Alexis Joannides

Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells

BACKGROUND Neural stem cells are a potential source of cells for drug screening or cell-based treatments for neurodegenerative diseases. However, ethical and practical considerations limit the availability of neural stem cells derived from human embryonic tissue. An alternative source of human neural stem cells is needed; a source that is readily accessible, easily expanded, and reliably induced to a neural fate. METHODS Dermis isolated from biopsy samples of adult human skin was cultured and expanded in the presence of the mitogens epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF 2), and then by serum. We used immunocytochemical techniques, clonal analysis, and physiological characterisation to assess neural differentiation after the treatment of expanded cells with novel induction media. FINDINGS Initial characterisation of skin samples confirmed the absence of nestin, a neural precursor marker. Sequential culture in EGF and FGF 2 followed by adherent expansion in serum, and re-exposure to mitogens in substrate-free conditions resulted in large numbers of nestin-positive/musashi-positive neural precursors. Subsequent exposure of these precursors to hippocampal-astrocyte-derived signals resulted in cells of neuronal morphology that had stable expression of markers of neuronal differentiation (neurofilament, beta tubulin). We also show the presence of voltage-dependent calcium transients, and demonstrate monoclonal neural potential. INTERPRETATION We describe the isolation and characterisation of cells derived from adult human dermis that can be expanded for extended periods of time in vitro, while retaining inducible neural potential. The generation of almost limitless numbers of neural precursors from a readily accessible autologous adult human source provides a platform for further experimental studies and has potential therapeutic implications.

A Highly Enriched Niche of Precursor Cells with Neuronal and Glial Potential Within the Hair Follicle Dermal Papilla of Adult Skin

Skin‐derived precursor cells (SKPs) are multipotent neural crest‐related stem cells that grow as self‐renewing spheres and are capable of generating neurons and myelinating glial cells. SKPs are of clinical interest because they are accessible and potentially autologous. However, although spheres can be readily isolated from embryonic and neonatal skin, SKP frequency falls away sharply in adulthood, and primary sphere generation from adult human skin is more problematic. In addition, the culture‐initiating cell population is undefined and heterogeneous, limiting experimental studies addressing important aspects of these cells such as the behavior of endogenous precursors in vivo and the molecular mechanisms of neural generation. Using a combined fate‐mapping and microdissection approach, we identified and characterized a highly enriched niche of neural crest‐derived sphere‐forming cells within the dermal papilla of the hair follicle of adult skin. We demonstrated that the dermal papilla of the rodent vibrissal follicle is 1,000‐fold enriched for sphere‐forming neural crest‐derived cells compared with whole facial skin. These “papillaspheres” share a phenotypic and developmental profile similar to that of SKPs, can be readily expanded in vitro, and are able to generate both neuronal and glial cells in response to appropriate cues. We demonstrate that papillaspheres can be efficiently generated and expanded from adult human facial skin by microdissection of a single hair follicle. This strategy of targeting a highly enriched niche of sphere‐forming cells provides a novel and efficient method for generating neuronal and glial cells from an accessible adult somatic source that is both defined and minimally invasive.

A scaleable and defined system for generating neural stem cells from human embryonic stem cells

The ability to differentiate human ESCs (hESCs) to defined lineages in a totally controlled manner is fundamental to developing cell‐based therapies and studying human developmental mechanisms. We report a novel, scaleable, and widely applicable system for deriving and propagating neural stem cells from hESCs without the use of animal products, proprietary formulations, or genetic manipulation. This system provides a definitive platform for studying human neural development and has potential therapeutic implications.

Automated Mechanical Passaging: A Novel and Efficient Method for Human Embryonic Stem Cell Expansion

There is a need for more standardized methods of maintenance and propagation of human embryonic stem cell (hESC) cultures. Enzymatic passaging currently represents the most widely used method for expansion of hESCs. Although rapid and straightforward, this technique results in variable‐sized cell clusters and significant cellular trauma, which may apply selective pressure in long‐term culture. Mechanical passaging has the potential advantages of defined colony fragment sizes, reduced cellular trauma, and the possibility of selecting undifferentiated colonies for transfer. However, manual dissection of individual colonies is a prohibitively time‐consuming process unsuitable for maintaining large numbers of hESCs without the use of additional chemical means. In this study we report an efficient automated method for mechanically passaging hESCs. We have used this method exclusively to maintain hESCs in long‐term undifferentiated culture without the use of enzymatic digestion for longer than 100 days. This automated technique can thus be used routinely to culture hESCs in the laboratory.

Embryonic stem cell derived neural progenitors display temporal restriction to neural patterning

Neural stem cells have considerable therapeutic potential because of their ability to generate defined neuronal cell types for use in drug screening studies or cell‐based therapies for neurodegenerative diseases. In this study, we differentiate mouse embryonic stem cells to neural progenitors with an initial forebrain identity in a defined system that enables systematic manipulation to generate more caudal fates, including motoneurons. We demonstrate that the ability to pattern embryonic stem cell‐derived neural progenitors is temporally restricted and show that the loss of responsiveness to morphogenetic cues correlates with constitutive expression of the basic helix‐loop‐helix transcription factors Olig2 and Mash1, epidermal growth factor receptor, and vimentin and parallels the onset of gliogenesis. We provide evidence for two temporal classes of embryonic stem cell‐derived putative radial glia that coincide with a transition from neurogenesis to gliogenesis and a concomitant loss of regional identity.

Myelin repair: the role of stem and precursor cells in multiple sclerosis

Multiple sclerosis is the most common potential cause of neurological disability in young adults. The disease has two distinct clinical phases, each reflecting a dominant role for separate pathological processes: inflammation drives activity during the relapsing–remitting stage and axon degeneration represents the principal substrate of progressive disability. Recent advances in disease-modifying treatments target only the inflammatory process. They are ineffective in the progressive stage, leaving the science of disease progression unsolved. Here, the requirement is for strategies that promote remyelination and prevent axonal loss. Pathological and experimental studies suggest that these processes are tightly linked, and that remyelination or myelin repair will both restore structure and protect axons. This review considers the basic and clinical biology of remyelination and the potential contribution of stem and precursor cells to enhance and supplement spontaneous remyelination.

Postnatal astrocytes promote neural induction from adult human bone marrow-derived stem cells

Neural stem cells (NSCs) have generated considerable interest because of their potential as a source of defined cells for drug screening or cell-based therapies for neurodegenerative diseases. Ethical and practical considerations limit the availability of human fetal-derived neural tissue and highlight the need to consider alternative sources of human NSCs. Because of their ready availability, their ability to be easily expanded, and reports of neural potential, bone marrow-derived populations have become the focus of intense study with regard to their potential clinical utility. However, recent identification of spontaneous cell fusion and limited neuronal differentiation has tempered initial optimism. In this study, we demonstrate the monoclonal neural and mesodermal potential of adult human bone marrow mesenchymal cells. Critically, we show that sequential treatment with the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) followed by postnatal hippocampal astrocyte conditioned medium significantly promotes the generation of neurofilament(+)/beta-tubulin(+) cells from bone marrow precursors. The ability to generate almost limitless numbers of neural precursors from a readily accessible autologous adult human source provides a platform for further studies and potentially has important therapeutic implications.

The management and outcome for patients with chronic subdural hematoma: a prospective, multicenter, observational cohort study in the United Kingdom

OBJECTIVE Symptomatic chronic subdural hematoma (CSDH) will become an increasingly common presentation in neurosurgical practice as the population ages, but quality evidence is still lacking to guide the optimal management for these patients. The British Neurosurgical Trainee Research Collaborative (BNTRC) was established by neurosurgical trainees in 2012 to improve research by combining the efforts of trainees in each of the United Kingdom (UK) and Irelands neurosurgical units (NSUs). The authors present the first study by the BNTRC that describes current management and outcomes for patients with CSDH throughout the UK and Ireland. This provides a resource both for current clinical practice and future clinical research on CSDH. METHODS Data on management and outcomes for patients with CSDH referred to UK and Ireland NSUs were collected prospectively over an 8-month period and audited against criteria predefined from the literature: NSU mortality < 5%, NSU morbidity < 10%, symptomatic recurrence within 60 days requiring repeat surgery < 20%, and unfavorable functional status (modified Rankin Scale score of 4-6) at NSU discharge < 30%. RESULTS Data from 1205 patients in 26 NSUs were collected. Bur-hole craniostomy was the most common procedure (89%), and symptomatic recurrence requiring repeat surgery within 60 days was observed in 9% of patients. Criteria on mortality (2%), rate of recurrence (9%), and unfavorable functional outcome (22%) were met, but morbidity was greater than expected (14%). Multivariate analysis demonstrated that failure to insert a drain intraoperatively independently predicted recurrence and unfavorable functional outcome (p = 0.011 and p = 0.048, respectively). Increasing patient age (p < 0.00001), postoperative bed rest (p = 0.019), and use of a single bur hole (p = 0.020) independently predicted unfavorable functional outcomes, but prescription of high-flow oxygen or preoperative use of antiplatelet medications did not. CONCLUSIONS This is the largest prospective CSDH study and helps establish national standards. It has confirmed in a real-world setting the effectiveness of placing a subdural drain. This study identified a number of modifiable prognostic factors but questions the necessity of some common aspects of CSDH management, such as enforced postoperative bed rest. Future studies should seek to establish how practitioners can optimize perioperative care of patients with CSDH to reduce morbidity as well as minimize CSDH recurrence. The BNTRC is unique worldwide, conducting multicenter trainee-led research and audits. This study demonstrates that collaborative research networks are powerful tools to interrogate clinical research questions.

Proposal for a prospective multi-centre audit of chronic subdural haematoma management in the United Kingdom and Ireland

Abstract Background. Chronic subdural haematoma (CSDH) is a common condition that increases in incidence with rising age. Evacuation of a CSDH is one of the commonest neurosurgical procedures; however the optimal peri-operative management, surgical technique, post-operative care and the role of adjuvant therapies remain controversial. Aim. We propose a prospective multi-centre audit in order to establish current practices, outcomes and national benchmarks for future studies. Methods. Neurosurgical units (NSU) in the United Kingdom and Ireland will be invited to enrol patients to this audit. All adult patients aged 16 years and over with a primary or recurrent CSDH will be eligible for inclusion. Outcome measures and analysis. The proposed outcome measures are (1) clinical recurrence requiring re-operation within 60 days; (2) modified Rankin scale (mRS) score at discharge from NSU; (3) morbidity and mortality in the NSU; (4) destination at discharge from NSU and (5) length of stay in the NSU. Audit standards have been derived from published systematic reviews and a recent randomised trial. The proposed standards are clinical recurrence rate < 20%; unfavourable mRS (4–6) at discharge from NSU < 30%; mortality rate in NSU < 5%; morbidity rate in NSU < 10%. Data will be submitted directly into a secure online database and analysed by the studys management group. Conclusions. The audit will determine the contemporary management and outcomes of patients with CSDH in the United Kingdom and Ireland. It will inform national guidelines, clinical practice and future studies in order to improve the outcome of patients with CSDH.

Proposal for establishment of the UK Cranial Reconstruction Registry (UKCRR).

Abstract Background. The increasing utilisation of decompressive craniectomy for traumatic brain injury and stroke has led to an increase in the number of cranioplasties undertaken. Cranioplasty is also undertaken following excision of tumours originating from or invading the skull vault, removal of bone flaps due to post-operative infection, and decompressive craniectomy for the management of rarer causes of brain oedema and/or refractory intracranial hypertension. The existing literature which mainly consists of single-centre, retrospective studies, shows a significant variation in practice patterns and a wide range of morbidity. There also exists a need to measure the outcome as perceived by the patients themselves with patient reported outcome measures (PROMs; functional outcome, quality of life, satisfaction with cosmesis). In the UK, the concept of long-term surveillance of neurosurgical implants is well established with the UK shunt registry. Based on this background, we propose to establish the UK Cranial Reconstruction Registry (UKCRR). Aim. The overarching aim of the UKCRR is to collect high-quality data about cranioplasties undertaken across the UK and Ireland in order to improve outcomes for patients. Methods. Any patient undergoing reconstruction of the skull vault with autologous bone, titanium, or synthetic material in participating units will be eligible for inclusion. Data will be submitted directly by participating units to the Outcome Registry Intervention and Operation Network secure platform. A Steering Committee will be responsible for overseeing the strategic direction and running of the UKCRR. Outcome measures. These will include re-operation due to a cranioplasty-related issue, surgical site infection, re-admission due to a cranioplasty-related issue, unplanned post-operative escalation of care, adverse events, length of stay in admitting unit, destination at discharge from admitting unit, mortality at discharge from admitting unit, neurological status and PROMs during routine follow-up. Conclusion. The UKCRR will be an important pillar in the ongoing efforts to optimise the outcomes of patients undergoing cranioplasty.