BACKGROUND: Astrocytes have recently gained attention as key players in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease. Numerous differentiation protocols have been developed to study human astrocytes in vitro. However, the properties of the resulting glia are inconsistent, making it difficult to select an appropriate method for a given research question. Therefore, we compared three approaches for the generation of iPSC-derived astrocytes. We performed a detailed analysis using a widely used long serum-free (LSFP) and short serum-free (SSFP) protocol, as well as a TUSP protocol using serum for a limited time of differentiation. RESULTS: We used RNA sequencing and immunochemistry to characterize the cultures. Astrocytes generated by the LSFP and SSFP methods differed significantly in their characteristics from those generated by the TUSP method using serum. The TUSP astrocytes had a less neuronal pattern, showed a higher degree of extracellular matrix formation, and were more mature. The short-term presence of FBS in the medium facilitated the induction of astroglia characteristics but did not result in reactive astrocytes. Data from cell-type deconvolution analysis applied to bulk transcriptomes from the cultures assessed their similarity to primary and fetal human astrocytes. CONCLUSIONS: Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol for solving specific research tasks or drug discovery studies with iPSC-derived astrocytes.

• Klíčová slova
• Astrocytes, Aβ42, Disease modeling, FBS, IPSC,
• Publikační typ
• časopisecké články MeSH

The selection of a highly-viable single embryo in assisted reproductive technology requires an acceptable predictive method in order to reduce the multiple pregnancy rate and increase the success rate. In this study, the metabolomic profiling of growing and impaired embryos was assessed on the fifth day of fertilization using capillary electrophoresis in order to find a relationship between the profiling and embryo development, and then to provide a mechanistic insight into the appearance/depletion of the metabolites. This unique qualitative technique exhibited the appearance of most non-essential amino acids and lactate, and depleting the serine, alanyl-glutamine and pyruvate in such a manner that the embryos impaired in their development secreted a considerably higher level of lactate and consumed a significantly higher amount of alanyl-glutamine. The different significant ratios of metabolomic depletion/appearance between the embryos confirm their potential for the improvement of the prospective selection of the developed single embryos, and also suggest the fact that pyruvate and alanyl-glutamine are the most critical ATP suppliers on the fifth day of blastocyst development.

• Klíčová slova
• capillary electrophoresis, culture media, embryo development, metabolomics,
• Publikační typ
• časopisecké články MeSH

The vitrification of human embryos is more and more frequently being utilized as a method of assisted reproduction. For this technique, gentle treatment of the embryos after thawing is crucial. In this study, the balance of amino acids released to/consumed from the cultivation media surrounding the warmed embryos was observed in the context of a cultivation environment, which was with the atmospheric oxygen concentration ≈20% or with a regulated oxygen level-hysiological (5%). It is the first time that total amino acid turnover in human embryos after their freezing at post compaction stages has been evaluated. During this study, progressive embryos (developed to blastocyst stage) and stagnant embryos (without developmental progression) were analyzed. It was observed that the embryos cultivated in conditions of physiological oxygen levels (5% oxygen) showed a significantly lower consumption of amino acids from the cultivation media. Progressively developing embryos also had significantly lower total amino acid turnovers (consumption and production of amino acids) when cultured in conditions with physiological oxygen levels. Based on these results it seems that a cultivation environment with a reduced oxygen concentration decreases the risk of degenerative changes in the embryos after thawing. Therefore, the cultivation of thawed embryos in an environment with physiological oxygen levels may preclude embryonal stagnation, and can support the further development of human embryos after their thawing.

• Klíčová slova
• amino acids, capillary electrophoresis, fluorescence detection, human embryo, in vitro cultivation, metabolic activity,
• Publikační typ
• časopisecké články MeSH