At present, most of the neurotoxicological analyses are based on in vitro and in vivo models utilizing animal cells or animal models. In addition, the used in vitro models are mostly based on molecular biological end-point analyses. Thus, for neurotoxicological screening, human cell-based analysis platforms in which the functional neuronal networks responses for various neurotoxicants can be also detected real-time are highly needed. Microelectrode array (MEA) is a method which enables the measurement of functional activity of neuronal cell networks in vitro for long periods of time. Here, we utilize MEA to study the neurotoxicity of methyl mercury chloride (MeHgCl, concentrations 0.5–500 nM) to human embryonic stem cell (hESC)-derived neuronal cell networks exhibiting spontaneous electrical activity. The neuronal cell cultures were matured on MEAs into networks expressing spontaneous spike train-like activity before exposing the cells to MeHgCl for 72 h. MEA measurements were performed acutely and 24, 48, and 72 h after the onset of the exposure. Finally, exposed cells were analyzed with traditional molecular biological methods for cell proliferation, cell survival, and gene and protein expression. Our results show that 500 nM MeHgCl decreases the electrical signaling and alters the pharmacologic response of hESC-derived neuronal networks in delayed manner whereas effects can not be detected with qRT-PCR, immunostainings, or proliferation measurements. Thus, we conclude that human cell-based MEA platform is a sensitive online method for neurotoxicological screening.

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

This review focuses on hydrogels and their patterning techniques in relation to central nervous system applications, with emphasis on synthetic and natural materials and chemical and topographical patterning techniques. We describe the properties of hydrogel materials and various techniques used in hydrogel patterning methods. Also, the applicability and utilization of patterned hydrogels with neural cells is discussed. Surface chemistry and topography significantly affect cell behaviour, including cell attachment, migration and maturation. Although several patterning techniques are described in the literature, a review of techniques applicable to hydrogel materials is needed. Use of these patterned cell-hydrogel constructs might provide novel ways to treat central nervous system deficits in the future.

Large numbers of neuronal cells are needed for regenerative medicine to treat patients suffering from central nervous system diseases and deficits such as Parkinson’s disease and spinal cord injury. One suggestion has been the utilization of human dental pulp stem cells (hDPSCs) for production of neuronal cells which would offer a patient-specific cell source for these treatments. Neuronal differentiation of hDPSCs has been described previously. Here, we tested the differentiation of DPSCs into neuronal cells with previously reported protocol and characterized the cells according to their morphology, gene and protein expressions and most importantly according to their spontaneous electrical functionality with microelectrode array platform (MEA). Our results showed that even though hDPSC-derived neural progenitor stage cells could be produced, these cells did not mature further into functional neuronal cells. Thus, utilization of DPSCs as a cell source for producing grafts to treat neurological deficits requires more efforts before being optimal

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SummaryThe possibilities of human pluripotent stem cell-derived neural cells from the basic research tool to a treatment option in regenerative medicine have been well recognized. These cells also offer an interesting tool for in vitro models of neuronal networks to be used for drug screening and neurotoxicological studies and for patient/disease specific in vitro models. Here, as aiming to develop a reductionistic in vitro human neuronal network model, we tested whether human embryonic stem cell (hESC)-derived neural cells could be cultured in human cerebrospinal fluid (CSF) in order to better mimic the in vivo conditions. Our results showed that CSF altered the differentiation of hESC-derived neural cells towards glial cells at the expense of neuronal differentiation. The proliferation rate was reduced in CSF cultures. However, even though the use of CSF as the culture medium altered the glial vs. neuronal differentiation rate, the pre-existing spontaneous activity of the neuronal networks persisted throughout the study. These results suggest that it is possible to develop fully human cell and culture-based environments that can further be modified for various in vitro modeling purposes.

The currently used cell culturing and differentiation procedures are both time- and laborintensive. Automation of some of these procedures will increase the efficiency of commonly used cell differentiation protocols. We used a particular cell culture platform to rapidly and efficiently screen the neuronal differentiation of human embryonic stem cells (hESC). Continuous live monitoring and analysis of non-labeled cells using this system allowed us to characterize neuronal populations over the entire neuronal differentiation process. The differentiation of individual cells from early progenitor cells to neurons and glial cells could be monitored continuously using this system with sub-confluent cell cultures. The imaged data was collected and analyzed with a specially designed cell recognition protocol, which resulted in a quantitative neuronal cell count. The analysis results were confirmed using conventional laboratory methods such as manual counting and flow cytometry. Our findings suggest that an automated culture platform combined with automated monitoring and analysis systems is a reliable method for developing enhanced cell differentiation procedures or as part of an automated quality control system for existing protocols

Wild boars (Sus scrofa) are often kept in enclosures for hunting or meat production purposes in Sweden. The sows are known to undergo behavioural changes in connection with farrowing and their natural behaviours may be compromised by the limited area of the enclosure. The aim of this study was to quantitatively describe wild boar sows' behaviour when farrowing in an enclosure. A field study was carried out in a hunting enclosure, where 1200 hours of behavioural recordings and data from 22 farrowings were collected. According to the results, the farrowing period could be divided into 3 phases: pre-farrowing, isolation and sociality phases (in relation to farrowing: day -14 to -1, day 1 to 8, day 9 to 14 respectively). The activity decreased during isolation and increased in the sociality phase (p<0.05), whereas the average distance to other individuals increased during isolation and decreased in the sociality phase (p<0.05). Nose contacts with other individuals increased in the isolation phase (p<0.05) and habitat use changed towards more protective habitats after farrowing. 68 % of the nests were situated in edges between 2 habitats of different vegetation density and 73% had some kind of protection to the north. We conclude that farrowing induces a number of changes in the activity, social behaviour and habitat preference in captive European wild boars. This may need attention when enclosures for this species are designed.