A Novel Protocol to Differentiate Induced Pluripotent Stem Cells by Neuronal microRNAs to Provide a Suitable Cellular Model

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Received Date : 23-Aug-2014 Revised Date : 15-Nov-2014 Accepted Date : 17-Nov-2014 Article type

: Research Article

A Novel Protocol to Differentiate Induced Pluripotent Stem Cells by Neuronal microRNAs to Provide a Suitable Cellular Model Mehrak Zare 2.5, Masoud Soleimani 3, Abolfazl Akbarzadeh6 , Behnaz Bakhshandeh4, Seyed Hamid Aghaee-Bakhtiari2, Mozhdeh Mohammadian8, and Nosratollah Zarghami 1,7*

1- Department of Clinical Biochemistry and Laboratory, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 2- Department of Cellular Biology, Stem Cell Technology Research Center, Tehran, Iran 3- Department of Haematology, School of Medicine, Tarbiat Modares University, Tehran, Iran 4- Department of Biotechnology ,College of Science, University of Tehran, Tehran, Iran 5- Neurosciences Research Center ,Tabriz University of Medical Sciences, Tabriz, Iran 6- Department of Medical Nanotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran

Correspondence: Nosratollah Zarghami, Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: 984133355788. Fax: 984133355789. E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12485 This article is protected by copyright. All rights reserved.

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7- Department of Clinical Biochmemistry, Radiopharmacy Lab, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 8- Amol Faculty of Paramedical Sciences, Mazandaran University of Medical Sciences, Sari, Iran.

Abstract Neurodegenerative diseases are one of the most challenging subjects in medicine. Investigation of their underlying genetic or epigenetic factors is hampered by lack of suitable models. Patient-specific induced pluripotent stem cells (iPS cells) represent a valuable approach to provide a proper model for poorly understood mechanisms of neuronal diseases and the related drug screenings. miR-124 and miR-128 are two brain-enriched miRNAs with different time points of expression during neuronal development. Herein we transduced human iPS cells with miR-124 and miR-128 harboring lentiviruses sequentially. The transduced plasmids contained GFP and puromycin antibiotic resistant genes for easier selection and identification. . Morphological assessment and immunocytochemistry (overexpressions of beta-tubulin and neuron specific enolase) confirmed that induced hiPScells by miR124 and miR128 represent similar characteristics to those of mature nuerons. In addition, the upregulation of neuron specific enolase, beta-tubulin, Map2, GFAP and BDNF were detected by quantitative real-time PCR. In conclusion, it seems that our novel protocol remarks the combinatorial effect of miR-124 and miR-128 on nueral differentiation in the absence of any extrinsic factor. Moreover such cellular models could be used in personalized

drug screening and applied for more effective therapies.

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Key words: Neuronal Differentiation, iPS cell, miR-124, miR-128, cellular model

1

Introduction

Neurodegenerative diseases result from progressive deterioration, extensive loss and dysfunction of neurons in the nervous system [1, 2]. The healing in nervous system is limited; so specific strategies are required to manage the neurological defects [3]. Non-human models cannot simulate the complexities of the human nervous system. [4]. In neurology, cellular modeling studies have resulted in generating in vitro disease models as well as large populations of healthy neurons to explore the therapeutic potential of transplantation[5, 6]. Ectopic expression of defined factors (Oct3/4, Sox2, Klf4, c-myc, Nanog or Lin28), leads to reprogram adult somatic cells into pluripotent cells which make it possible to generate patient-specific induced pluripotent stem cells (iPS cells) for autologous cell therapies [7, 8]. Similar to ESCs, iPS cells could differentiate into all three germ layers without ethical restrictions and immune rejection [9-11]. Numerous protocols have described successful generations of neuronal models using differentiation of pluripotent cells; embryoid body formation, stromal feeder co-culture, or selective survival conditions [4, 12, 13]. In this study we focused on human iPS cell to provide a neuronal suitable model. There is interesting evidence that dysregulation of microRNAs (miRNAs) networks is involved in the onset and development of neurodegenerative diseases [1, 14]. miRNAs as small non-coding RNAs, regulate the gene expression by translational repression or mRNA degradation using partial complementarity of the miRNA/mRNA duplex [15-17]. miR-124, known as a brain-enriched miRNA, first rose to fame when it was transfected into non-neuronal HeLa cells and resulted in a shift in the expression profile toward brain genes

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[18,19]. Being highly conserved from worm to human [20], miR-124 promotes neuronal differentiation of committed neuronal precursors [21]. During neurogenesis, miR-124 expression is very low in neuronal progenitors while its increasing expression is detected in mature neurons [22]. In neurogenesis of embryonic stem cells, miR-124 was expressed at late neural precursor stage and reaches maximum level in day5 of neural differentiation stage [23]. Moreover this microRNA helps maintain the gene expression profile of the brain by repressing unwanted transcripts [24]. miR-128, as another brain-enriched miRNA, plays important roles in migration and maturation of neurons [25]. Mature miR-128 transcripts are increasingly detected during brain development in vivo with close association with neuronal differentiation [26]. A research group observed that overexpression of miR-128 inhibited proliferation of glioma cells [27]. Therefore, we selected mir-124 and mir-128 for neurogenic induction in iPS cells. With the aim of providing a cellular model for drug screening and simulating neurodegenerative diseases, we produced human iPS cells and induced neuronal differentiation in them by mir-124 and mir-128 artificial overexpression.

2 2.1

Materials and Methods Cell Culture

The iPS cells (a gift from Stem Cells Technology Research Center (Tehran, Iran), supplementary Doc. 1)[9] were cultured on previously mitomycin-inactivated SNL cells, in a 60mm culture dish coated with gelatin 0.1%. They were maintained in DMEM F12 (Gibco) supplemented with 15% ES (Gibco) (v/v), 1% non-essential amino acids (Sigma), 4 ng/ml bFGF (peprotech) and 1mM L-glutamine (Gibco) with every 5 day passaging. In order to investigate any spontaneous differentiation, iPS cells dissociated by collagenase IV (Gibco) 1mg/ml and diluted in iPS medium lacking bFGF. 25 µL drops (each containing

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approximately 150 cells) were placed on the inside of a petri dish lid. Two days later the embryoid bodies (EBs) were plated in 6-well non-adherent petri dishes and cultured for 2 more days in suspension medium. On fifth day, the EBs in DMEM F12 supplemented with 5% FBs were transferred to 0.1% gelatin coated plates. 2.2

Lentivirus Production and Titration

To produce lentivirus particles each of the miRNA harboring plasmids pLentiIIImiR-124GFP or pLentiIIImiR-128GFP and pLentiIII-GFP backbone (abm) were cotransfected to the Hek293 cells with psPax as packaging plasmid and PMD2G as an envelope plasmid using calcium phosphate transfection method. After 16 hours, culture medium was replaced with fresh DMEM and 10% FBS. Then the supernatant (containing viral particles) was collected 2 times with 24 hours intervals. The viral supernatant was then filtered through 0.45 µm membranes and mixed over night with 5% PEG-8000 and 0.15 M NaCl by mild shaking. The next day, viruses were centrifuged at 4100 g for 10 minutes and suspended in EB medium. The viral titration was performed using serial dilutions of HEK transfected cells and subsequent FACS analysis for GFP expression evaluation. 2.3

Neuronal Differentiation

Hanging drop method was used for sequential neuronal-miRNA transduction except that in each 25 µL drop, the concentrated virus particles in EB medium for pLentiIIImiR-124GFP or pLentiIIImiR-128GFP or pLentiIII-GFP backbone (abm) with the MOI of 30, were added to the iPS cells which led to transduction during EBs formation. In details, on first day virus containing pLentiIIImiR-124GFP was added to the drops of iPS cells; on fifth day, transduced cells were selected by 1 µg/ml puromycin supplementation. On 8th day, DMEM F12 medium supplemented with 5% FBS and 10⁵ pLentiIIImiR-128GFP virus particles, for each iPS clone (approximately 4000 cells), was added to the cells; after 8 day culture, iPS cells were analyzed for neuronal differentiation. This article is protected by copyright. All rights reserved.

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2.4

Real time PCR

Total RNA of iPS cells was extracted using Trizol reagent (Invitrogen). cDNAs were generated using Vivantis reverse transcriptase. The specific primers were designed for neuronal marker genes; NSE, beta-tubulin, Nestin, Map2, GFAP and BDNF (table 1). Gene expressions were normalized to β-actin for neuronal genes and to SNORD for miRNAs as internal controls. Real Time PCR was performed on RotorGene6000 (Corbett, Australia). 2.5

Immunocytochemistry

The cells were rinsed twice with PBS and fixed with 4% paraformaldehyde (in PBS) for 20 minutes in 4°C and 10 more minutes in room temperature. Since both neuronal markers (NSE and beta-tubulin) are intracellular, after 2 time washing, the cells were permeabilized with 0.1% triton x-100 for 10 minutes. Goat serum 5% was used as blocking solution for 1 hour in room temperature. Then cells were incubated overnight in 4 °C with appropriate dilution of first antibodies (beta-tubulin 1:50, NSE 1:100, both from santa cruiz bio) in 0.2% PBS/BSA. Afterward, the cells were incubated with second antibody (PE conjugated goat anti-mouse IgG, SCB) in PBS containing 0.1% BSA for 1 hour at 37°C. Nuclei were stained with DAPI (1 µ/ml) for 1 minute at room temperature. 2.6

Statistics

The student t-test or one way ANOVA were used for comparison of two or more than two groups, respectively. P value < 0.05 was considered as significantly different in all cases. All experiments were done in triplicate, unless otherwise stated and data were shown as mean ± standard deviation (SD).

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3 3.1

Results Quantitative Evaluation of mir-124 and mir-128 Expression

As illustrated in figure 1, on day 16,the amounts of mir-124 and mir-128 in induced iPS cells were both elevated significantly compared to un-transduced self-differentiated iPS cells . The miRNA over expression observed near 20 fold for miR124 and about 5 fold for miR128 suggesting the stable gene transfer after 16 days of differentiation. Little amount of microRNAs were expressed intrinsically by intact iPScells . . 3.2

Morphological Changes of Differentiating iPScells

Since the plasmids contained GFP-gene marker, GFP was expressed along with the miRNA transcription; so by fluorescent microscopy, the morphological alterations of the iPS cells were investigated. As shown in figure 2, on 4th day, The fully green spherical shapes in suspension indicates EB formation and well transduction. When EBs flattened and attached to dishes, some untransducted cells appear on day 5 . The comparison of the shininess of the bodies in day 5 (before antibiotic application) and day 8 (after antibiotic application) clearly represented this deletion and enrichment of transduced cells. On day 10, rosette-like structures were obvious. (Figure 2). In addition, from day 12, more differentiated cells with distinct out-growth neurites were observable.

3.3

Transcriptional Evaluation of Neuronal Markers

The transcription of some markers (NSE, beta-tubulin, Nestin, Map2, GFAP and BDNF) was quantitatively evaluated in four groups: mir-124-transduced cells, mir-128-transduced cell, both mir-124 and mir-128 transduced cells and backbone-plasmid transduced cells (selfdifferentiated ones) on day 16 of culture. As presented in figure 3, almost all of the neuronal This article is protected by copyright. All rights reserved.

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markers, were up regulated significantly in miRNAs-transduced cells. Interestingly Nestin was up regulated in self-differentiated cells whilst this marker was down-regulated in mirtransduced cells significantly. The reduction in Nestin expression as a progenitor cell marker reminds passing the stemness fate in miRNA transduced cells which could be observed in spontaneous neural differentiated control group. GFAP had a mild expression in all three groups. Although compare d to beta Tubulin its expression was much lower. It may concern the existence of the astrocytes in our cell population.

3.4

Translational Evaluation of Neuronal Markers Immunocytochemichal analysis performed to verify whether coexpression of miR-124 and

miR-128 could promote neural characteristics in iPSCs. Immunofluorescence staining using anti beta-tubulin showed that out growth neurites obviously become longer and more detectable in bitransducted cells comparing to single miR124 and untransdused iPScells. The beta-III tubulin protein extending through the microtubule projections represent nueral like cells. (Figure 4)To confirm this data the expression of a cytoplasmic neural protein NSE was also investigated. The anti-NSE staining indicated the positive cells for this neural marker just in miR-124 and miR128 transduced group. No staining was observed in the iPScells overexpressing miR124 and the untransduced group. (figure5)The immunoreactivity of beta-tubulin and NSE were consistent with their up regulation in transcriptional level.

4

Discussion

Establishment of neuronal cellular model helps to address critical issues such as i) the possibility to better study human neurogenesis in vitro; ii) the creation of neurons from the undifferentiated stem cells that could provide a continuous supply of these precious and hardto-come-by cells; iii) novel drug discovery testing for regenerative medicine, and the study of

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human neuronal disease. miRNAs are all expressed endogenously and are supposed to be safe for normal cells [28]. Therefore any neurogenic induction protocol, based on RNA, is safer and thus, more applicable in clinical treatments than the protocols based on growth hormones. MiR-124 and miR-128 are two brain-enriched miRNAs with different expression time points during neuronal development [4, 21, 25]. The aim was to provide a cellular model for neuronal drug screening and simulation of neuronal disorders that can be obtained by further complementary electrophysiological evalations. In this study human iPS cells were transduced sequentially with miR-124 and then with miR-128 harboring lentiviruses in order to evaluate the ability of these miRNAs in neuronal differentiation of iPS cells. The ordered transduction was designed because of the time points of mir-124 and mir-128 upregulations in neuronal development [21]. Koehler et al reported that extended passaging increased the efficiency of neuronal differentiation in iPS cells neurons due to their stability after sufficient divisions [29]. Hence, in this study the iPS cells of 15th passage were utilized. Since EBs have spherical shapes with masked inner cells, ectopically lentiviral transduction by hanging drop method during EBs formation ensured us about the exposure of most of the cells to lentiviral particles. On day 16, quantitative evaluation of mir-124 and mir-128 confirmed their overexpression in the transduced cells compared to control (selfdifferentiated cells) (figure 1). Investigation of the GFP marker gene in the backbone plasmid represented the rosette-like structures (day 10) and distinct out-growth neurites (day 16) in transduced iPS cells (figure 2). Moreover puromycin selection helped to enrichment of transduced cells which was validated by shinier EBs on day 8 (after antibiotic application) in comparison to day 5 (before antibiotic application).

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We evaluated neuronal markers in miR-transduced cells both transcriptionally and translationally. Quantitative evaluations of neuronal markers in transcription level in four groups (mir-124-transduced cells, mir-128-transduced cell, mir-124 and mir-128 transduced cells and self-differentiated cells) were shown in figure 3 that illustrated the significant upregulations. The size of the colonies and thus, the cell number was not changed significantly after miR128 transduction; meanwhile BDNF was down regulated after miR128 transduction. In order to explain these findings, the role of mir-128 in cell proliferation through NTRK3 regulation (a receptor for nuerotrophin family) could be mentioned. Moreover, proliferation reduction of neural cells maybe caused by Bcl2 upregulation by miR128 overexpression which arrests the cells in G0 phase of cell cycle [30]. The highest level of beta-tubulin was seen in combinatorial transduction of miR-124 and miR-128, which demonstrated the synergism effect of these two miRNAs on beta-tubulin transcription. This finding was in congruence with immunocytochemistry result of betatubulin (figure 4). The clear out-growth neurites in co-transduced mir-124 and mir-128 suggests a posterior neuronal maturation stage. Furthermore, immunostaining illustrated the cell connections which are required for more effective differentiation. In addition, over expression in NSE, as another neuronal marker, in co-transduced mir-124 and mir-128 cells on day 16 is represented in figure 5. To investigate the self-tendency of iPS cells, we examined the self-differentiated ones that were cultured for 16 days with back-bone plasmid transduction (figure 3). Although the GFAP (a glial marker) was overexpressed in all groups but its overexpression in selfdifferentiated ones was dramatically high. The variations in neuronal differentiation capacity of iPS cells [31] could explain the upregulation of GFAP in them. Possibly GFAP+ population could provide a more resemble micro-environment for neuronal differentiation.

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Figures legends

Figure 1. Relative expression of mir-124 and mir-128 in bitransduced and untransduced iPSCs on day 16 of culture. Snord was evaluated as internal miRNA (control). All data were normalized to day 1 of culture.Data were reported as mean±SD (n=3); *p