Differentiation of human epidermal neural crest stem cells (hEPI-NCSC) into virtually homogenous populations of dopaminergic neurons.

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFß receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC.

Emergency caesarean section: influences on the decision-to-delivery interval.

RCOG/NICE guidelines recommend that, for fetal compromise in labour, delivery should be accomplished ideally within 30 minutes. In this study, we investigated the factors which affect the decision-to-delivery (DD) intervals for emergency caesareans. To achieve this, prospective data were collected for all grade 1 and 2 caesareans performed on a busy labour ward over 12 months. We found that the ratio of labouring women to midwives had a significant effect on the DD intervals, which were significantly prolonged when 1 : 1 care was not provided (P < 0.001). The observed effect resulted exclusively from a prolonged transfer time to theatre. General anesthesia use shortened the DD interval for grade 1 caesareans (P < 0.001) and was more likely to be used during the day shift (P < 0.009). We conclude that midwifery staffing levels and the form of anaesthesia employed influence on DD intervals for the most urgent caesarean sections.

Human epidermal neural crest stem cells (hEPI-NCSC)--characterization and directed differentiation into osteocytes and melanocytes.

Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine.

Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells.

Recently we reported that the expression of the protein kinase A (PKA) regulatory subunit RIIα is dynamically regulated in human smooth muscle cells of the uterus. We showed that expression levels of mRNA/protein were substantially increased during pregnancy and decreased upon labour, changes that were mirrored by particulate type II PKA activity. This implied an important role for RIIα in maintaining uterine quiescence during pregnancy. Consequently the purpose of the present study was to identify potential mechanisms by which expression of the RIIα gene was regulated in this tissue. We indicate here that the three SpI-III (GC) binding domains within the proximal promoter region of the human RIIα gene may play important roles in modulating expression of the gene in human myometrial cells. We show that all three GC binding domains are involved in binding Sp1, Sp3, histone deacetylase (HDACs) 1/2 and RbAp48 transcriptional complexes. The functional significance of these binding domains was further analysed employing in vitro luciferase reporter assays with full-length/truncated RIIα promoter constructs. Importantly we show that treatment of primary human myometrial cell cultures with the general class I/II HDAC inhibitor trichostatin A results in an increase in mRNA/protein levels. Moreover the increase in mRNA levels appeared to be preceded by an increase in aH3, PolIIa, Sp3 and HDAC 2 binding to the three SpI-III (GC) binding sites within the RIIα promoter. These results enable us to provide a model whereby RIIα expression is epigenetically regulated in human myometrial smooth muscle cells by histone deacetylase(s) activity within the GC-rich proximal promoter region of the gene.

The management of pregnancy in paroxysmal nocturnal haemoglobinuria on long term eculizumab.

In Paroxysmal nocturnal haemoglobinuria (PNH), pregnancy is associated with increased maternal and foetal complications to such an extent that the condition has been considered relatively contra-indicated in PNH. Eculizumab has revolutionized the treatment of PNH. We evaluate its use in pregnancy to date. We report on seven patients exposed to eculizumab at different stages of pregnancy including the first two patients to receive the drug from conception to delivery. There was no evidence of complement blockade from cord blood samples taken at delivery. Eculizumab appears safe to use in this setting and is likely to prevent many of the complications usually observed.

Recombinant Activated Factor VII (rFVIIa) in the Management of Major Obstetric Haemorrhage: A Case Series and a Proposed Guideline for Use.

Major obstetric haemorrhage remains a significant cause of maternal morbidity and mortality. Previous case reports suggest the potential benefit of recombinant activated factor VII (rFVIIa: NovoSeven(R)) as a haemostatic agent. We performed a retrospective review of the use of rVIIa in major obstetric haemorrhage in the Northern Region between July 2004 and February 2007. Fifteen women received rFVIIa. The median patient age was 34 years. Major haemorrhage occurred antepartum (5 patients), intrapartum (1), and postpartum (9). All women received an initial dose of 90 mcg/kg rFVIIa and one received 2 further doses. Bleeding stopped or decreased in 12 patients (80%). Additional measures included antifibrinolytic and uterotonic agents, Rusch balloon insertion, uterine curettage/packing, and vessel embolisation. Eight patients required hysterectomy. All women survived to discharge from hospital. No adverse events, including thrombosis, were recorded. This study provides further support for the safety and efficacy of rFVIIa as adjunct therapy in major obstetric haemorrhage.

Characterization of cellular retinoid-binding proteins in human myometrium during pregnancy.

Many complementary or competing signalling pathways bear an influence on the myometrium at any one time, and because the retinoic acid signalling pathway influences differentiation of a wide array of human tissues, this may be one of the determinants of myometrial differentiation during pregnancy. We have explored the novel hypothesis that the retinoids may act as important regulators in controlling the differentiated state of the human myometrium during pregnancy by characterizing the expression profiles for cellular retinoid-binding proteins CRBPI, CRABPI and CRABPII in non-pregnant, pregnant (non-labouring) and labouring human myometrium taken from the functionally distinct upper and lower uterine segments. In addition, we have investigated the effect of all-trans retinoic acid (ATRA) on the expression of several retinoic acid response genes including cyclooxygenase-2 (COX-2) and connexin-43 (Cx-43). Different spatial and temporal patterns of expression were observed for CRBPI, CRABPI and CRABPII within the upper and lower uterine segments through the three trimesters of pregnancy and in labour. Furthermore, the expression of COX-2, Cx-43, CRABPI, the transcription factor c-Jun and the retinoic acid receptor RARbeta altered in response to different concentrations of ATRA, suggesting that the differential expression of cellular retinoid-binding proteins may lead to different levels of retinoic acid being delivered to its nuclear targets, leading to the differential expression of specific target genes within the myometrium during pregnancy.