Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

Thoracoscopic operations in children.

Over the past two decades there has been an exponential growth in the use of thoracoscopy in children. Indeed, many advanced procedures-including lobectomy, repair of tracheoesophageal fistula, excision of mediastinal tumours, and diaphragmatic hernia repairs-can now be performed by this means in advanced paediatric surgical centres in the world. This review describes the historical perspectives and the current state of thoracoscopic surgery, including potential benefits and challenges, in children.

Internet Health Resources on Nocturnal Enuresis: A Readability, Quality, and Accuracy Analysis.

INTRODUCTION: Nocturnal enuresis is a common yet quality-of-life-limiting pediatric condition. There is an increasing trend for parents to obtain information on the disease's nature and treatment options via the internet. However, the quality of health-related information on the internet varies greatly and is largely uncontrolled and unregulated. With this study, a readability, quality, and accuracy evaluation of the health information regarding nocturnal enuresis is carried out. MATERIALS AND METHODS: A questionnaire was administered to parents and patients with nocturnal enuresis to determine their use of the internet to research their condition. The most common search terms were determined, and the first 30 websites returned by the most popular search engines were used to assess the quality of information about nocturnal enuresis. Each site was categorized by type and assessed for readability using the Gunning fog score, Simple Measure of Gobbledygook (SMOG) index, and Dale-Chall score; for quality using the DISCERN score; and for accuracy by comparison to the International Children's Continence Society guidelines by three experienced pediatric urologists and nephrologists. RESULTS: A total of 30 websites were assessed and classified into five categories: professional (n = 13), nonprofit (n = 8), commercial (n = 4), government (n = 3), and other (n = 2). The information was considered difficult for the public to comprehend, with mean Gunning fog, SMOG index, and Dale-Chall scores of 12.1 ± 4.3, 14.1 ± 4.3, and 8.1 ± 1.3, respectively. The mean summed DISCERN score was 41 ± 11.6 out of 75. Only seven (23%) websites were considered of good quality (DISCERN score > 50). The mean accuracy score of the websites was 3.2 ± 0.6 out of 5. Commercial websites were of the poorest quality and accuracy. Websites generally scored well in providing their aims and identifying treatment benefits and options, while they lacked references and information regarding treatment risks and mechanisms. CONCLUSION: Online information about nocturnal enuresis exists for parents; however, most websites are of suboptimal quality, readability, and accuracy. Pediatric surgeons should be aware of parents' health-information-seeking behavior and be proactive in guiding parents to identify high-quality resources.

Glycogen synthase kinase 3ß and activin/nodal inhibition in human embryonic stem cells induces a pre-neuroepithelial state that is required for specification to a floor plate cell lineage.

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3ß and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6- expression, we describe as "pre-neuroepithelial." Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA2+ cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage.

In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.

Pluripotent stem cells are a potential source of autologous cells for cell and tissue regenerative therapies. They have the ability to renew indefinitely while retaining the capacity to differentiate into all cell types in the body. With developments in cell therapy and tissue engineering these cells may provide an option for treating tissue loss in organs which do not repair themselves. Limitations to clinical translation of pluripotent stem cells include poor cell survival and low cell engraftment in vivo and the risk of teratoma formation when the cells do survive through implantation. In this study, implantation of human induced-pluripotent stem (hiPS) cells, suspended in Matrigel, into an in vivo vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. Differentiation of cells in the chamber environment was shown by teratoma formation, with all three germ lineages evident within 4 weeks. The rate of teratoma formation was higher with partially differentiated hiPS cells (as embryoid bodies) compared to undifferentiated hiPS cells (100% versus 60%). In conclusion, the in vivo vascularized tissue engineering chamber supports the survival through implantation of human iPS cells and their differentiated progeny, as well as a novel platform for rapid teratoma assay screening for pluripotency.

Gli1 is an inducing factor in generating floor plate progenitor cells from human embryonic stem cells.

Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer.

Small-molecule induction of neural crest-like cells derived from human neural progenitors.

Neural crest (NC) cells are stem cells that are specified within the embryonic neuroectodermal epithelium and migrate to stereotyped peripheral sites for differentiation into many cell types. Several neurocristopathies involve a deficit of NC-derived cells, raising the possibility of stem cell therapy. In Hirschsprung's disease the distal bowel lacks an enteric nervous system caused by a failure of colonization by NC-derived cells. We have developed a robust method of producing migrating NC-like cells from human embryonic stem cell-derived neural progenitors using a coculture system of mouse embryonic fibroblasts. Significantly, subsequent exposure to Y27632, a small-molecule inhibitor of the Rho effectors ROCKI/II, dramatically increased the efficiency of differentiation into NC-like cells, identified by marker expression in vitro. NC-like cells derived by this method were able to migrate along NC pathways in avian embryos in ovo and within explants of murine bowel, and to differentiate into cells with neuronal and glial markers. This is the first study to report the use of a small molecule to induce cells with NC characteristics from embryonic stem cells that can migrate and generate neurons and support cells in complex tissue. Furthermore, this study demonstrates that small-molecule regulators of ROCKI/II signaling may be valuable tools for stem cell research aimed at treatment of neurocristopathies.

Lysophosphatidic acid inhibits neuronal differentiation of neural stem/progenitor cells derived from human embryonic stem cells.

Lysophospholipids are signaling molecules that play broad and major roles within the nervous system during both early development and neural injury. We used neural differentiation of human embryonic stem cells (hESC) as an in vitro model to examine the specific effects of lysophosphatidic acid (LPA) at various stages of neural development, from neural induction to mature neurons and glia. We report that LPA inhibits neurosphere formation and the differentiation of neural stem cells (NSC) toward neurons, without modifying NSC proliferation, apoptosis, or astrocytic differentiation. LPA acts through the activation of the Rho/ROCK and the phosphatidylinositol 3-kinase/Akt pathways to inhibit neuronal differentiation. This study is the first demonstration of a role for LPA signaling in neuronal differentiation of hESC. As LPA concentrations increase during inflammation, the inhibition of neuronal differentiation by LPA might contribute to the low level of neurogenesis observed following neurotrauma.

Application of anchoring stitch prevents rectal prolapse in laparoscopic assisted anorectal pullthrough.

BACKGROUND: Rectal prolapse has been reported after laparoscopic assisted anorectal pullthrough in children with anorectal malformation. We report our clinical outcome and study the application of an anchoring stitch to tack the rectum to the presacral fascia and the occurrence of rectal prolapse. MATERIAL AND METHODS: A retrospective review of all children who had undergone laparoscopic assisted anorectal pullthrough for anorectal malformation from 2000 to 2015 was performed. Patients were divided into two groups (group I: with anchoring stitch, group II: without anchoring stitch). Outcome measures including rectal prolapse, soiling, voluntary bowel control, and constipation, and Kelly Score were analyzed. RESULTS: There were thirty-four patients (group I, n=20; group II, n=14) undergoing laparoscopic assisted anorectal pullthrough during the study period. The median follow up duration for group I and group II was 60months and 168months, respectively. All patients had stoma performed prior to the operation. Both groups consisted of patients with high type (30% vs 57%, p=0.12) and intermediate type (70% vs 43%, p=0.12) anorectal malformation. Seven (35%) patients in group I and 3 (21%) in group II had concomitant vertebral and spinal cord pathologies (p=0.408). The mean operative time was significantly shorter in group I (193±63min vs 242±49min, p=0.048). Rectal prolapse occurred less in group I, 4 (20%) vs 9 (64%) patients in group II and was statistically significant (p=0.008). Median time to development of rectal prolapse was 7months in group I and 5months in group II (p=0.767). Mucosectomy was performed in 15% of group I and 36% of group II (p=0.171). Soiling occurred less in group I (55% vs 79%, p=0.167). Voluntary bowel control (85% vs 93%, p=0.499) and constipation (55% vs 64%, p=0.601) were comparable in both groups. 75% in group I and 71% in group II achieved a Kelly score of 5 or above (p=0.823). CONCLUSIONS: Our study showed application of anchoring stitch reduces rectal prolapse and soiling in laparoscopic assisted anorectal pullthrough. Treatment Study-Level III.

Hypothermia protects human neurons.

BACKGROUND AND AIMS: Hypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury. METHODS: Human embryonic stem cells were cultured and differentiated to provide a source of neurons expressing ß III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H2 O2 -induced oxidative stress were used to induce relevant injury. RESULTS: Hypothermia to 33°C protected these human neurons against H2 O2 -induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% (P ≤ 0·0001; 95% confidence interval 34·8-71·04) and 42% (P ≤ 0·0001; 95% confidence interval 27·5-56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3-71·3 and 26%, P ≤ 0·001; 95% confidence interval 12·4-52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H2 O2 -induced oxidative stress even when hypothermia was initiated six-hours after onset of injury (P ≤ 0·05; 95% confidence interval 0·57-43·1). CONCLUSION: We conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H2 O2 -induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances.

Functional characterization of Friedreich ataxia iPS-derived neuronal progenitors and their integration in the adult brain.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.

An in vitro model of developmental synaptogenesis using cocultures of human neural progenitors and cochlear explants.

In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.

Late passage human fibroblasts induced to pluripotency are capable of directed neuronal differentiation.

It is possible to generate induced pluripotent stem (iPS) cells from mouse and human somatic cells by ectopic expression of defined sets of transcription factors. However, the recommendation that somatic cells should be utilized at early passages for induced reprogramming limits their therapeutic application. Here we report successful reprogramming of human fibroblasts after more than 20 passages in vitro, to a pluripotent state with four transcription factors: Oct4, Sox2, Klf4, and c-Myc. The late passage-derived human iPS cells resemble human embryonic stem cells in morphology, cell surface antigens, pluripotent gene expression profiles, and epigenetic states. Moreover, these iPS cells differentiate into cell types representative of the three germ layers in teratomas in vivo, and directed neuronal differentiation in vitro.

Generation of induced pluripotent stem cell lines from Friedreich ataxia patients.

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibroblasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results in the expression of frataxin protein in differentiated neurons. As such, iPS cell lines derived from FRDA patients, following correction of the mutated gene, could provide a useful source of immunocompatible cells for transplantation therapy.

Acute effect of endothelins on intercellular communication of human embryonic stem cells.

Endothelin (ET) family comprises three isoforms, ET-1, ET-2 and ET-3 that bind to two receptors ET-A and ET-B. Upon hESC differentiation, ET-1 and ET-B are respectively up- and down-regulated, suggesting a potential role of ETs in hESC biology. Here we show expression of ET receptors in hESC and demonstrate that ET-1 and ET-2 inhibit gap junctional intercellular communication (GJIC), while ET-3 does not. Pre-incubation of the cell cultures with the two specific antagonists of ET-A and ET-B, BQ123 and BQ788 respectively, demonstrate that inhibition of GJIC by ETs is mediated by ET-A. Long-term treatment of hESC with ET-1 indicates no visible effect on hESC maintenance of pluripotency markers, as assessed by expression of the hESC markers Oct-4, GCTM-2 and TG-30. Altogether these data show that hESC are target cells of ETs.

Cognitive behavioural therapy for clients with schizophrenia: implications for mental health nursing practice.

1. This paper discusses the application of cognitive behavioural therapy (CBT) in the treatment of clients with schizophrenia and the implications for mental health nursing practice. 2. The literature shows that CBT has positive effects for clients in improving mental state and reducing relapse rate. 3. Because of their direct client contact, mental health nurses are in the best position to assess, address and manage clients' psychotic symptoms and problems. 4. A nursing care plan is used to illustrate how the principles of CBT could be incorporated into nursing care. 5. Education and training are important to equip nurses with the necessary knowledge and skills to implement the therapy. 6. Nurses have to take into account cultural issues related to the use of CBT. 7. Culturally sensitive research needs to be conducted to assess the outcome of the therapy.