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The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following Wnt signaling modulation cardiac differentiation.

ABSTRACT: Owing to the beneficial properties of amniotic fluid-derived stem cells (AFSCs), including pluripotency and the lack of ethical issues associated with embryonic stem cells (ESCs), they should be a promising cell source for regenerative medicine. However, how to differentiate AFSCs into contracting cardiomyocytes has not been established. In this study, a well-established, direct cardiac differentiation protocol involving the modulation of Wnt signaling was used to differentiate Oct 3/4+ AFSCs into cardiomyocytes. By day 14 of cardiomyocyte differentiation, these AFSCs expressed cardiac-specific genes (i.e., cardiac troponin T and myosin light chain 2v) and proteins but could not spontaneously contract. Using the patch-clamp technique, we further characterized the electrophysiological properties of human ESC-derived cardiomyocytes (hESC-CMs) and differentiated AFSCs. We used different configurations to investigate membrane potentials and ion currents in differentiated AFSCs and hESC-CMs. Under cell-attached voltage- or whole-cell current-clamp modes, we recorded spontaneous action currents (ACs) or action potentials (APs) in hESC-CMs but not in differentiated AFSCs. Compared to hESC-CMs, differentiated AFSCs showed significantly diminished activity of both BKCa and IKCa channels, which might lead to a lack of spontaneous ACs and APs in differentiated AFSCs. These results indicated that this well-established Wnt signaling modulating cardiac differentiation protocol was insufficient to induce the differentiation of functional cardiomyocytes from Oct 3/4+ AFSCs. Therefore, AFSC may not be an ideal candidate for cardiomyocyte differentiation.

SUBMITTER: Liu YW

PROVIDER: S-EPMC6349909 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following Wnt signaling modulation cardiac differentiation.

Liu Yen-Wen YW Fang Yi-Hsein YH Su Chi-Ting CT Hwang Shiaw-Min SM Liu Ping-Yen PY Wu Sheng-Nan SN

Cell death discovery 20190128

Owing to the beneficial properties of amniotic fluid-derived stem cells (AFSCs), including pluripotency and the lack of ethical issues associated with embryonic stem cells (ESCs), they should be a promising cell source for regenerative medicine. However, how to differentiate AFSCs into contracting cardiomyocytes has not been established. In this study, a well-established, direct cardiac differentiation protocol involving the modulation of Wnt signaling was used to differentiate Oct 3/4<sup>+</su ...[more]

PMID: 30701091

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Project description: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.

Dataset Information

The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following Wnt signaling modulation cardiac differentiation.

Publications

The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following Wnt signaling modulation cardiac differentiation.

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