Multimodal imaging of hair follicle bulge-derived stem cells in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) is a devastating event for which current therapies are limited. Stem cell transplantation may lead to recovery of function via different mechanisms, such as cell replacement through differentiation, stimulation of angiogenesis and support to the microenvironment. Adult hair follicle bulge-derived stem cells (HFBSCs) possess neuronal differentiation capacity, are easy to harvest and are relatively immune-privileged, which makes them potential candidates for autologous stem cell-based therapy. In this study, we apply in vivo multimodal, optical and magnetic resonance imaging techniques to investigate the behavior of mouse HFBSCs in a mouse model of TBI. HFBSCs expressed Luc2 and copGFP and were examined for their differentiation capacity in vitro. Subsequently, transduced HFBSCs, preloaded with ferumoxytol, were transplanted next to the TBI lesion (cortical region) in nude mice, 2 days after injury. Brains were fixed for immunohistochemistry 58 days after transplantation. Luc2- and copGFP-expressing, ferumoxytol-loaded HFBSCs showed adequate neuronal differentiation potential in vitro. Bioluminescence of the lesioned brain revealed survival of HFBSCs and magnetic resonance imaging identified their localization in the area of transplantation. Immunohistochemistry showed that transplanted cells stained for nestin and neurofilament protein (NF-Pan). Cells also expressed laminin and fibronectin but extracellular matrix masses were not detected. After 58 days, ferumoxytol could be detected in HFBSCs in brain tissue sections. These results show that HFBSCs are able to survive after brain transplantation and suggest that cells may undergo differentiation towards a neuronal cell lineage, which supports their potential use for cell-based therapy for TBI.

The association of fatigue, comorbidity burden, disease activity, disability and gross domestic product in patients with rheumatoid arthritis. Results from 34 countries participating in the Quest-RA program.

OBJECTIVES: The aim is to assess the prevalence of comorbidities and to further analyse to which degree fatigue can be explained by comorbidity burden, disease activity, disability and gross domestic product (GDP) in patients with rheumatoid arthritis (RA). METHODS: Nine thousands eight hundred seventy-four patients from 34 countries, 16 with high GDP (>24.000 US dollars [USD] per capita) and 18 low-GDP countries (<24.000 USD) participated in the Quantitative Standard monitoring of Patients with RA (QUEST-RA) study. The prevalence of 31 comorbid conditions, fatigue (0-10 cm visual analogue scale [VAS] [10=worst]), disease activity in 28 joints (DAS28), and physical disability (Health Assessment Questionnaire score [HAQ]) were assessed. Univariate and multivariate linear regression analyses were performed to assess the association between fatigue and comorbidities, disease activity, disability and GDP. RESULTS: Overall, patients reported a median of 2 comorbid conditions of which hypertension (31.5%), osteoporosis (17.6%), osteoarthritis (15.5%) and hyperlipidaemia (14.2%) were the most prevalent. The majority of comorbidities were more common in high-GDP countries. The median fatigue score was 4.4 (4.8 in low-GDP countries and 3.8 in high-GDP countries, p<0.001). In low-GDP countries 25.4% of the patients had a high level of fatigue (>6.6) compared with 23.0% in high-GDP countries (p<0.001). In univariate analysis, fatigue increased with increasing number of comorbidities, disease activity and disability in both high- and low-GDP countries. In multivariate analysis of all countries, these 3 variables explained 29.4% of the variability, whereas GDP was not significant. CONCLUSIONS: Fatigue is a widespread problem associated with high comorbidity burden, disease activity and disability regardless of GDP.

Class III ß-tubulin, a novel biomarker in the human melanocyte lineage.

It is generally thought that class III ß-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III ß-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III ß-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.

Response to glucocorticoids at 2 weeks predicts the effectiveness of DMARD induction therapy at 3 months: post hoc analyses from the tREACH study.

OBJECTIVE: To investigate if a glucocorticoid (GC) response at 2 weeks, defined by EULAR response criteria, can predict active disease (Disease Activity Score (DAS)>2.4) at 3 months. METHODS: For this study, data of the Treatment in the Rotterdam Early Arthritis Cohort study (tREACH), an ongoing clinical trial that evaluates different induction therapies in early rheumatoid arthritis, were used. We selected patients who had a high probability of progressing to persistent arthritis (>70% based on the prediction model of Visser). All patients within the high-probability stratum, who had a baseline DAS>2.2 and a DAS assessment at 2 weeks after randomisation, were included (n=120). Besides GC response at 2 weeks, we investigated which other factors were associated with active disease (DAS>2.4) after 3 months of disease-modifying antirheumatic drug (DMARD) treatment. All variables with a p≤0.25 were assessed in our logistic regression model with backward selection. Variables were eliminated until all remaining variables had a significant association (p<0.05). RESULTS: Patients who did not respond to GC bridging therapy at 2 weeks had an overall OR of having active disease at 3 months of 10.29 (95% CI 3.34 to 31.64; p<0.001) in comparison with responders. The corrected OR was 14.00 (95% CI 3.31 to 59.21; p<0.001). Our final model predicting response at 3 months included the following variables: gender, GC response, induction therapy arms and baseline DAS, which had an explained variance of 39%. CONCLUSIONS: GC response at 2 weeks is a useful tool for recognising those patients who will probably have active disease (DAS>2.4) after 3 months of DMARD treatment.

Back to the Future: From Appendage Development Toward Future Human Hair Follicle Neogenesis.

Hair disorders such as alopecia and hirsutism often impact the social and psychological well-being of an individual. This also holds true for patients with severe burns who have lost their hair follicles (HFs). HFs stimulate proper wound healing and prevent scar formation; thus, HF research can benefit numerous patients. Although hair development and hair disorders are intensively studied, human HF development has not been fully elucidated. Research on human fetal material is often subject to restrictions, and thus development, disease, and wound healing studies remain largely dependent on time-consuming and costly animal studies. Although animal experiments have yielded considerable and useful information, it is increasingly recognized that significant differences exist between animal and human skin and that it is important to obtain meaningful human models. Human disease specific models could therefore play a key role in future therapy. To this end, hair organoids or hair-bearing skin-on-chip created from the patient's own cells can be used. To create such a complex 3D structure, knowledge of hair genesis, i.e., the early developmental process, is indispensable. Thus, uncovering the mechanisms underlying how HF progenitor cells within human fetal skin form hair buds and subsequently HFs is of interest. Organoid studies have shown that nearly all organs can be recapitulated as mini-organs by mimicking embryonic conditions and utilizing the relevant morphogens and extracellular matrix (ECM) proteins. Therefore, knowledge of the cellular and ECM proteins in the skin of human fetuses is critical to understand the evolution of epithelial tissues, including skin appendages. This review aims to provide an overview of our current understanding of the cellular changes occurring during human skin and HF development. We further discuss the potential implementation of this knowledge in establishing a human in vitro model of a full skin substitute containing hair follicles and the subsequent translation to clinical use.

Building an Artificial Stem Cell Niche: Prerequisites for Future 3D-Formation of Inner Ear Structures-Toward 3D Inner Ear Biotechnology.

In recent years, there has been an increased interest in stem cells for the purpose of regenerative medicine to deliver a wide range of therapies to treat many diseases. However, two-dimensional cultures of stem cells are of limited use when studying the mechanism of pathogenesis of diseases and the feasibility of a treatment. Therefore, research is focusing on the strengths of stem cells in the three-dimensional (3D) structures mimicking organs, that is, organoids, or organ-on-chip, for modeling human biology and disease. As 3D technology advances, it is necessary to know which signals stem cells need to multiply and differentiate into complex structures. This holds especially true for the complex 3D structure of the inner ear. Recent work suggests that although other factors play a role, the extracellular matrix (ECM), including its topography, is crucial to mimic a stem cell niche in vitro and to drive stem cells toward the formation of the tissue of interest. Technological developments have led to the investigation of biomaterials that closely resemble the native ECM. In the fast forward moving research of organoids and organs-on-chip, the inner ear has hardly received attention. This review aims to provide an overview, by describing the general context in which cells, matrix and morphogens cooperate in order to build a tissue, to facilitate research in 3D inner ear technology. Anat Rec, 303:408-426, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea.

Stem-cell-based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe-to-profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non-invasive in vivo visualization of cells and long-term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co-expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase-expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427-440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Use of risk stratification to target therapies in patients with recent onset arthritis; design of a prospective randomized multicenter controlled trial.

BACKGROUND: Early and intensive treatment is important to inducing remission and preventing joint damage in patients with rheumatoid arthritis. While intensive combination therapy (Disease Modifying Anti-rheumatic Drugs and/or biologicals) is the most effective, rheumatologists in daily clinical practice prefer to start with monotherapy methotrexate and bridging corticosteroids. Intensive treatment should be started as soon as the first symptoms manifest, but at this early stage, ACR criteria may not be fulfilled, and there is a danger of over-treatment. We will therefore determine which induction therapy is most effective in the very early stage of persistent arthritis. To overcome over-treatment and under-treatment, the intensity of induction therapy will be based on a prediction model that predicts patients' propensity for persistent arthritis. METHODS: A multicenter stratified randomized single-blind controlled trial is currently being performed in patients 18 years or older with recent-onset arthritis. Eight hundred ten patients are being stratified according to the likelihood of their developing persistent arthritis. In patients with a high probability of persistent arthritis, we will study combination Disease Modifying Antirheumatic Drug therapy compared to monotherapy methotrexate. In patients with an intermediate probability of persistent arthritis, we will study Disease Modifying Antirheumatic Drug of various intensities. In patients with a low probability, we will study non-steroidal anti-inflammatory drugs, hydroxychloroquine and a single dose of corticosteroids. If disease activity is not sufficiently reduced, treatment will be adjusted according to a step-up protocol. If remission is achieved for at least six months, medication will be tapered off. Patients will be followed up every three months over two years. DISCUSSION: This is the first rheumatological study to base treatment in early arthritis on a prediction rule. Treatment will be stratified according to the probability of persistent arthritis, and different combinations of treatment per stratum will be evaluated. Treatment will be started early, and patients will not need to meet the ACR-criteria for rheumatoid arthritis. TRIAL REGISTRATION: This trial has been registered in Current Controlled Trials with the ISRCTN26791028.

Uncomplicated differentiation of stem cells into bipolar neurons and myelinating glia.

Epidermal neural crest stem cells (EPI-NCSCs), derived from the bulge of hair follicles, appear to be promising donor stem cell candidates. In the current work, EPI-NCSCs were harvested from rodents and humans. Isolation procedures revealed high levels of nestin-positive neural stem cells and the percentage of human neural stem cells (95+/-0.6%) is even higher than the percentage found in cultures of hair follicles from rodents (90+/-0.9%). Furthermore, differentiation of EPI-NCSCs into bipolar neurons, myelinating Schwann cells and oligodendrocytes occurred by applying a simple and straightforward method. Bipolar neurons could be obtained by culturing on a collagen matrix and are of great interest for auditory neuron regeneration since auditory neurons are bipolar. We propose that this type of stem cells, would make an excellent model for autologous transplantation and offers great potential for neural regeneration in diseases, such as Parkinson's and Alzheimer's disease.

Transforming growth factor beta and wound healing in human cholesteatoma.

OBJECTIVE: Cholesteatoma is a nonmalignant, destructive lesion of the temporal bone that gradually expands and causes complications by the erosion of the adjacent bony structures. The consequences can be as severe as facial paralysis and intracranial complications. Until now, surgery has been the only treatment of choice. The pathogenesis of cholesteatoma remains controversial. Current concepts postulate that cholesteatoma may be a wound-healing process, although formal proof is lacking as of yet. Several reports provide evidence for the involvement of transforming growth factor (TGF)beta in both normal and abnormal wound healing. STUDY DESIGN: The expression of TGFbeta, the activated form of its intracellular effector, phosphorylated-Sma-Mad (pSmad)2, its natural inhibitor, Smad7, and target gene extra domain A-positive fibronectin (EDA-FN) were examined. METHODS: Quantitative immunohistochemical analysis was performed using an image analysis system. RESULTS: In 12 cholesteatoma and control samples, protein expressions showed consistent relationships among TGFbeta, nuclear pSmad2, and Smad7. We found concordant expressions of TGFbeta and nuclear pSmad2 in cholesteatoma epithelium and its control. Epithelial Smad7 expression was significantly reduced in cholesteatoma when compared with control epithelium (P = .04). In cholesteatoma extracellular matrix (ECM), a significantly increased TGFbeta, and nuclear pSmad2 was demonstrated (P < .01). Smad7 expression in the ECM was comparable in cholesteatoma and its control. EDA-FN deposition in cholesteatoma ECM was excessive, whereas EDA-FN expression was absent in controls. CONCLUSION: Our results confirm reports of in vitro experiments and support the concept that cholesteatoma behaves as a chronic wound healing process.

Efficacy of Hydroxychloroquine in Hand Osteoarthritis: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To determine the symptom-modifying effect of hydroxychloroquine (HCQ) in hand osteoarthritis (OA). METHODS: In this randomized, double-blind, multicenter trial, patients with symptomatic hand OA received either HCQ 400 mg once a day or placebo during 24 weeks. The primary outcome was change of pain measured on a 100-mm visual analog scale (VAS) at 24 weeks. Secondary outcomes included decrease of pain at weeks 6 and 12 and change in Australian Canadian Hand Osteoarthritis Index (AUSCAN) and Arthritis Impact Measurement Scale 2 short form (AIMS2-SF) total scores. RESULTS: A total of 196 patients was included (placebo n = 98, HCQ n = 98). Mean ± SD age was 58.0 ± 7.6 years, and 86% were female. Baseline mean ± SD pain VAS was 44.9 ± 22.9 mm in the placebo group and 43.2 ± 22.3 mm in the HCQ group. At 24 weeks, change in pain VAS was not significantly different between both groups (imputed mean VAS 42.7 in the HCQ group versus 45.3 in the placebo group after 24 weeks), as was the case in pain VAS at weeks 6 and 12. Changes in AUSCAN total score and AIMS2-SF total score in both groups were similar between the groups. In total, 24 patients in the placebo group and 21 patients in the HCQ group reported ≥1 adverse event. In the HCQ group, 3 patients reported a severe allergic reaction. Fifteen patients withdrew from the study (5 placebo, 10 HCQ group) due to adverse events. CONCLUSION: Treatment with HCQ at 24 weeks is not effective in reducing the symptoms of hand OA compared to placebo.

Ouabain Does Not Induce Selective Spiral Ganglion Cell Degeneration in Guinea Pigs.

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with 1 mM or 10 mM ouabain showed shifts in hearing thresholds, corresponding with varying histological changes in their cochleas. Most cochleas exhibited complete outer hair cell loss in the basal and middle turns, while some had no changes, together with either moderate or near-complete loss of SGCs. Neither loss of inner hair cells nor histological changes of the stria vascularis were observed in any of the animals. Cochleas in Category 1 had normal aABRs and morphology. On average, in Category 2 OHC loss was 46.0±5.7%, SGC loss was below threshold, ABR threshold shift was 44.9±2.7 dB, and ABR wave II amplitude was decreased by 17.1±3.8 dB. In Category 3 OHC loss was 68.3±6.9%, SGC loss was 49.4±4.3%, ABR threshold shift was 39.0±2.4 dB, and ABR amplitude was decreased by 15.8±1.6 dB. Our results show that ouabain does not solely destroy type I SGCs in the guinea pig cochlea.

Survival signaling and terminal differentiation in cholesteatoma epithelium.

CONCLUSION: There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. OBJECTIVES: Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. MATERIALS AND METHODS: Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. RESULTS: Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p < or = 0.05).

MPO-ANCA associated vasculitis with mononeuritis multiplex following influenza vaccination.

BACKGROUND: Although influenza vaccines are generally safe and effective, a variety of autoimmune phenomena have been reported after vaccination over the past years, such as Guillain-Barre syndrome, rheumatoid arthritis, pemphigus vulgaris, psoriasis, giant cell arteritis and anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV). CASE REPORT: We describe the case of a 67-year old man who presented with a myeloperoxidase-ANCA associated vasculitis with renal involvement and mononeuritis multiplex after seasonal influenza vaccination. He was initially treated with intravenous cyclophosphamide and high-dose prednisolone followed by maintenance treatment consisting of azathioprine and prednisolone. CONCLUSION: We hypothesize that seasonal influenza vaccination triggered a systemic immune response in a susceptible patient to develop AAV with renal involvement and vasculitic neuropathy. In general, seasonal influenza vaccinations are considered to be safe, however, clinicians should be aware of this rare phenomenon.

Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants.

Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 µm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair.

Terminal differentiation and mitogen-activated protein kinase signaling in human cholesteatoma epithelium.

OBJECTIVES: To investigate whether--in cholesteatoma epithelium--terminal differentiation, resulting in high involucrin expression, is associated with mitogen-activated protein kinase (MAPK) signaling. BACKGROUND: Alterations in specific signal transduction pathways may explain abnormal differentiation of the keratinocytes in cholesteatoma. Signaling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses converge on activated mitogen-activated protein kinases, mainly extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38. METHODS: Tissue samples were taken from 16 patients with acquired cholesteatoma. Histologic examination showed that 12 of the 16 cholesteatomas were inflamed. Immunohistochemical methods were used to determine expressions of involucrin and the activated form of p38, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase proteins. The results obtained from cholesteatoma tissue were compared with paired control samples from retroauricular skin. RESULTS: We demonstrated increased levels of involucrin and increased levels of the activated forms of p38 and ERK1/2 in cholesteatoma epithelium when compared with control samples. No abnormality was found in the activation and expression of JNK1/2. A positive correlation was found between p38, pERK1/2, and involucrin expression (p<0.05). CONCLUSION: Our results demonstrate that signaling via the mitogen-activated protein kinases ERK1/2 and p38 is increased in cholesteatoma epithelium when compared with control skin. The correlations between involucrin-and phosphorylated pERK1/2 expression and between involucrin-and phosphorylated p38 expression indicates that terminal differentiation in cholesteatoma epithelium proceeds via activation of these mitogen-activated protein kinase signaling pathways. We discussed whether this increased mitogen-activated protein kinase-driven terminal differentiation is probably part of a keratinocyte survival program or caused by an inflammation-induced cellular stress response.

Lentiviral transduction and subsequent loading with nanoparticles do not affect cell viability and proliferation in hair-follicle-bulge-derived stem cells in vitro.

The application of stem cells in the treatment of various degenerative diseases is highly promising. However, cell-based therapy could be limited by the problem of low viability of grafted cells and uncertainty about their fate. The combination of molecular imaging and contrast-enhanced MRI may give more insight into the survival and behavior of grafted stem cells. We explore hair-follicle-bulge-derived stem cells (HFBSCs) as a potential candidate for autologous cell-based therapy. HFBSCs are transduced with a lentiviral construct with genes coding for bioluminescent (Luc2) and fluorescent (copGFP) reporter proteins, and subsequently loaded with magnetic nanoparticles to enable MRI visualization. Thus, we investigate for the first time if lentiviral transduction and cellular loading with nanoparticles have a cytotoxic effect upon these stem cells. Transduction efficiency, proliferation rate, cell viability and reporter protein co-expression during long-term culture of transduced HFBSCs were studied using fluorescence and bioluminescence microscopy. In addition, the effect of TMSR50 nanoparticles on proliferation and viability was investigated using the MTS assay and bioluminescence microscopy. The amount of TMSR50-loaded HFBSCs needed to reach signal threshold for MRI was assessed using an agarose phantom. Transduction with the Luc2-copGFP construct did not influence senescence, proliferation, doubling time, and differentiation of the HFBSCs. CopGFP expression was visible immediately after transduction and persisted for at least 15 passages, concomitantly with Luc2 expression. Cellular loading with TMSR50 nanoparticles did not affect cell viability and proliferation. The results imply that combined MRI and bioluminescence imaging may enable in vivo localization and long-term monitoring of grafted viable HFBSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Isolation, expansion and neural differentiation of stem cells from human plucked hair: a further step towards autologous nerve recovery.

Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2α and SOX9, and negative for SOX10. Each bulge yielded approximately 1 × 10(4) cells after three passages. Doubling time was 3.3 (±1.5) days. Cellular viability did not differ significantly from control cells after shear stress. The cells expressed class III ß-tubulin (TUBB3) and synapsin-1 after 3 weeks of neuronal differentiation. Glial differentiation yielded KROX20- and MPZ-immunopositive cells after 2 weeks. We demonstrated that human hair follicle bulge-derived stem cells can be cultivated easily, expanded efficiently and kept frozen until needed. After cryopreservation, the cells were viable and displayed both neuronal and glial differentiation potential.

Sustained extracellular signal-regulated kinase1/2 mitogen-activated protein kinase signalling is related to increased p21 expression in cholesteatoma epithelium.

CONCLUSION: These results show for the first time that the RAS/RAF/ERK1/2 MAPK signalling pathway is active and involved in p21-mediated cell cycle arrest in human cholesteatoma epithelium. OBJECTIVE: In a previous report we have demonstrated that the epithelium in human cholesteatoma is characterized by high p53-dependent p21 expression. The RAS/RAF/extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signalling pathway can induce p21 expression and subsequent cell cycle arrest via p53-dependent or -independent mechanisms. We designed the present study to investigate whether the RAS/RAF/ERK1/2 MAPK signalling pathway is involved in p53-dependent and p21-mediated cell cycle arrest in human cholesteatoma. MATERIAL AND METHODS: A total of 18 cholesteatoma samples and 18 paired control retro-auricular skin samples were immunohistochemically stained for p53, p21, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2. Positive cells were counted by means of digital image analysis. Double-label fluorescence immunohistochemistry was performed to demonstrate co-expression of p21 and pERK1/2. RESULTS: Protein expression of p53, p21 and pERK1/2 differed significantly between cholesteatoma epithelium and retro-auricular skin (p <0.01). In cholesteatoma, co-expression of p21 and pERK1/2 was prominent, whereas in retro-auricular skin there was hardly any co-expression. Positive correlations were found between p53 and p21 (p =0.003) and between p21 and pERK1/2 (p =0.013).

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss.

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9-W18) and show the cochlear expression dynamics of key potassium-regulating proteins. At W12, MITF+/SOX10+/KIT+ neural-crest-derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+-ATPase-positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy.