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

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.

A transcriptomic analysis of serial-cultured, tonsil-derived mesenchymal stem cells reveals decreased integrin α3 protein as a potential biomarker of senescent cells

“…Previously, we isolated MSCs from tonsillar tissue of children who had undergone a tonsillectomy [37]. TMSCs have been implied for various tissue engineering of the bone [18,38], parathyroid gland [17], pancreatic islet [39], Schwan cells [22], tendon [21], nerve cell [20,23], and skeletal muscle [19] regeneration. For practical applications of cell therapy, it is necessary to obtain a sufficient number of cells through a long-term serial culture [38].…”

“…The replicative senescence induced by the serial culture of TMSCs decreased not only replicative capacity but also the differentiation potential of TMSCs. An examination of three mesodermal differentiation potentials-adipogenesis, osteogenesis, and chondrogenesisrevealed that early-passage TMSCs (passages 5-8) have a superior differentiation potential compared with latepassage TMSCs (passages [20][21][22][23][24][25]. It has also been reported that the quality of other MSCs, such as adipose tissue- [43], bone marrow- [36], and Wharton's jellyderived MSCs [2], were also shown to progressively decline with donor age [27].…”

“…The growth medium was changed every 3 days. In vitro-cultured TMSCs were divided into two groups as follows: control TMSCs (passages 5-8) and culture-aged TMSCs (passages [20][21][22][23][24][25]. Morphological changes in the TMSCs were monitored daily by examination under an inverted microscope (Olympus, Tokyo, Japan).…”

“…3a-d). [20][21][22][23][24][25]. c Transcriptomic microarray analysis of differential gene expression between control and culture-aged TMSCS .d ECM-receptor interaction-related genes were identified by KEGG pathway analysis.…”

“…Human tonsil-derived MSCs (TMSCs) are obtained from discarded children's tonsillar tissues after tonsillectomy. TMSCs have been considered as a potential therapeutic tool in the application of tissue engineering and regenerative medicine [14][15][16], because of their high regenerative capacity and multipotency to differentiate into various cell types, such as the bone [17], cartilage [17], adipose [18], muscle [19,20], tendon [21], stroma, and neuronal cells [22,23]. Unfortunately, a long-term culture of TMSCs could lead to replicative senescence, decreasing the stemness and multi-differentiation potential of TMSCs.…”