Endothelial Nitric Oxide Synthase Knockdown in Human Stem Cells Impacts Mitochondrial Biogenesis and Adipogenesis: Live-Cell Real-Time Fluorescence Imaging.

Sylvia Lee-Huang,Philip Lin Huang,Paul Lee Huang

doi:10.3390/jcm10040631

Sylvia Lee-Huang, Philip Lin Huang + Show 1 more

Open Access

https://doi.org/10.3390/jcm10040631

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Abstract

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

Highlights

Endothelial nitric oxide synthase-derived nitric oxide (NO) regulates many important functions, including vascular tone and regional blood flow, vascular smooth muscle cell proliferation, leukocyte–endothelial interactions, and thrombosis [1,2,3,4,5]
The aim of this study is to investigate the impact of Endothelial nitric oxide synthase (eNOS) knockdown (KD) in human stem cells on mitochondrial biogenesis and adipogenesis using live-cell real-time fluorescence imaging
Expression was unaffected, and there was no impact on mitochondrial biogenesis/function. These results show that the knockdown process, the insertion, and the expression of the green fluorescence protein reporter gene in the human mesenchymal stem cells (hMSCs) had no effect on mitochondrial biogenesis and function

Summary

Introduction

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) regulates many important functions, including vascular tone and regional blood flow, vascular smooth muscle cell proliferation, leukocyte–endothelial interactions, and thrombosis [1,2,3,4,5]. The effect of eNOS-derived NO on mitochondria and its links to insulin resistance and obesity have been mostly studied in animal models [6,7,8,9,10]. Few studies have been performed in human stem cells. We reported that eNOS-derived NO plays distinct and separable roles in white and brown adipogenesis in human stem cells [11]. ENOS-derived NO upregulates the expression of the thermogenic genes.

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