Live cell imaging for the transdifferentiation of mesenchymal stem cells into neurons

Abstract

Purpose: Mesenchymal stem cells (MSCs) have been of great interest for cell-replacement therapy in neurodegenerative disease due to substantial merit capable of directly using patient own cells. Developing the imaging system for tracking dynamic change of the neuronal differentiation process from MSCs is important to evaluate the transdifferentiation efficacy of grafted MSCs. In this study, we aimed to assess real-time live cell imaging for detection of differentiation process by the small molecule known as compound 1 (C18H17N3O3) to facilitate neuronal transdifferentiation induced from MSCs using a neuronal promoter-driven fluorescence imaging system. Materials and methods: Rat bone marrow-derived MSCs were treated with 20 μM compound 1 to induce differentiation of MSCs into neuronal cells. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed for evaluation of neuronal marker expression. Plasmid vector containing red fluorescence reporter genes under the control of the tubulin α1 (Tα1) promoter (Tα1p-DsRed2) were transfected into MSCs to examine the change of fluorescence signal during neuronal differentiation. Static microscopic imaging and time-lapse live cell imaging were performed for monitoring of neuronal differentiation progress from MSCs by compound 1 using confocal microscopy and fluorescence live cell microscope imaging device, respectively. Results: Two days after treatment with compound 1, MSC cells showed changes of neuron-like phenotype changes, as well as increased expression of neuron-specific markers such as neuron specific enolase (NSE), class III β-tubulin (Tuj 1), and synaptophysin. Immunofluorescence staining results revealed that Tuj 1 expression level was increased after induction of neuronal differentiation by compound 1. When Tα1p-DsRed2 reporter vector was transfected into MSCs, immunostaining data displayed that the enhanced fluorescence signals were detected in the cytoplasm region of MSCs after treatment of compound 1. In vitro monitoring in MSC transfected with Tα1p-DsRed2 reporters showed progressively increased fluorescence signals by 30 h after treatment of compound 1, corresponding with progressive neuronal differentiation from MSCs. Conclusions: We examined an efficient neuronal differentiation pattern by compound 1, and monitored neuronal differentiation process by neuron specific promoter-based fluorescence reporter system in cellular level. These findings will be helpful in understanding the efficacy of neuronal differentiation derived from MSCs for stem cell-based therapy.

중간엽줄기세포(mesenchymal stem cells, 이하 MSCs) 는 인체 내 다른 줄기세포 들에 비해 획득이 용이하여 이를 이용한 신경 퇴행성 질환의 세포 치환 치료의 개발에 관심이 모아지고 있다. 이 연구에서는 중간엽줄기세포의 신경세포 교차분화를 촉진시키는 compound 1의 분화 유도능을 확인하고 신경세포 분화를 추적 관찰하기 위한 reporter imaging system의 성능을 평가하고자 하였다. 쥐의 MSCs에 compound 1을 처리하여 신경세포 교차분화의 양상을 확인하였고 reverse transcription polymerase reaction (RT-PCR) 및 면역조직화학염색을 통해 신경세포 교차분화 후 신경세포 특이 유전자 발현의 증가를 확인하였다. Tubulin α1 (이하 Tα1) promoter를 이용한 fluorescence reporter gene을 MSCs에 이입하여 신경 분화 과정에 따른 형광 신호의 증가 양상을 confocal microscopy와 live cell imaging을 통해 확인하였다. Compound 1 처리 2일 후에 대부분의 MSCs들은 신경세포와 유사한 외형의 변화를 보였으며 신경세포 특이 유전자 (NSE, Tuj 1, synaptophysin) 발현의 증가를 동반하였다. 또한, 면역염색방법을 통해 compound 1으로 신경세포 분화를 유도시켰을 때, 세포골격단백질인 β-tubulin 염색을 통해 세포모양의 변화를 확실히 구분할 수 있었고, Tuj 1 단백질을 발현이 증가됨을 확인할 수 있었다. 신경세포 분화과정을 실시간으로 관찰하기 위해 Tα1 promoter의 조절을 받는 형광리포터 유전자를 MSC 세포에 이입하였을 때 Compound 1을 처리한 MSCs에서 신경 분화의 진행에 따라 점차적으로 형광시그널이 증가함을 관찰할 수 있었다. 이 연구에서는 MSCs로부터 신경세포로의 분화를 Compound 1이라는 화합물을 이용하여 관찰하였고, 신경세포 분화용 리포터유전자를 이용하여 분화과정을 영상화하였다. 중간엽줄기세포로부터 신경세포로의 분화영상법의 개발은 줄기세포 치료 관점에서 분화효율을 추적하거나, 세포치료 메커니즘을 이해하는 데 중요한 도구가 될 것이라고 생각한다.