Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis

Background and Aims: Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. Results: Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4% ± 5.5% of cells. Conclusions: We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma

Therapeutic potential of somatic cell nuclear transfer for degenerative disease caused by mitochondrial DNA mutations

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations

Comparison of development to the blastocyst stage between the enclosed workstations and conventional open-fronted cabinets.

<p>(<b>A</b>) Images show examples of early, expanded and hatched human blastocysts indicating the trophectoderm and the ICM. (<b>B</b>) Development of embryos to the blastocyst stage was compared between the open and enclosed systems. A significantly higher proportion of embryos developed to the blastocyst stage in the enclosed system (P<0.05). (<b>C</b>) Three point moving average of development to the blastocyst stage in consecutive cohorts of twenty embryos. The increase in the proportion of embryos developing to the blastocyst stage coincides with the switch from the open to the enclosed workstations. The two dark bars represent the intermediate period when the cohorts of 20 consisted of embryos cultured in the open and enclosed systems. (<b>D</b>) The proportions of blastocysts graded as early (black), expanded (grey) and hatched (white) on day 6. A significantly higher proportion of blastocysts had undergone hatching by day 6 after culture in the enclosed system compared to the open system. There was a corresponding reduction in the proportion of early blastocysts in the enclosed system (P<0.05). (<b>E</b>) Nuclear counts were performed to compare the total number of cells contained in blastocysts cultured to day 6 or 7 in the open system or enclosed system. The cell count of embryos cultured in the enclosed system was significantly higher on day 6 (128.3±38.6 <i>vs</i> 92.1±43.7; P<0.05) and day 7 (167.7±69.7 vs 106.7±48.8; P<0.01) compared with those cultured in the open system. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031010#pone.0031010.s001" target="_blank">Figure S1A</a>–C for an example of nuclear staining in a blastocyst and comparison of nuclear counts between different operators and imaging systems.</p

Comparison of patient profiles.

<p>Comparison of patient profiles between the open and enclosed systems for women ≤37 years with >10 follicles undergoing their first cycle of treatment. Groups 1 and 2 were processed in the open system but in different laboratories. Group 3 was processed in the enclosed system. There was no statistical difference between groups for any of the parameters measured.</p

Layout of the enclosed workstations.

<p>(<b>A</b>) Schematic illustrating the layout of the enclosed workstations showing the workflow from the entry of oocytes into the system until the embryos are replaced in the uterus. The incubators act as pass-through hatches linking adjacent workstations. (<b>B</b>) Schematic diagram showing the layout of 2 enclosed workstations connected by the double-doored incubation chambers. (<b>C</b>) Image showing the main features of an enclosed workstation with a built-in stereomicroscope and incubators. There are separate hatches to allow introduction of consumables and removal of waste. (<b>D</b>) Image shows the control panel for setting the temperature of the incubator, workstation and hotplate and the %CO₂ in the incubator and workstation. An alarm sounds when the environmental parameters deviate from the set-points.</p

Effect of the enclosed system on IVF and ICSI clinical outcome measures.

<p>Comparison of the effect of the enclosed system on clinical outcome for consecutive groups of women aged ≤37 years with >10 ovarian follicles undergoing their first cycle of either IVF or ICSI treatment. Embryos from Groups 1 and 2 patients were cultured in the conventional open-fronted system but in different laboratories. Group 3 were conducted in the enclosed system. Letters indicate statistically significant difference. (<b>A</b>) The proportion of transferred embryos resulting in successful implantation, as determined by the presence of a fetal heartbeat at 7 weeks, was significantly higher in Group 3 (31.8%) compared with Group 1 (19.8%) or Group 2 (21.6%); P<0.05. (<b>B</b>) The clinical pregnancy rate per oocyte retrieval was 45.3% for Group 3 compared with 32.2% for Group 1 and 35.6 for Group 2; P<0.05. (<b>C</b>) Comparison of implantation rates in IVF and ICSI treatments in the open and enclosed system.</p

Comparison of temperature and pH stability between the enclosed workstations and conventional open-fronted cabinets.

<p>(<b>A</b>) A pre-equilibrated organ culture dish was removed from the incubator and placed on the viewing area of either the enclosed workstation (purple) or Class II hood (blue). Time point zero indicates the first measurement taken immediately after placing the dish on the viewing area. The temperature was then measured at 3-minute intervals. The temperature in the Class II cabinet dropped from 36°C±0.1 to 35.2°C±0.3 and then stabilised at 34.9–35.0°C±0.2. In the enclosed workstation, the starting temperature dropped from 37.0–36.7°C±0.1 and stabilised at 36.5–36.6°C±0.1. The lower starting temperature in the Class II cabinet is most likely due to heat loss during transfer from the incubator. In each case 3 independent experiments were conducted. (<b>B</b>) A pre-equilibrated organ culture dish was removed from the incubator and placed on the viewing area of either the enclosed workstation (purple) or Class II cabinet (blue). Time point zero indicates the first measurement taken immediately after placing the dish on the viewing area. There was a gradual increase in pH of the media in the Class II cabinet over the time course of the experiment. By contrast the pH of the media in the enclosed workstation remained in the range 7.30–7.32. In each case 3 independent experiments were conducted. (<b>C</b>) After completing the measurements for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031010#pone-0031010-g002" target="_blank">Figure 2B</a>, the dish was returned to the incubator and pH measurements taken at 5 minute intervals for 90 minutes. The pH failed to return to 7.31 after 90 minutes in the incubator.</p