www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 1), pp: 576-590 Research Paper Liver X receptors agonist promotes differentiation of rat bone marrow derived mesenchymal stem cells into dopaminergic neuron-like cells Oumei Cheng1,2,*, Xiaoyan Tian1,*, Ying Luo1, Shaoshan Mai1, Yang Yang1, Shengnan Kuang1, Qi Chen1, Jie Ma1, Beibei Chen2, Rong Li2, Lu Yang1, Huan Li1, Congli Hu1, Jiahua Zhang1, Zhihao Chen1, Yuke Li1, Hui Xia1, Ying Xu3 and Junqing Yang1 1Department of Pharmacology, Chongqing Medical University, The Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing 400016, China 2Department of Neurology, The First Affiliated China Hospital, Chongqing Medical University, Chongqing, 400016, China 3Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA *These authors share co-first authorship Correspondence to: Junqing Yang, email: [email protected] Keywords: Parkinson’s disease; dopaminergic neurons; liver X receptors; bone marrow derived mesenchymal stem cells and cell differentiation Received: August 23, 2017 Accepted: November 14, 2017 Published: December 09, 2017 Copyright: Cheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Dopaminergic (DA) neurons derived from bone marrow derived mesenchymal stem cells (BMSCs) maybe a valuable source for cell replacement therapy in Parkinson disease. Recent studies showed that new functions of LXR and their ligands have been proposed to prevent PD in the adult nervous system. The present study was designed to observe the effect of liver X receptors (LXR) agonist on differentiation of rat BMSCs into DA neurons. Expressions of the neuronal markers (Tuj1 and Nestin), the specific marker of DA neurons (tyrosine hydroxylase, TH), LXR α and LXR β were measured by immunocytochemical assay and TH/Tuj1 positive cells were determined by quantitative cell count analyses. mRNA expressions of LXR α, LXR β, TH, DAT, Nurr1, Pitx3, En1 and Lmx1b were measured by qPCR. Compared with growth factors (GF) treated group, combined use of LXR and GF induced rat BMSCs to TH-expressing cells with 87.42% of efficiency in 6 days of period of induction. LXR agonist alone did not induce the differentiation. Compared with GF alone, combined use of LXR and GF increased expressions of LXR α and LXR β protein and mRNA and TH, DAT, Nurr1, and Pitx3 mRNA, decreased expressions of En1 and Lmx1b mRNA. Our experimental results indicated that LXR activation leads to improve induction efficiency and shorten induction period of rat BMSCs into DA neuron-like cells through regulating DA development-related genes expressions and that LXR can be considered as a candidate target for drug development to improve differentiation of BMSCs into DA neurons. INTRODUCTION after Alzheimer’s disease [1]. It is characterized by a progressive and extensive loss of dopaminergic (DA) Parkinson’s disease (PD) is a chronic progressive neurons in the substantia nigra pars compacta (SNpc) neurodegenerative disease that affects over 1% of the and their terminals in the striatum, which results in population over 65 years of age and has been positioned debilitating movement disorders. Currently available as the second most common neurodegenerative disorder pharmacological and surgical therapies ameliorate www.impactjournals.com/oncotarget 576 Oncotarget clinical symptoms in the early stages of disease, but they RESULTS cannot stop or reverse degeneration of DA neurons and have some serious side effects with long-term use [2]. Characterization of rat BMSCs Cell replacement therapy (CRT) offers great promise as the future of regenerative medicine in PD [3]. There are Undifferentiated rat BMSCs in expansion cultures many reports about the derivation of DA neurons from were examined at 3rd passage for the expression of cell embryonic stem cells (ESCs) [4, 5], neural stem cells surface antigens by flow-cytometry, which commonly (NSCs) [6] and mesenchymal stem cells (MSCs) [7]. used in the characterization of rat BMSCs populations In contrast to ESCs and NSCs, MSCs can be derived [22]. Rat BMSCs expressed CD29, CD44 and CD90 and from the patient’s own bone marrow and easy to isolate. were negative for CD45 and CD11b (Figure 1). So, bone marrow derived MSCs (BMSCs) represent a potential source for autologous cell transplantation Effects of combined use of LXR agonist with GF in avoid ing or reducing immunological rejections. on differentiation of rat BMSCs into DA neurons BMSCs usage for CRT circumvents the ethical problems concerning fetal tissue usage, thus makes itself attractive Immunocytochemistry was performed to evaluate for regenerative medicine research [8]. Much effort has the expressions of the neuronal markers (Tuj1 and Nestin) been laid to compare the effect of naive MSCs and MSCs and the specific marker of DA neurons (TH). DA neurons differentiated into tyrosine hydroxylase (TH)-positive are classically identified by expression of TH, the rate- cells on transplanting in PD animal models. Although limiting enzyme in the biosynthesis of DA [23]. both naive and differentiated MSCs evoked behavioral recovery, the effect of the differentiated cells was more Determination for the adding time of LXR pronounced [2, 9–11]. agonist Differentiation of BMSCs can be induced by exposure to growth factors (GF) [7], chemical inducers Almost cells in control group probed with TH and [12] and also by lentiviral transduction of DA transcription Tuj1 antibodies were negative and only show the nuclear factors [13]. By means of in vitro manipulation with stain DAPI. Cells in LXR+GF group displayed merged growth factors (sonic hedgehog and fibroblast growth images of double staining with Tuj1 (dylight 488, green) factors, SHH and FGFs), Trzaska KA et al. succeeded and TH (dylight 549, red). Colocalization was identified by in inducing adult human BMSCs to DA neurons with the yellow fluorescence in the merged images. Expressions an 67% of efficiency in 12 days, which is the highest of Tuj1 and TH reached the peak at simultaneous addition induction efficiency among the previous research reports of LXR agonist and GF (Day 0). Accompanying with [7]. However, the diverse differentiation methods, low the adding time of LXR agonist delayed, expressions of production rate, and low survival rate after transplantation Tuj1 and TH were decreased gradually. Thus, the optimal are still obstacles that need to be overcome before adding time of LXR agonist was at Day 0 (Figure 2). the application of BMSCs to treat PD [14] and the mechanisms involved in differentiation of BMSCs is not Determination for induction period of LXR yet well understood. agonist The liver X receptors (LXR α and LXR β, encoded by NR1H3 and NR1H2, respectively) are ligand-dependent Cells in GF group and LXR+GF (Day 0) group transcription factors belonging to the nuclear hormone revealed the appearance of extended long cellular receptor superfamily [15, 16]. The most well-known processes and retracted cell bodies, which were the typical functions of LXR are to regulate lipid metabolism and neuronal morphology, and the amount of cells in LXR+GF homeostasis [17, 18], increase cholesterol efflux from (Day 0) group were increased and later decreased, and the cells and protect from cholesterol overload and reached the peak at day 6 in the bright-field images toxicity [19]. LXR α/β double knockout mice showed a (Figure 3A). The growth rate of cells in GF group and progressive accumulation of lipids in the brain and loss LXR+GF (Day 0) group reached the peak at day 6 and of adult spinal cord motor neurons and ventral midbrain the growth rate of cells in LXR+GF (Day 0) group was (VM) DA neurons [20, 21]. These researches suggested consistent with images in the bright-field (Figure 3B). that LXR is highly correlated to the development of DA Cells in GF group and LXR+GF (Day 0) group neurons in vivo. However, it remains unknown whether displayed merged images of double staining with LXR have effect on differentiation of adult rat BMSCs Tuj1 (dylight 488, green) and TH (dylight 549, red). into DA neurons. Colocalization was identified by the yellow fluorescence The present study was designed to investigate the in the merged images. Expressions of Tuj1 and TH were effect of LXR agonist (TO901317) on the differentiation of gradually increased in GF group and maximal expressions rat BMSCs into DA neurons and its possible mechanism. were obtained at 12 days (Figure 4). Thus, the time for www.impactjournals.com/oncotarget 577 Oncotarget induction period of GF was 12 days and GF group is evaluate the amount of TH positive neurons. Almost cells GF-treated-12day group for short. Expressions of Tuj1 in control group probed with TH and Tuj1 antibodies were and TH reached the peak at day 6 in LXR+GF group. negative and only show the nuclear stain DAPI (according Thus, the time for induction period of LXR agonist was to Figure 3C). Compared with control group, of a total of 6 days (Figure 3C&D) and it was increased significantly Tuj1 positive cells counted, 62.61 ± 2.334% of these cells compared with those of GF group (#P < 0.05 and ##P were positive for TH (according to Figure 3C) and the < 0.01, respectively). Compared with control group, amount of TH positive neurons was significantly increased expressions of Tuj1 and TH were increased significantly in GF group (**P < 0.01). Compared with GF group, of a in GF group (**P < 0.01). total of Tuj1 positive cells counted, 87.42 ± 7.573% of these cells were positive for TH (according to Figure 3C) Determination for the concentration of LXR and the amount of TH positive neurons was significantly agonist increased in LXR+GF group (##P < 0.01) (Figure 7).