Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells.

Mi-Sun Lim,Chun-Hyung Kim,Soo Young Lee,Sang-Hun Lee,Youl-Hee Cho,Jeong-Kyu Hoh,Yang-Ki Minn,Min-Seop Shin,Chang-Hwan Park,Dong-Wook Kim,Austin John Cooney

doi:10.1371/journal.pone.0138460

Mi-Sun Lim, Chun-Hyung Kim + Show 9 more

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https://doi.org/10.1371/journal.pone.0138460

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Abstract

Directed methods for differentiating human embryonic stem cells (hESCs) into dopaminergic (DA) precursor cells using stromal cells co-culture systems are already well established. However, not all of the hESCs differentiate into DA precursors using these methods. HSF6, H1, H7, and H9 cells differentiate well into DA precursors, but CHA13 and CHA15 cells hardly differentiate. To overcome this problem, we modified the differentiation system to include a co-culturing step that exposes the cells to noggin early in the differentiation process. This was done using γ-irradiated noggin-overexpressing CF1-mouse embryonic fibroblasts (MEF-noggin) and MS5 stromal cells (MS5-noggin and MS5-sonic hedgehog). After directed differentiation, RT-PCR analyses revealed that engrailed-1 (En-1), Lmx1b, and Nurr1, which are midbrain DA markers, were expressed regardless of differentiation stage. Moreover, tyrosine hydroxylase (Th) and an A9 midbrain-specific DA marker (Girk2) were expressed during differentiation, whereas levels of Oct3/4, an undifferentiated marker, decreased. Immunocytochemical analyses revealed that protein levels of the neuronal markers TH and TuJ1 increased during the final differentiation stage. These results demonstrate that early noggin exposure may play a specific role in the directed differentiation of DA cells from human embryonic stem cells.

Highlights

Neurodegenerative disorders including Parkinson’s disease (PD), Alzheimer’s disease, and Huntington’s disease are characterized by progressive neural loss and dysfunction [1]
After differentiation on the MS5 feeder layer, CHA Human embryonic stem cells (hESCs) still expressed high levels of the pluripotent marker, Oct3/4, whereas rosette (ZO1) and neural ectoderm markers (Sox1 and Otx2) were expressed at much lower levels than in H9 and HSF6 hESCs (Fig 1F). These results indicated that not all of the hESCs differentiated into DA precursor cells using the classical stromal cell co-culture system
The main goal of this study is to develop a strategy to enhance the induction of hESC-derived neural precursor cells (NPCs)/DA neurons in vitro using a stromal cell co-culturing method

Summary

Introduction

Neurodegenerative disorders including Parkinson’s disease (PD), Alzheimer’s disease, and Huntington’s disease are characterized by progressive neural loss and dysfunction [1]. Various cell sources have been used for cell transplantation, for example fetal and adult neural precursor cells (NPCs). Several differentiation methods have been shown to generate tyrosine hydroxylase (TH)-positive neurons from hESCs and hiPSCs in vitro, the differentiation efficiency was low, and the transplanted cells were unable to survive in the host brain [7,8,9,10]. To improve the directed differentiation of hESCs and hiPSCs to appropriate NPCs/DA neurons, several studies have used the BMP signaling antagonist noggin to promote neural differentiation from the ectoderm [11] through suppressed SMAD signaling [12,13,14] and rostral induction. As a result early noggin exposure markedly reduced pSMAD protein expression and induced rosette-like structure formation by CHA hESCs

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