species are capable of impressive regeneration of CNS structures. How did this situation arise through evolution? Increasing cellular and molecular understanding of regeneration in different animal species coupled with studies of adult neurogenesis in mammals is providing a basis for addressing this question. Here we compare CNS regeneration among vertebrates and speculate on how this ability may have emerged or been restricted.

Chondrogenic differentiation of adipose tissue-derived stem cells within nanocaged POSS-PCU scaffolds: a new tool for nanomedicine.

Scaffold cellularization for cartilage engineering can aid implant properties, their retention and minimize repeated intervention, particularly in paediatric reconstructive craniofacial surgery. We developed novel bionanoscaffolds using paediatric adipose tissue-derived stem cells (hADSCs), an accessible autologous cell source, and POSS-PCU. Little is known about cellular responses to this nanomaterial, though it was used in human. We assessed: 1) POSS-PCU cellularization and bioaffinity to hADSCs; 2) hADSC chondrogenic differentiation ability in POSS-PCU; 3) whether bionanoscaffolds became encased within a vascular network and/or vascularised. POSS-PCU supported ADSC survival and proliferation and their migration and differentiation into cartilage within the nanoscaffold. Furthermore, after CAM-grafting, bionanoscaffolds were rapidly surrounded by blood vessels without any apparent negative reaction and erythrocytes of host origin were detected inside the scaffold, suggesting invasion from some capillaries. Altogether, this study demonstrates that POSS-PCU displays excellent bioactivity and hADSC/POSS-PCU bionanoscaffolds offer much promise for autologous cell-based tissue engineering for clinical applications. FROM THE CLINICAL EDITOR: In this study, human adipose tissue derived stem cells were used in combination with POSS-PCU nanoscaffolds to generate cartilage tissue demonstrating excellent bioactivity for autologous cell-based tissue engineering for clinical applications.

PolyQ length-dependent metabolic alterations and DNA damage drive human astrocyte dysfunction in Huntington's disease.

Huntington's Disease (HD) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the Huntingtin gene. Astrocyte dysfunction is known to contribute to HD pathology, however our understanding of the molecular pathways involved is limited. Transcriptomic analysis of patient-derived PSC (pluripotent stem cells) astrocyte lines revealed that astrocytes with similar polyQ lengths shared a large number of differentially expressed genes (DEGs). Notably, weighted correlation network analysis (WGCNA) modules from iPSC derived astrocytes showed significant overlap with WGCNA modules from two post-mortem HD cohorts. Further experiments revealed two key elements of astrocyte dysfunction. Firstly, expression of genes linked to astrocyte reactivity, as well as metabolic changes were polyQ length-dependent. Hypermetabolism was observed in shorter polyQ length astrocytes compared to controls, whereas metabolic activity and release of metabolites were significantly reduced in astrocytes with increasing polyQ lengths. Secondly, all HD astrocytes showed increased DNA damage, DNA damage response and upregulation of mismatch repair genes and proteins. Together our study shows for the first time polyQ-dependent phenotypes and functional changes in HD astrocytes providing evidence that increased DNA damage and DNA damage response could contribute to HD astrocyte dysfunction.

Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability.

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.

Aryl Hydrocarbon Receptor (AhR)-Mediated Signaling in iPSC-Derived Human Motor Neurons.

Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants.

Mechanical and morphological properties of parietal bone in patients with sagittal craniosynostosis.

Limited information is available on the effect of sagittal craniosynostosis (CS) on morphological and material properties of the parietal bone. Understanding these properties would not only provide an insight into bone response to surgical procedures but also improve the accuracy of computational models simulating these surgeries. The aim of the present study was to characterise the mechanical and microstructural properties of the cortical table and diploe in parietal bone of patients affected by sagittal CS. Twelve samples were collected from pediatric patients (11 males, and 1 female; age 5.2 ± 1.3 months) surgically treated for sagittal CS. Samples were imaged using micro-computed tomography (micro-CT); and mechanical properties were extracted by means of micro-CT based finite element modelling (micro-FE) of three-point bending test, calibrated using sample-specific experimental data. Reference point indentation (RPI) was used to validate the micro-FE output. Bone samples were classified based on their macrostructure as unilaminar or trilaminar (sandwich) structure. The elastic moduli obtained using RPI and micro-FE approaches for cortical tables (ERPI 3973.33 ± 268.45 MPa and Emicro-FE 3438.11 ± 387.38 MPa) in the sandwich structure and diploe (ERPI1958.17 ± 563.79 MPa and Emicro-FE 1960.66 ± 492.44 MPa) in unilaminar samples were in strong agreement (r = 0.86, p < .01). We found that the elastic modulus of cortical tables and diploe were correlated with bone mineral density. Changes in the microstructure and mechanical properties of bone specimens were found to be irrespective of patients' age. Although younger patients are reported to benefit more from surgical intervention as skull is more malleable, understanding the material properties is critical to better predict the surgical outcome in patients <1 year old since age-related changes were minimal.

A single-point mutation in FGFR2 affects cell cycle and Tgfbeta signalling in osteoblasts.

Fgf and Tgfbeta are key regulators of bone development. It is not known, however, whether there is a relationship between defective Fgf signalling, resulting in a premature cranial suture fusion, and Tgfbeta signalling. We used mouse calvaria osteoblasts carrying a mutation (hFGFR2-C278F) associated with Crouzon and Pfeiffer syndromes to investigate effects of this mutation on cell growth and possible mechanisms underlying it. Mutated osteoblasts displayed reduced S-phase, increased apoptosis and increased differentiation. As Tgfbeta signalling appeared to be required in an autocrine/paracrine manner for osteoblast proliferation, we tested the hypothesis that reduced growth might be due, at least in part, to an altered balance between FGF and Tgfbeta signalling. Tgfbeta expression was indeed decreased in mutated osteoblasts, as compared to osteoblasts carrying the wild type hFGFR2. Treatment with Tgfbeta, however, neither increased proliferation in mutated osteoblasts, unlike in controls, nor rescued proliferation in control osteoblasts treated with an Erk1/2 inhibitor. Significantly, Erk2, that is important for proliferation, was reduced relatively to Erk1 in mutated cells. Altogether this study suggests that the hFGFR2-C278F mutation affects the osteoblast ability to respond to Tgfbeta stimulation via the Erk pathway and that the overall effect of the mutation is a loss of function.

Is there a relationship between adult neurogenesis and neuron generation following injury across evolution?

All vertebrates can produce new neurons postnatally in discrete regions of their nervous system, but only some lower vertebrates (fish and amphibians) can significantly repair several neural structures, including brain, spinal cord, retina, olfactory and auditory-vestibular system, to compensate for neural tissue loss and recover significant functionality. Some regenerative ability, however, is found also in reptiles and birds, and even in mammals. The recognition that neurogenesis indeed occurs in the CNS of all adult vertebrates challenges the view that there is a simple relationship between maintenance of neurogenic regions in the adult CNS and regenerative capability. The aim of this review is to revisit this relationship in the light of recent literature focusing on selected examples of neurogenesis and regeneration, and discuss possible frameworks that may help to elucidate the relationship between adult neurogenesis and regeneration. This could provide useful paradigms for harnessing regeneration in the human CNS.

Eocene bipolar glaciation associated with global carbon cycle changes.

The transition from the extreme global warmth of the early Eocene 'greenhouse' climate approximately 55 million years ago to the present glaciated state is one of the most prominent changes in Earth's climatic evolution. It is widely accepted that large ice sheets first appeared on Antarctica approximately 34 million years ago, coincident with decreasing atmospheric carbon dioxide concentrations and a deepening of the calcite compensation depth in the world's oceans, and that glaciation in the Northern Hemisphere began much later, between 10 and 6 million years ago. Here we present records of sediment and foraminiferal geochemistry covering the greenhouse-icehouse climate transition. We report evidence for synchronous deepening and subsequent oscillations in the calcite compensation depth in the tropical Pacific and South Atlantic oceans from approximately 42 million years ago, with a permanent deepening 34 million years ago. The most prominent variations in the calcite compensation depth coincide with changes in seawater oxygen isotope ratios of up to 1.5 per mil, suggesting a lowering of global sea level through significant storage of ice in both hemispheres by at least 100 to 125 metres. Variations in benthic carbon isotope ratios of up to approximately 1.4 per mil occurred at the same time, indicating large changes in carbon cycling. We suggest that the greenhouse-icehouse transition was closely coupled to the evolution of atmospheric carbon dioxide, and that negative carbon cycle feedbacks may have prevented the permanent establishment of large ice sheets earlier than 34 million years ago.

Considering the Cellular Composition of Olfactory Ensheathing Cell Transplants for Spinal Cord Injury Repair: A Review of the Literature.

Olfactory ensheathing cells (OECs) are specialized glia cells of the olfactory system that support the continual regeneration of olfactory neurons throughout adulthood. Owing to their pro-regenerative properties, OECs have been transplanted in animal models of spinal cord injuries (SCI) and trialed in clinical studies on SCI patients. Although these studies have provided convincing evidence to support the continued development of OEC transplantation as a treatment option for the repair of SCI, discrepancies in the reported outcome has shown that OEC transplantation requires further improvement. Much of the variability in the reparative potential of OEC transplants is due to the variations in the cell composition of transplants between studies. As a result, the optimal cell preparation is currently a subject of debate. Here we review, the characterization as well as the effect of the cell composition of olfactory cell transplantation on therapeutic outcome in SCI. Firstly, we summarize and review the cell composition of olfactory cell preparations across the different species studied prior to transplantation. Since the purity of cells in olfactory transplants might affect the study outcome we also examine the effect of the proportions of OECs and the different cell types identified in the transplant on neuroregeneration. Finally, we consider the effect of the yield of cells on neuroregeneration by assessing the cell dose of transplants on therapeutic outcome.

Mislocalization of Nucleocytoplasmic Transport Proteins in Human Huntington's Disease PSC-Derived Striatal Neurons.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene (HTT). Disease progression is characterized by the loss of vulnerable neuronal populations within the striatum. A consistent phenotype across HD models is disruption of nucleocytoplasmic transport and nuclear pore complex (NPC) function. Here we demonstrate that high content imaging is a suitable method for detecting mislocalization of lamin-B1, RAN and RANGAP1 in striatal neuronal cultures thus allowing a robust, unbiased, highly powered approach to assay nuclear pore deficits. Furthermore, nuclear pore deficits extended to the selectively vulnerable DARPP32 + subpopulation neurons, but not to astrocytes. Striatal neuron cultures are further affected by changes in gene and protein expression of RAN, RANGAP1 and lamin-B1. Lowering total HTT using HTT-targeted anti-sense oligonucleotides partially restored gene expression, as well as subtly reducing mislocalization of proteins involved in nucleocytoplasmic transport. This suggests that mislocalization of RAN, RANGAP1 and lamin-B1 cannot be normalized by simply reducing expression of CAG-expanded HTT in the absence of healthy HTT protein.

Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord.

The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.