Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells.

Zhenqiang Zhao,Xinfeng Liu,Qian Liu,Zhibin Chen,Weimin Jiang,Yanlin Ma,Deyan Kong,Yanhong Yu,Tan Wang,Kunxiong Yuan,Qi Li,Xiaowu Chen,Lan Hu,Yanan Peng

doi:10.3389/fncel.2016.00291

Abstract

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.

Highlights

Parkinson’s disease (PD) is a chronically and progressively neurodegenerative disorder, and it is characterized by loss of the dopaminergic (DA) neurons in the substantial nigra pars compacta (SNpc) (Damier et al, 1999)
G-banding technique was applied to examine the karyotype and the results showed that these new human embryonic stem cells (hESCs) lines had a normal karyotype (Figure 1G)
P96-derived cell population exhibited significantly higher expression levels of FOXA2, tyrosine hydroxylase (TH), PITX3, and NURR1, demonstrating 14.9-fold, 1.7-fold, 5.9-fold, and 6.5-fold increase, respectively, in comparison with HN4-derived cells (Figure 5B). These results indicated that feeder cells had no effects on neural induction process; instead they played a significant role in dopaminergic differentiation potential of hESCs line

Summary

Introduction

Parkinson’s disease (PD) is a chronically and progressively neurodegenerative disorder, and it is characterized by loss of the dopaminergic (DA) neurons in the substantial nigra pars compacta (SNpc) (Damier et al, 1999). Human embryonic stem cells (hESCs) provide a potential resource for cells transplantation to replace the extensive loss of dopaminergic neurons. Dopaminergic neurons in the midbrain are divided into three distinct clusters termed the A8, A9, and A10 groups (Dahlstroem and Fuxe, 1964). Different midbrain DA neurons project to distinct regions and modulate specific functions. DA neurons in SNpc (A9 group) projected to the dorsal striatum and control voluntary movement, and the loss of these neurons is the pathological hallmark of PD (Lees et al, 2009). It is important to understand the electrophysiological properties of hESCs-derived DA neurons before they are used for transplantation. The electrophysiological criteria of hESCs-derived DA neurons are still not established

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