Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.

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Recently, due to the changing geopolitical situation, economic instability and vulnerability, practicing auditors are facing new challenges that affect the identification and assessment of risks of material misstatement of financial statements. The purpose of the research is to identify the main factors affecting the risk of material misstatement and specific risks (fraud, accounting estimates, and going concern) that auditors should consider when deciding on the level of the risk of material misstatement of financial statements. To achieve the goal, two tasks were set: to compile lists of factors affecting the risk of material misstatement and specific risks; conduct a questionnaire survey to confirm the compiled lists. The following research methods were used during the research: content analysis and systematization of scientific and normative literature, comparison, logical analysis, questionnaire survey and summarization. As a result of the research, lists of the main factors determining the risk of material misstatement and specific risks were compiled, the completeness of which was confirmed during a questionnaire survey of auditors and auditor assistants working in Lithuanian audit companies. The results of the research show the necessary need for a more thorough assessment of specific risks. Compiled lists of factors determining the risk of material misstatement and specific risks can be useful for auditors of audit companies to assess the risk of material misstatement of financial statements.

Cryopreservation of placenta tissue for long-term storage provides the opportunity in the future to isolate mesenchymal stromal cells that could be used for cell therapy and regenerative medicine. Despite being widely used, the established cryopreservation protocols for freezing and thawing still raise concerns about their impact on molecular characteristics, such as epigenetic regulation. In our study, we compared the characteristics of human placental mesenchymal stromal cells (hPMSCs) isolated from fresh (native) and cryopreserved (cryo) placenta tissue. We assessed and compared the characteristics of native and cryo hPMSCs such as morphology, metabolic and differentiation potential, expression of cell surface markers, and transcriptome. No significant changes in immunophenotype and differentiation capacity between native and cryo cells were observed. Furthermore, we investigated the epigenetic changes and demonstrated that both native and cryo hPMSCs express only slight variations in the epigenetic profile, including miRNA levels, DNA methylation, and histone modifications. Nevertheless, transcriptome analysis defined the upregulation of early-senescence state-associated genes in hPMSCs after cryopreservation. We also evaluated the ability of hPMSCs to improve pregnancy outcomes in mouse models. Improved pregnancy outcomes in a mouse model confirmed that isolated placental cells both from native and cryo tissue have a positive effect on the restoration of the reproductive system. Still, the native hPMSCs possess better capacity (up to 66%) in comparison with cryo hPMSCs (up to 33%) to restore fertility in mice with premature ovarian failure. Our study demonstrates that placental tissue can be cryopreserved for long-term storage with the possibility to isolate mesenchymal stromal cells that retain characteristics suitable for therapeutic use.