Impact of the dynamic culture system for 3D high cell density neural differentiation of hESC in electrospun PCL scaffolds

The ability of pluripotent stem cells to differentiate into any of the three germ layers have sparked an array of investigations in developmental biology, tissue replacement, drug screening and cellular interactions. A demand has risen for 3-dimensional technologies to improve scalability and better mimic the stem cells niche. With the increasingly complex cultivation platforms and larger scales a limit is rapidly reached in terms of nutrient and waste exchange to the cells. These limitations can be overcome with the use of dynamic culture systems such as bioreactors. However, a culture platform that allows for differentiation of pluripotent stem cells to neural lineages to homogenous macro scale tissue cultures is still challenging. We have previously developed a protocol for the culture of pluripotent cells in 3D electrospun PCL scaffolds in multi-well format [1]. The purpose of the present was to investigate the potential of different dynamic systems to support the neural differentiation of pluripotent stem cells in 3D electrospun polycaprolactone (PCL) scaffold disks of 35 mm diameter. The dynamic systems were a bioreactor with culture medium flow either longitudinal or orthogonal to the scaffold, alternatively a system with orbital shaking, and absence of agitation. Human embryonic stem cells, hESC, (line HS980 received from Prof. O. Hovatta (Karolinska University Hospital, Huddinge, Sweden), transferred to KTH and used in agreement with ethics approval issued to us by the Regional Ethics Board, Stockholm, Sweden (Ethical Permission Dnr 2013/493-31, amended 2015/824-39)) were submitted to a 3-week neural differentiation protocol. The cells were cultured in 3D Mimetix Air electrospun polycaprolactone (PCL) scaffold disks of 35 mm diameter in small stainless steel bioreactors using a modified version of the dual-SMAD differentiation protocol with small molecular inhibitors. The levels of Sox2, Nestin, ki67, Map2, Doublecortin and beta III tubulin were measured. Applying dynamic culture conditions for the differentiation of hESC towards neural lineages in 3D electrospun PCL scaffolds greatly increased the cell yield and had a large beneficial impact on the marker expression compared to a static system. Static cultivation resulted in a heterogeneous cell population with cells expressing both early and late differentiation markers. A culture with a flow orthogonal to the scaffold did not sustain a consistent cell culture. By using a flow longitudinal to the scaffold, the cells grew homogeneously across the scaffold and expressed early differentiation markers at a higher level compared to orbital shaking. This latter system increased the cell yield to the highest level, i.e. 120x106 cells/scaffold, and led to homogeneously differentiated cells with an increased level of mature neuronal markers. To our knowledge this was the first time that pluripotent stem cells have been differentiated to neural fate in a 3D scaffold of connected homogeneous macroscopic scale (\u3e cm). Reference: Leino M, Åstrand C, Hughes-Brittain N, Robb B, McKean R, Chotteau V. J Biomed Mater Res B Appl Biomater. 2018. 106:1226-1236. doi: 10.1002/jbm.b.33928

The 1st Verified Software Competition, Extended Experience Report

We, the organizers and participants, report our experiences from the 1st Veried Software Competition, held in August 2010 in Edinburgh at the VSTTE 2010 conferenc

The 1st Verified Software Competition, Extended Experience Report

We, the organizers and participants, report our experiences from the 1st Veried Software Competition, held in August 2010 in Edinburgh at the VSTTE 2010 conferenc

Advancing DNA-based proximity methods

Cellular functions are governed by intricate chains of interactions between proteins. In order to properly understand cellular biology one must look into, not only protein function, but also its interacting network. Furthermore, due to the large heterogeneity between cells within cultures and tissue samples it is important to retain spatial information to enable investigations on a single cell level. In order to achieve this, in situ methods play a large importance in further elucidation of these interacting networks. In order to investigate interactions between macromolecules such as proteins and nucleic acids, many outstanding methods have been developed. Some focusing on larger scale analysis, some on live cell imaging and some on detecting novel interactions. Our own group has focused on in situ methods utilizing DNA conjugated antibodies. DNA itself is a great macromolecule to work with, it can be produced synthetically, DNA hybridization is highly predictable and there is a large repertoire of DNA-modifying enzymes. This has been used in the development of methods such as proximity ligation assay (PLA) and Proximity-dependent initiation of hybridization chain reaction (ProxHCR). Both methods utilize antibodies conjugated with DNA in order to detect proximity events between two proteins. PLA utilizes ligation to confirm proximity, while ProxHCR utilizes a chain of strand displacements to do the same. Both methods work well, but no method is beyond further optimization. For PLA, a general concern lies in the formation of incorrectly interacting probes, resulting in incorrect ligations that yield linear fragments, incapable of producing visible signal. As a result PLA can produce a substantial amount of false negatives. To address this, we produced a similar method, Unfold, to streamline the probe interactions and ligations to improve efficiency. For ProxHCR the original method required overly stringent reactions conditions to allow for efficient strand displacements and thus strong signal. Furthermore, signal strength was further compromised by oligonucleotide quality. To improve these issues, the ProxHCR method was completely redesigned and oligonucleotide quality along with signal strength was improved by further purification. Both optimizations resulted in more efficient and versatile methods suitable for routine lab work and potential diagnostic use

Advancing DNA-based proximity methods

Cellular functions are governed by intricate chains of interactions between proteins. In order to properly understand cellular biology one must look into, not only protein function, but also its interacting network. Furthermore, due to the large heterogeneity between cells within cultures and tissue samples it is important to retain spatial information to enable investigations on a single cell level. In order to achieve this, in situ methods play a large importance in further elucidation of these interacting networks. In order to investigate interactions between macromolecules such as proteins and nucleic acids, many outstanding methods have been developed. Some focusing on larger scale analysis, some on live cell imaging and some on detecting novel interactions. Our own group has focused on in situ methods utilizing DNA conjugated antibodies. DNA itself is a great macromolecule to work with, it can be produced synthetically, DNA hybridization is highly predictable and there is a large repertoire of DNA-modifying enzymes. This has been used in the development of methods such as proximity ligation assay (PLA) and Proximity-dependent initiation of hybridization chain reaction (ProxHCR). Both methods utilize antibodies conjugated with DNA in order to detect proximity events between two proteins. PLA utilizes ligation to confirm proximity, while ProxHCR utilizes a chain of strand displacements to do the same. Both methods work well, but no method is beyond further optimization. For PLA, a general concern lies in the formation of incorrectly interacting probes, resulting in incorrect ligations that yield linear fragments, incapable of producing visible signal. As a result PLA can produce a substantial amount of false negatives. To address this, we produced a similar method, Unfold, to streamline the probe interactions and ligations to improve efficiency. For ProxHCR the original method required overly stringent reactions conditions to allow for efficient strand displacements and thus strong signal. Furthermore, signal strength was further compromised by oligonucleotide quality. To improve these issues, the ProxHCR method was completely redesigned and oligonucleotide quality along with signal strength was improved by further purification. Both optimizations resulted in more efficient and versatile methods suitable for routine lab work and potential diagnostic use

Purification of DNA oligonucleotides to improve hybridization chain reaction performance

Hybridization chain-reaction (HCR) is technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on that every hairpin is metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which places a strong demand on oligonucleotide quality. In this paper we show how further purification can greatly increase polymerization potential. We found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR

From paper to plastic – En studie om tidningens egenskaper och läsarnas användningsmönster

E-papperet är ett medie som i framtiden kan komma att användas för att läsa dagstidningar. Istället för att bläck trycks på papperet så visar e-papperet informationen genom att små partiklar på papperets yta byter färg vilket innebär att samma e- pappersark kan innehålla och visa ett stort antal sidor. Nya medier blir sällan accepterade utan att det funnits länkar till tidigare eller existerade tekniker. Denna studie ämnar kartlägga tidningsläsarnas användningsmönster och papperstidningens egenskaper för att understödja utformningen av morgondagens e-papperstidning. Genom att kombinera litteratur med egna undersökningar presenteras riktlinjer för vilka egenskaper och användningsmönster som framtidens e-papper bör stödja. Studien lyfter exempelvis fram att gränssnitt och navigation bör utformas i åtanke att majoriteten av tidningsläsarna läser tidningar vi köksbord och att formatet på e-pappret bör påminna om dagens tabloid format. Resultatet som presenteras skall kunna komma till nytta för företag och organisationer som skall utforma framtidens e-papperstidning. Rapporten beskriver med utgångspunkt från bland annat "diffusion theory" en kvalitativ studie där 400 enkäter skickades ut till prenumeranter av Sundsvalls tidning för att avslutas med en diskussion där förslag på fortsatt forskning och övriga slutsatser behandlas. Studien lyfter fram de viktigaste faktorerna som bör följas, samt ger förslag på hur de kan lösas samtidigt som nya frågor och diskussioner föds ur resultaten och diskussionerna

The method developer's guide to oligonucleotide design

Introduction Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described. Areas covered Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results. Expert opinion Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution

Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling

The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor beta-receptor (PDGFR-beta) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-beta preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-beta by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-beta activation, and that this link is not shared with the epidermal growth factor receptor