Differentiation of Motor Neuron-Like Cells from Tonsil-Derived Mesenchymal Stem Cells and Their Possible Application to Neuromuscular Junction Formation.

Saeyoung Park,Seoha Myung,Ji Yeon Kim,Namhee Jung,Yeonzi Choi,Sung-Chul Jung

doi:10.3390/ijms20112702

Abstract

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.

Highlights

Human tonsil-derived mesenchymal stem cells (T-MSCs) have typical features of MSCs that can differentiate into adipocytes, chondrocytes, and osteocytes, and that mediate immunomodulation [1,2,3]
The T-MSC-neural progenitor cells (NPCs) were differentiated into T-MSC-MMoottoorr neuron-lliikkee cells (MNCs) by culture in the motor neuronal induction medium (MNM)
To induce MN differentiation, 1.5–2 × 104/cm2 T-MSC-NPCs were placed into motor neuronal induction medium (MNM), which comprised low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2.5% fetal bovine serum (FBS), 1% N2 supplement (Gibco, Life Technologies, Burlington, ON, Canada), 1 μM retinoic acid RA (Sigma-Aldrich), 10 ng/mL BDNF (R&D Systems, Minneapolis, MN, USA), 10 ng/mL NGF (R&D Systems), and 0.1 ng/mL Shh (R&D Systems)

Summary

Introduction

Human tonsil-derived mesenchymal stem cells (T-MSCs) have typical features of MSCs that can differentiate into adipocytes, chondrocytes, and osteocytes, and that mediate immunomodulation [1,2,3]. T-MSCs are a useful source for cell therapy because they can be separated from human tonsil tissue that is discarded after surgery. They are affected by donor age and can be cultured long-term (15 passages) and cryopreserved while maintaining their morphology, cell surface markers, and differentiation potential [4,5]. T-MSCs have been reported to differentiate into the three primary germ layers, i.e., mesodermal lineage (including fat, cartilage, bone, and muscle cells), endodermal lineage (including the liver and insulin-secreting and parathyroid hormone-secreting cells), and ectodermal lineage (including Schwann and neuronal cells) [4,6,7,8,9,10,11,12]. Some MNDs are hereditary, and nonhereditary or sporadic MND are related to environmental, toxic, viral, or genetic factors [13,14]

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