Early mouse neural stem cells (NSCs) first appear in embryonic day E5.5 and express pluripotency markers Oct4, Sox2, Nanog and early neural marker Sox1. Early NSCs are a good model for understanding the role of various pathways that control initial neural commitment. However, a protocol for differentiation of mouse embryonic stem cells (ESCs) into early NSCs by adherent monolayer culture has not yet been established. Hence, in this study, we identified the combination of growth factors and small molecules that differentiated mouse ESCs into early NSCs and supported their proliferation. Leukaemia inhibitory factor (LIF) was the first factor to be tested and it was found that ESCs can differentiate into early neurogenic lineage in the presence of LIF. However, we found that the induction is weaker in the presence of LIF as compared to cells differentiated in its absence. GSK-3 inhibitor, along with BMP and TGF-β pathway inhibitor (dual SMAD inhibition), are commonly used to sequentially direct ESCs towards NSCs. However, when we used this combination, mouse ESCs failed to differentiate into early NSCs. We observed that by adding Wnt inhibitor to the combination of GSK-3 inhibitor, BMP inhibitor, TGF-β inhibitor and LIF, it was possible to differentiate ESCs into early NSCs. qRT-PCR analysis of early NSCs illustrated that they expressed key pluripotency genes Oct4 and Nanog, albeit at levels lower than non-differentiated ESCs, along with early neural markers Sox1 and Pax6.
The cellular components of the satellite cell niche participate in the regulation of skeletal muscle regeneration. Beside myogenic cells at different developmental stages, this niche is formed by cells of the immune system, the interstitial connective tissue and the vascular ystem. Unambiguous determination of the origin of these cell types could contribute to optimization of the cell-based therapy of skeletal muscle disorders. In our work, we intravenously transplanted mouse GFP+ unseparated bone marrow cells into whole-body lethally irradiated immunocom-petent mice four weeks before cardiotoxin-induced injury of the recipients’ skeletal muscles. Seven and 28 days after the toxin injection, the injured regenerating and contralateral intact muscles were examined for identification of GFP+ bone marrow-derived cells by direct fluorescence, protein immunohistochemistry and immunogold transmission electron microscopy. In both the intact and injured muscles, GFP positivity was determined in immune cells, mainly in macrophages, and in interstitial spindle-shaped cells. Moreover, in the injured muscles, rare GFP+ endothelial cells of the blood vessels and newly formed myotubes and muscle fibres were present. Our results confirmed the ability of bone marrow-derived cells to contribute to the cellular component of the satellite cell niche in the intact and regenerating skeletal muscle. These cells originated not only from haematopoietic stem cells, but obviously also from other stem or progenitor cells residing in the bone marrow, such as multipotent mesenchymal stromal cells and endothelial progenitors. and Corresponding author: Dana Čížková
The present review brings the survey of the most frequently used behavioural tests in experimental models of Parkinson's disease (PD). Although there is no spontaneous occurrence of parkinsonism in animals, several experimental animal models of PD have been developed to achieve the same clinical features in animals. The techniques employing neurotoxins in lesioning the nigrostriatal dopaminergic (DA) system have a large selectivity and reproducibility. The most frequently used neurotoxins are l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA). MPTP-lesioned monkeys mimic best the symptomatology of PD in human patients while rats appear to be refractory to MPTP. For that reason, 6-OHDA is used to damage the substantia nigra in a rodent model. Behavioural tests of animals with nigrostriatal lesion represent valuable non- invasive methods for assessing the influence of damaged DA system on locomotor activity. The most frequently used experimental model of PD is the drug-evoked rotation in 6-OHDA unilaterally lesioned rats. This model produces well-defined and stable behavioural deficits. The rotation test is a useful parameter for evaluating imbalances of dopamine in both striata of the hemi-parkinsonian rat model. T-maze, treadmill running test or sensorimotor tests are used to evaluate spontaneous locomotor activity of lesioned animals. Skilled motor tasks measure the influence of dopamine-depleting lesions on complex motor acts. Transplantation of DA tissue into the striatum offers a new approach to the treatment of PD. Experimental models and behavioural tests are used to evaluate the extent of graft-induced recovery of MPTP- or 6-OHDA-lesioned animals. Different results obtained after the use of different tests reflect the level of graft integration into the host circuitry.
Here we describe a comparative study of phenotypic properties of hepatic cells in situ and in vitro. We analyzed the expression levels and distribution patterns of ABC transporters MRP2 and MDR1, pan-cytokeratin, cytokeratin 18, albumin, alpha-fetoprotein and the specific hepatocyte marker OCH1E5 in the fetal and adult rat as well as human liver tissue and in human fetal hepatocytes of WRL 68 cell line using peroxidase immunohistochemistry or immunofluorescence. Transporters MRP2 and MDR1 were expressed in all examined liver tissues, except rat ED13 embryo. The immunopositivity of these proteins was localized to the canalicular membrane of differentiating and mature hepatocytes but in the later developmental stages and in the adult liver tissues it was also found in the apical membrane of cholangiocytes. In WRL 68 cells, MRP2 and MDR1 immunoreactivity appeared after 5-6 days of cultivation and both transporters were fully expressed in the plasmalemma and in the cytoplasm 9 days after the passage. In conclusion, we observed only moderate variances reflecting diverse ontogenetic phases between the fetal and adult liver tissue. To study functions of hepatocytes in vitro, WRL 68 cells have to differentiate prior to the examination. Our findings indicate that WRL 68 cells can undergo differentiation in vitro and their antigenic profile closely resembles hepatocytes in the human liver.
The authors examined the influence of acetylcholinesterase inhibitor (neostigmine) on the in vitro reactivity of urinary bladder smooth muscle (UBSM) in guinea pigs. The aim of the present study was to determine the participation of pharmacokinetic properties of acetylcholine and carbachol in different UBSM reactivity to these mediators. In vitro method of organ baths was used and reactivity of UBSM strips to cumulative doses of acetylcholine and carbachol was tested before and after the incubation with neostigmine (10-4 mol.l-1). Neostigmine caused a significant increase of UBSM reactivity to acetylcholine. The UBSM reactivity to acetylcholine was significantly higher at concentrations of 10-5 and 10-4 mol.l-1 compared to carbachol at the same concentrations. These findings indicate that in addition to different mediator affinity to muscarinic receptors and to their different intrinsic activity, the pharmacokinetic properties of acetylcholine and carbachol also participate in UBSM reactivity.
The extracellular matrix (ECM) consists of proteins,
glycosaminoglycans and glycoproteins, that support the dynamic
interactions between cells, including intercellular communication,
cell attachment, cell differentiation, cell growth and migration. As
such, the ECM represents an essential and very sensitive system
within the tissue microenvironment that is involved in processes
such as tissue regeneration and carcinogenesis. The aim of the
present review is to evaluate its diversity through Ca2+ signaling
and its role in muscle cell function. Here, we discuss some
methodological approaches dissecting Ca2+ handling mechanisms
in myogenic and non-myogenic cells, e.g. the importance of Ca2+
and calpains in muscle dystrophy. We also consider the
reconstruction of skeletal muscle by colonization of decellularized
ECM with muscle-derived cells isolated from skeletal muscle.
Therefore, it is necessary to establish new methodological
procedures based on Ca2+ signaling in skeletal muscle cells and
their effect on ECM homeostasis, allowing the monitoring of
skeletal muscle reconstruction and organ repair.