Table_1_Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells.DOCX

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.

人羊水干细胞（AFSC）作为治疗应用中极具吸引力和巨大潜力的干细胞来源，备受瞩目。本研究旨在探讨短时间小分子治疗对提升干细胞特性及分化能力的影响。为此，我们采用了表观遗传活性化合物，如组蛋白脱乙酰化酶抑制剂三尖杉素A（TSA）和丁酸钠（NaBut），以及天然来源的多功能分子，如视黄酸（RA）和维生素C（vitC）。观察发现，这些化合物的组合触发了多能性相关基因（KLF4、OCT4、NOTCH1、SOX2、NANOG、LIN28a、CMYC）的上调，但蛋白质表达变化轻微，仅观察到Notch1的显著下调。此外，通过流式细胞术检测到的细胞表面标志物表达的变化中，CD105和CD117的表达变化最为显著。利用Seahorse分析仪进行的细胞能量学分析以及与细胞代谢和呼吸相关的基因表达评估（NRF1、HIF1α、PPARGC1A、ERRα、PKM、PDK1、LDHA、NFKB1、NFKB2、RELA、RELB、REL），揭示了小分子治疗能够刺激AFSCs向更具能量活跃的表型转变。为诱导细胞向神经发生谱系分化，我们采用了包括商业补充剂N2和B27以及RA在内的多种不同方案，并与添加了由小分子（vitC、TSA和RA）组成的预诱导步骤的同分化方案进行了比较。在分化过程中，分析了多个神经标记基因的表达（Nestin、MAP2、TUBB3、ALDH1L1、GFAP、CACNA1D、KCNJ12、KCNJ2、KCNH2），并观察到小分子治疗对分化潜能的积极作用，表现为基因表达的上调。分化还通过TUBB3、NCAM1和维甲酸蛋白的染色得到证实，并通过BDNF的分泌进行评估。本研究结果为短时间小分子治疗在改善AFSCs特性及增强其分化潜能方面的潜在应用提供了宝贵见解。