Neuromesodermal progenitors and the making of the spinal cord

Neuromesodermal progenitors (NMps) contribute to both the elongating spinal cord and the adjacent paraxial mesoderm. It has been assumed that these cells arise as a result of patterning of the anterior neural plate. However, as the molecular mechanisms that specify NMps in vivo are uncovered, and as protocols for generating these bipotent cells from mouse and human pluripotent stem cells in vitro are established, the emerging data suggest that this view needs to be revised. Here, we review the characteristics, regulation, in vitro derivation and in vivo induction of NMps. We propose that these cells arise within primitive streak-associated epiblast via a mechanism that is separable from that which establishes neural fate in the anterior epiblast. We thus argue for the existence of two distinct routes for making central nervous system progenitors

Neural Differentiation of Embryonic Stem Cells In Vitro: A Road Map to Neurogenesis in the Embryo

Background: The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. Methodology/Principal Findings: In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expresse

Communication Skills in Foreign Languages in Engineering

This article is devoted to innovative methods of teaching ESL. The factors of development of engineering activities are shown and the prerequisites for the component composition of a foreign language professional communicative competence are identified. The first positions in achieving the effectiveness of business communication are the verbal activities of the engineer

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells:application in serum free culture

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former.CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.</p

Distributed automated manufacturing of pluripotent stem cell products

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site–decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing ‘live’ corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs i. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (incl. scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Generation and characterization of a novel mouse embryonic stem cell line with a dynamic reporter of Nanog expression.

The pluripotent state in embryonic stem (ES) cells is controlled by a core network of transcription factors that includes Nanog, Oct4 and Sox2. Nanog is required to reach pluripotency during somatic reprogramming and is the only core factor whose overexpression is able to oppose differentiation-promoting signals. Additionally, Nanog expression is known to fluctuate in ES cells, and different levels of Nanog seem to correlate with ES cells' ability to respond to differentiation promoting signals. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is therefore critical to develop a better understanding of the pluripotent state.We describe the generation and validation of a mouse ES cell line with a novel Nanog reporter (Nd, from Nanog dynamics), containing a BAC transgene where the short-lived fluorescent protein VNP is placed under Nanog regulation. We show that Nanog and VNP have similar half-lives, and that Nd cells provide an accurate and measurable read-out for the dynamic levels of Nanog. Using this reporter, we could show that ES cells with low Nanog levels indeed have higher degree of priming to differentiation, when compared with high-Nanog cells. However, low-Nanog ES cells maintain high levels of Oct4 and Sox2 and can revert to a state of high-Nanog expression, indicating that they are still within the window of pluripotency. We further show that the observed changes in Nanog levels correlate with ES cell morphology and that Nanog dynamic expression is modulated by the cellular environment.The novel reporter ES cell line here described allows an accurate monitoring of Nanog's dynamic expression in the pluripotent state. This reporter will thus be a valuable tool to obtain quantitative measurements of global gene expression in pluripotent ES cells in different states, allowing a detailed molecular mapping of the pluripotency landscape

Morphology and Nanog:VNP levels correlation.

<p>(A) Bright field images of Nd cells grown in different culture media: serum; serum/LIF; 2i; and 2i with reduced inhibitors (FGF/ERK and GSK3ß inhibitors) concentration (1∶10, 1∶5 and 1∶2). The addition of increasing amounts of inhibitors results in more tightly packed morphology of ES cell colonies and in the reduction in flattened differentiated cells and a more tightly packed morphology of ES cell colonies. When ES cells are grown in serum alone, the inverse is observed. (B) Representative FC histograms of Nanog:VNP expression for Nd cells grown in different culture media.The addition of increasing amounts of inhibitors results in the increasing expression of Nanog:VNP (to up 95%), while cell grown in serum alone decreases significantly Nanog:VNP expression (to around 15%). The negative control cells (E14tg2a) are represented in gray. (C) Nanog:VNP expression quantifications for Nd cells grown in different culture media (n = 3). Statistically significant differences (p-value <0.002) observed between “serum/LIF” and other conditions are denoted with (*).</p

Protein expression analysis.

<p>n.d. = not determined.</p><p>Expression of Nanog, Oct4, Sox2, VNP and GFP proteins in Nd, E14tg2a and TNG-A cells, grown in serum/LIF or 2i media, and measured by IF, FC-IS or FC in non-fixed cells. Averages and standard deviations of at least 3 independent experiments are depicted (except for TNG-A cells, where only two biological replicates were performed). Nanog expression is not statistically different between different cell lines grown in the same media, although IF measurements provide slightly lower values than FC-IS, probably due to higher sensitivity of cytometry analysis. ES cells grown in 2i media significantly increase Nanog expression (determined by FC-IS) when compared to serum/LIF conditions for all cell lines. In Nd cells, VNP mimics Nanog expression in both culture conditions. In contrast, the percentage of GFP-positive cells in TNG-A ES cells is much higher than the percentage of Nanog-positive cells in serum/LIF conditions, most likely due to reporter stability. Oct4 and Sox2 are always detected in more than 90% of cells in both media and for all ES cell lines, with no statistically significant differences being observed. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059928#pone.0059928.s007" target="_blank">Table S3</a> for statistical data analysis (p-values).</p

Protein and RNA half-lives of different pluripotency and reporter genes.

<p>(A) Western blot analysis for VNP/GFP, Nanog, Oct4and a-Tub proteins in Nd, E14tg2a and TNG-A ES cell lines, over 6 hours after protein synthesis inhibition by cycloheximide (6h DMSO corresponds to the control without cycloheximide addition). Column on the right indicates the decay times of the targeted proteins. (B) Representative quantitative RT-PCR graphs for Vnp, Nanog, Oct4 and Sox2 mRNAs, calculated over 6 hours after transcription inhibition with actinomycin D (averages and standard deviations of at least 2 independent experiments are shown). Half-lives calculated using these data are depicted on each graph in brackets. No statistically significant differences were observed between Nd and E14tg2a cell lines (p-values >0.15).</p