Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells

Abstract

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.

Amniotic fluid stem cells: A novel treatment for necrotizing enterocolitis

“…These molecules can improve stem cell characteristics to further reinforce their therapeutic potential and efficiency. A study assessed the effect of various concentrations and mixtures of small molecules known to participate in cell repair and regeneration on AFSCs, such as surface marker and pluripotency associated gene expression ( 67 ). HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaBut) promote somatic cell reprogramming, while multifunctional molecules retinoic acid (RA) and vitamin C (vitC) synergistically boost pluripotency.…”

“…These small molecules in combination with each other have no cytotoxic effects but do increase gene expression patters of pluripotency markers and neurogenic transcription factors (Oct4, Nanog, Lin28a, Cmyc, Notch1, Sox2). Surface level markers are also affected (SSEA4, CD117, Tra-1-81, CD105) ( 67 ). Lastly, the effect on metabolic phenotype was significantly increased.…”