Correlation between receptor-interacting protein 140 expression and directed di ff erentiation of human embryonic stem cells into neural stem cells

Zhu-ran Zhao, Wei-dong Yu, Cheng Shi, Rong Liang, Xi Chen, Xiao Feng, Xue Zhang Qing Mu Huan Shen, Jing-zhu Guo

1 Department of Pediatrics, Peking University People’s Hospital, Beijing, China

2 Institute of Clinical Molecular Biology, Peking University People’s Hospital, Beijing, China

3 Department of Obstetrics and Gynecology, Peking University People’s Hospital, Beijing, China

4 Department of Pediatrics, Peking University International Hospital, Beijing, China

Correlation between receptor-interacting protein 140 expression and directed di ff erentiation of human embryonic stem cells into neural stem cells

Zhu-ran Zhao1,#, Wei-dong Yu2,#, Cheng Shi3, Rong Liang3, Xi Chen3, Xiao Feng4, Xue Zhang1, Qing Mu1, Huan Shen3, Jing-zhu Guo1,＊

1 Department of Pediatrics, Peking University People’s Hospital, Beijing, China

2 Institute of Clinical Molecular Biology, Peking University People’s Hospital, Beijing, China

3 Department of Obstetrics and Gynecology, Peking University People’s Hospital, Beijing, China

4 Department of Pediatrics, Peking University International Hospital, Beijing, China

How to cite this article:Zhao ZR, Yu WD, Shi C, Liang R, Chen X, Feng X, Zhang X, Mu Q, Shen H, Guo JZ (2017) Correlation between receptor-interacting protein 140 expression and directed di ff erentiation of human embryonic stem cells into neural stem cells. Neural Regen Res 12(1):118-124.

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Graphical Abstract

RIP140 involves neural di ff erentiation of human embryonic stem cellsviathe ERK1/2 signaling

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orcid: 0000-0003-1720-8927 (Jing-zhu Guo)

Overexpression of receptor‐interacting protein 140 (RIP140) promotes neuronal di ff erentiation of N2a cellsviaextracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural di ff erentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural di ff erentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlat‐ed with stem cell markers Oct4 and Sox2 during early stages of neural di ff erentiation, and positively correlated with the neural stem cell marker Nestin during later stages.us, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural di ff erentiation to eventually a ff ect the number of neurons produced.

nerve regeneration; receptor-interacting protein 140; neural stem cells; human embryonic stem cells; directed di ff erentiation; Oct4; Sox2; Nestin; extracellular regulated kinase 1/2 signaling pathway; neural regeneration

Introduction

During neurogenesis, neural di ff erentiation is controlled by a selective spatiotemporal gene expression pro fi le produced by switching expression of di ff erent genes on or o ff. Tempo‐ral expression of neural di ff erentiation markers is the foun‐dation of normal nervous system function. Abnormal neu‐ronal di ff erentiation, such as occurs with Down’s syndrome or Alzheimer’s disease, can lead to an exceptional number, density, morphology, and/or connection of neurons and syn‐apses, as well as a low ratio of neural to glial cells and changes neural network building, resulting in a series of abnormal clinical manifestations involving the central nervous system (Greber et al., 1999; Martin et al., 2012; Moya‐Alvarado et al., 2016). It has been demonstrated that Oct4, Sox2 and Nanog are core factors for stem cell maintenance and self‐renewal (Niwa, 2007). As changes in the expression of these genes areclosely related to stem cell differentiation (Wu et al., 2014), their expression can be used to identify stages of neural dif‐ferentiation. In addition, Notch, Wnt, repressor element‐1‐si‐lencing transcription factor/neuron restrictive silence factor, sonic hedgehog, extracellular regulated kinase 1/2 (ERK1/2), and phosphatidyl inositol 3‐kinase/protein kinase B pathways simultaneously play important roles in neural di ff erentiation (Li et al., 2007, 2008; Canzonetta et al., 2008; Woo et al., 2009; Topol et al., 2015; Yan et al., 2015; Gao et al., 2016).

Receptor‐interacting protein 140 (RIP140, also known as NRIP1) is a negative regulatory transcription factor locat‐ed in the nucleus. RIP140 participates in the regulation of retinoic acid, thyroid hormones, glucocorticoids and other hormones (Subramaniam et al., 1999; Heim et al., 2009; Park et al., 2009). Our previous studies confirmed that RIP140 exhibits a dynamic spatiotemporal expression pattern during normal murine brain development (Li et al., 2007). Mean‐while, RIP140 overexpression promotes mouse neuroblasto‐ma cell di ff erentiation (Feng et al., 2015), while the RIP140/ LSD1 complex fi ne‐tunes the neural di ff erentiation marker Pax6 during mouse neuronal differentiation (Wu et al., 2016). However, expression of RIP140 and its potential in‐volved in directed di ff erentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs) remains unclear. In this study, we investigated the spatiotemporal expression of RIP140 and its correlation with NSC markers during direct‐ed di ff erentiation of hESCs.

Materials and Methods

Cell culture

The hESC line H1 was obtained from Peking University School of Life Sciences (Beijing, China) and cultured in Dul‐becco’s Modified Eagle’s Medium (DMEM)/F12 (Hyclone, Logan, UT, USA) supplemented with 20% KnockOutTMSe‐rum Replacement (Invitrogen, Carlsbad, CA, USA), 2 mM GlutaMAXTM(Invitrogen), 100 U/mL non‐essential amino acids, 100 U/mL penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA), 0.55 mM β‐mer‐captoethanol, and 20 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA). hESCs were grown on a feeder cell layer of mouse embryonic fi broblasts, purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) and treated with 0.01 mg/mL of mitomycin C (Roche, Basel, Switzerland), and maintained in a 5% CO2humidi fi ed incubator (ermo) at 37°C. Mouse embryonic fi broblasts were maintained in DMEM/high glucose media supplemented with 10% fetal bovine serum (Gibco), 100 U/ mL non‐essential amino acids, and 100 U/mL penicillin/ streptomycin.

In vitrodirected neural di ff erentiation

High‐quality hESCs (with minimal or no differentiated colonies) were cultured on Matrigel®(Becton Dickinson, Franklin Lakes, NJ, USA). When hESCs reached 70—80% confluency, clones were dislodged with Dispase II (Roche) to generate cell clumps, which were then plated in Matri‐ gel‐coated 6‐well plates at a density of 2.5—3 × 105hESCs per well. One day after hESC plating, culture medium was replaced with 2.5 mL of pre‐warmed complete PSC Neural Induction Medium (Gibco). Medium was refreshed every other day and the amount was doubled on days 4 and 6 of neural induction. Non‐neural differentiated cells were re‐moved with a Pasteur glass pipette (Corning Incorporated, Corning, NY, USA). Cells were collected on days 0, 3.5, and 7 of neural induction from both control (cultured in normal growth medium containing maintenance growth factors) and differentiation (cultured in neural induction medium containing directed induction factors) groups. Images of cell morphology were acquired using a Nikon Eclipse TE300 In‐verted Microscope (Sendai, Japan).

Real-time polymerase chain reaction (PCR)

Total RNA was isolated from cell lines using an RNeasy®Mini Kit (Qiagen, Hilden, Germany). cDNA was reverse transcribed from 0.5 μg of RNA using ReverTra ACE®qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Ja‐pan). Real‐time PCR was performed using SYBR®Green PCR Master Mix (Applied Biosystems, Grand Island, NY, USA) in a Bio‐Rad Opticon®2 Real Time PCR System (Her‐cules, CA, USA). Cycling conditions were as follows: 94°C for 2 minutes; 40 cycles of 94°C for 20 seconds, 60°C for 20 seconds, and 72°C for 30 seconds (Feng et al., 2015). Primer pairs were designed as described in Supplementaryable 1 (Woo et al., 2009, Birket et al., 2011). Reaction speci fi city was con fi rmedviamelting curve analysis.e housekeeping gene β‐actin was used as an internal standard. Fold‐changes between gene expression in control and differentiated cells were determined using the ΔΔCt method. All experiments were conducted at least in triplicate.

Immuno fl uorescence analysis

Cells were placed on glass coverslips (Fisher, Pittsburgh, PA, USA) and then fi xed in 4% paraformaldehyde in phos‐phate‐bu ff ered saline (PBS). Cell membranes were permea‐bilized with 0.2% Triton X‐100, washed with PBS and then blocked with 5% goat serum (Applygen, Beijing, China). Samples were then incubated with primary antibodies against Oct4 (1:200; Rabbit mAb; Sigma Aldrich, St. Louis, MO, USA), Sox2 (1:150; Mouse mAb; Sigma), Nestin (1:200; Rabbit mAb; Sigma), and/or RIP140 (1:50; Rabbit mAb; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Oct4 and Sox2 are markers for stem cells, while Nestin is a marker for NSCs. Following primary antibody incubation, samples were washed with PBS and incubated with rhodamine‐ or FITC‐conjugated goat anti‐rabbit IgG antibody (1:100; Millipore, Billerica, MA, USA) at 37°C for 2 hours protected from light. Aer additional washes, nu‐clei were counterstained with 4′,6‐diamidino‐2‐phenylindole (Beyotime, Shanghai, China). Images were acquired with an Olympus IX70 Inverted Fluorescence Microscope (To‐kyo, Japan). All experiments were conducted in triplicate and quanti fi ed in a blind manner. Corresponding quanti‐tative results are described in Supplementaryable 2.

Figure 1 Morphologic changes of cell clones.

Figure 2 Expression of neural di ff erentiation markers changed dynamically during neural induction from day 0 to day 7 of hESCs.

Figure 3 RIP140 expression increased during directed di ff erentiation of hESCs.

Figure 4 p-ERK1/2 expression increased during directed di ff erentiation of hESCs.

Western blot assay

Protein lysates were obtained by scraping and subjecting cells to 8% sodium dodecyl sulfate‐polyacrylamide gel electro‐phoresis (Sigma), and then transferring onto nitrocellulose membranes (ermo). Membranes were blocked in a TBST solution consisting of 20 mM Tris‐HCl (pH 7.5), 137 mM NaCl, and 0.05% TWEEN®20, containing 5% non‐fat milk powder for 1 hour at 25°C, then incubated with antibodies against RIP140 (1:500; Rabbit mAb; Santa Cruz Biotechnol‐ogy) or phosphorylated ERK1/2 (p‐ERK1/2, 1:500; MousemAb; Cell Signaling Technology, Danvers, MA, USA) or β‐actin (1:1,500; Mouse mAb; Cell Signaling Technology) overnight at 4°C. After TBST washes, membranes were incubated with horseradish peroxidase‐conjugated goat anti‐rabbit or anti‐mouse IgG antibodies (1:2,000; Cell Signaling Technology) for 1 hour at 25°C. Protein expres‐sion was subsequently detected and visualized using an Enhanced Chemilumenescence substrate (ermo) (Feng et al., 2015). Results were quanti fi ed by ImageJ 2× soware (National Institutes of Health, Stapleton, NY, USA). All experiments were conducted in triplicate.

able 1 Correlation between RIP140 and markers for neural di ff erentiation

able 1 Correlation between RIP140 and markers for neural di ff erentiation

RIP140: Receptor‐interacting protein 140.

Days 0—7 Oct4 –0.836 0.005 ＜ 0.05 Days 0—7 Sox2 –0.931 ＜ 0.001 Days 0—7 Nestin 0.410 0.272 Days 0—3.5 Oct4 –0.833 0.039 ＜ 0.05 Days 0—3.5 Sox2 –0.847 0.033 ＜ 0.05 Days 0—3.5 Nestin –0.480 0.336 Days 3.5—7 Oct4 –0.395 0.439 Days 3.5—7 Sox2 –0.902 0.014 ＜ 0.05 Days 3.5—7 Nestin 0.984 ＜ 0.001

Statistical analysis

Data are expressed as the mean ± standard deviation. Statis‐tical analyses were performed using SPSS 20.0 soware (IBM, Armonk, NY, USA). Statistical significance of differences between groups was determined by one‐way analysis of vari‐ance followed by a Dunnett’spost-hoctest. Correlations were analyzed using Pearson correlation analysis.Pvalues of less than 0.05 were considered statistically signi fi cant.

Results

Morphological changes of cell clones during directed neural di ff erentiation of hESCs into NSCs

During directed differentiation, circular cell clones be‐came irregular and reached con fl uency later.e edges of cell clones developed one or more branches. Scattered cells were observed to have oval, triangular, or stellate shapes, thus presenting NSC‐like morphological characteristics (Figure 1).

Spatiotemporal expression of neural markers on days 0, 3.5, and 7 of neural induction

At the mRNA level, relative expression of Oct4 and Sox2 ex‐hibited decreasing trends over days 0, 3.5, and 7 in the di ff er‐entiated group (Figure 2A, B). Nestin expression decreased slightly from days 0 to 3.5, and then increased sharply in the differentiation group (Figure 2C). All three markers were significantly different in the differentiation group compared with the control group by day 7 and thereaer. Expression of Oct4 (P= 0.003) and Sox2 (P= 0.021) was lower, while Nestin (P= 0.001) was higher in the di ff eren‐tiation group. At the immunoreactive level, Oct4 showed high fl uorescence intensity on day 0, but was barely visible on days 3.5 and 7, representing a decreasing trend (Figure 2D). However, the fl uorescence intensity of Sox2 did not substantially change (Figure 2E). Nestin increased dra‐matically on day 7, coinciding with real‐time PCR results (Figure 2F). While Oct4 and Sox2 localized to the nucle‐us, Nestin localized to the cytoplasm during neural induc‐tion (Figure 2G—I). Changes in marker expression in the control group are presented in Supplementary Figure 1A; whereas, localization patterns are outlined in Supplementary Figure 2A.

RIP140 increased during neural induction

RIP140 mRNA (Figure 3A) and fluorescence intensity (Figure 3B) gradually increased with prolonged induction time. Meanwhile, a significant increase in RIP140 mRNA expression was observed in the di ff erentiation group on day 7 compared with the control group (P＜ 0.001). Levels of RIP140 protein, which localized to the nucleus throughout neural di ff erentiation (Figure 3C and Supplementary Figure 2), also increased as neural differentiation progressed (Figure 4A, C).

Expression of RIP140 and neural di ff erentiation markers were highly correlated at di ff erent phases of directed neural di ff erentiation

Throughout neural differentiation, RIP140 expression showed a strongly negative correlation with that of Oct4 and Sox2 (P＜ 0.05 andP＜ 0.001, respectively), but had no significant correlation with Nestin expression (P＞ 0.05). During early differentiation (days 0—3.5), expression of RIP140 exhibited a strongly negative correlation with Oct4 and Sox2 (P＜ 0.05); moreover, during late differentiation (days 3.5—7), RIP140 exhibited a strongly negative correla‐tion with Sox2 expression (P＜ 0.05) and strongly positive correlation with Nestin expression (P＜ 0.001;able 1).

RIP140 may a ff ect directed di ff erentiation of hESCs into NSCsviathe ERK1/2 pathway

Increasing evidence has pointed toward an important role for ERK1/2 activation during neuronal differentiation ((Li et al., 2006; Chan et al., 2013).us, we investigated levels of phosphorylated ERK1/2 (p‐ERK1/2) expression during directed di ff erentiation of hESCs into NSCs. Interestingly, an increasing trend in ERK1/2 phosphorylation was observed to mirror that of RIP140 (Figure 4A, B).

Discussion

Directed neural differentiation is essential during neuro‐genesis. Alterations in neural differentiation can lead to an aberrant number of neurons, resulting in the formation of abnormal cortical circuits that contribute to cognitive de fi cits such as those seen in Down Syndrome (Yabut et al., 2010; Haydar and Reeves, 2012).

Temporal expression of stem cell and neural markers, such as Oct4, Sox2, Nestin, Pax6, and Nanog, determines the phase of directed neural di ff erentiation and serves as an es‐tablished standard of inductionin vitro(Wu et al., 2014). As such, induction models can be used to detect the expression and correlation between RIP140 and other genes in directed di ff erentiation from hESCs into NSCsin vitro.

RIP140 is an auxiliary adjustment factor for nuclear receptor transcription. Initial studies of RIP140 primarily concentrated on aspects of energy metabolism and the female reproductive system (Nautiyal et al., 2013). How‐ever, our recent studies have suggested that RIP140 has a close relationship with neural differentiation induced by retinoic acid, as RIP140 promoted mouse neuroblastoma cell (N2a) di ff erentiationviathe ERK1/2 pathway, there‐by leading to stem cell depletion, a reduction of nerve cells, and subsequent participation in a series of abnormal phenotypes during central nervous system development (Feng et al., 2014, 2015). A previous study also indicated that the RIP140/LSD1/Pit‐1 complex could repress Pax6 expression, thereby participating in retinoic acid‐induced neuronal differentiation of mouse embryonic stem cells (mESCs) (Wu et al., 2016). We established that increased RIP140 expression strongly correlated with neural di ff er‐entiation markers during early neural induction.us, we hypothesized that RIP140 might also have an important role in directed neural di ff erentiation of human cells. Use of human stem cells as a model of neural differentiation more faithfully recapitulatesin vivoneurogenesis than use of mESCs or cancer cells. However, during different phases of induction, RIP140 might correlate with di ff erent markers via various pathways. ERK1/2 pathway has been demonstrated to play a signi fi cant role in neural di ff eren‐tiation (Lim et al., 2008; Hosseini Farahabadi et al., 2015, Hu et al., 2016). Meanwhile, we observed an increasing trend of ERK1/2, which may be a key pathway in N2a neu‐ral differentiation (Feng et al., 2015), and found similar variations in this study; namely, increased expression of p‐ERK1/2 was also observed during di ff erentiation.us, we concluded that ERK1/2 may also be a critical pathway in directed di ff erentiation of hESCs.

Compared with animal cell models, hESCs are able to overcome species‐speci fi c di ff erences and, thus, more close‐ly reflect RIP140 expression trends during normal human embryonic development. However, to determine the exact functions and mechanisms involved in directed neural dif‐ferentiation, hESC models of RIP140 overexpression, knock‐down and/or knockout are needed. Moreover, improvement in the extremely low e ffi ciency of establishing hESC lines is urgently needed.

Taken together, our data support the participation of RIP140 in directed differentiation of hESCs into NSCs. We hypothesize that this may be mediated through the ERK1/2 pathway. Establishing normal expression patterns is the goal of studying the function of RIP140 and other genes, as this information provides a theoretical basis for diagnosis of neurodevelopmental disorders that may also become potential targets for clinical intervention. Finally, the capacity of RIP140 to affect the number, structure, function, or proportion of neural and glial cells during NSC maturation into terminal neurons requires further investigation.

Author contributions:ZRZ participated in study concept, de fi nition of intellectual content, literature search, experimental studies, data acquisition, data analysis, statistical analysis and paper preparation. WDY participated in study concept and design, de fi nition of intellectual content, literature search, data analysis, paper preparation, edition and review, and served as a guarantor. CS participated in study concept and design, and experimental studies. RL and XC participated in study concept and experimental studies. XF, XZ and QM participated in literature search. HS participated in paper review and served as a guarantor. JZG participated in study concept and design, de fi nition of intellectual content, literature search, experimental studies, data acquisition, paper preparation, edition and review, and served as a guarantor. All authors approved the fi nal version of this paper.

Con fl icts of interest:None declared.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

Peer review:

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Copyedited by Van Deusen AL, Norman C, Li CH, Qiu Y, Song LP, Zhao M

10.4103/1673-5374.198997

Accepted: 2016-12-10

＊Correspondence to: Jing-zhu Guo, Ph.D., gjzhxx@sina.com.cn.