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

Aistė Zentelytė,Veronika V Borutinskaitė,Rūta Navakauskienė,Deimantė Žukauskaitė,Ieva Jacerytė

doi:10.3389/fbioe.2021.623886

Aistė Zentelytė, Veronika V Borutinskaitė + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2021.623886

Copy DOI

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.

Highlights

Nowadays, alternative sources of potent stem cells are of great interest and human amniotic fluid could be an attractive option
We investigated the impact of the following small molecules on primary stem cell lines: HDAC inhibitors trichostatin A (TSA), sodium butyrate (NaBut) and multifunctional molecules of natural origin retinoic acid (RA) and vitamin C
Amniotic fluid stem cells (AFSC) were strongly positive for mesenchymal cell surface markers, such as CD44, CD90, and CD105 and immunological marker HLA-ABC, negative (

Summary

Introduction

Alternative sources of potent stem cells are of great interest and human amniotic fluid could be an attractive option. Amniotic fluid stem cells (AFSC) possess the ability to self-renew, differentiate into cell lineages from all three germ layers, they do not form teratomas in vivo and have low immunogenicity (Bossolasco et al, 2006; De Coppi et al, 2007; Roubelakis et al, 2007; Da Sacco et al, 2011). These cells are autogenous to the fetus and semiallogeneic to each parent and are said to be more potent than stem cells from other sources, such as bone marrow, umbilical cord blood, endometrium and other (Yan et al, 2013; Bonaventura et al, 2015; Alessio et al, 2018). This study demonstrates that short treatments with small molecule combinations could be used as pre-induction steps to improve differentiation efficiency

Objectives

Methods

Results

Discussion

Conclusion