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. 2019 Sep 5;71(5):977–988. doi: 10.1007/s10616-019-00339-w

The ERK signaling pathway is involved in cardiotrophin-1-induced neural differentiation of human umbilical cord blood mesenchymal stem cells in vitro

Changhui Lang 1, Xiaomei Shu 1,✉, Longying Peng 1, Xiaohua Yu 1
PMCID: PMC6787130  PMID: 31489528

Abstract

Central nervous system diseases remain the most challenging pathologies, with limited or even no therapeutic possibilities and a poor prognosis. This study aimed to investigate the differentiation properties of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) transfected with recombinant adenovirus expressing enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) and the possible mechanisms involved. Cells were isolated, and MSC immunophenotypes were confirmed. The resulting differentiated cells treated with Adv-EGFP-CT-1 and cultured in neural induction medium (NIM) expressed higher levels of Nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) markers than cells in other treatments. Expression of glycoprotein 130/leukemia inhibitory factor receptor β (gp130/LiFRβ), Raf-1, phosphorylated Raf-1 (p-Raf-1), extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) increased gradually within 72 h after transfection with Adv-EGFP-CT-1 and NIM culture. Additionally, inhibition of extracellular signal-regulated kinase kinase (MEK) abrogated expression of p-ERK1/2, Nestin, GFAP and NeuN. Thus, the ERK1/2 pathway may contribute to CT1-stimulated neural differentiation of hUCB-MSCs.

Keywords: Cardiotrophin-1, Umbilical cord blood mesenchymal stem cells, Neuron-like cells, ERK/MAPK signaling pathway

Introduction

Mesenchymal stem cells (MSCs) are defined as self-renewing, multipotent progenitor cells with the capacity to differentiate into several types of mature cells, including neurons, adipocytes, and chondrocytes (Malgieri et al. 2010). Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are an excellent candidate for cell therapy because they are easily accessible, pose no ethical problems for basic research and exhibit low immunogenicity (Lee et al. 2014; Horn et al. 2011). Indeed, hUCB-MSCs are being explored for regeneration of damaged tissues and treatment of nervous system diseases such as spinal cord injury (Forostyak et al. 2013; Yang et al. 2018; Park et al. 2012) and multiple sclerosis (Connick et al. 2012; Harris et al. 2018; Ng et al. 2014).

However, in the brain, MSCs show poor maintenance of sufficient levels of neuronal differentiation. Several studies have applied neurotrophins to address the problem, and the potential therapeutic applications of MSCs treated with neurotrophins have become important for research on neurological diseases.

Cardiotrophin-1 (CT-1) is a member of the interleukin-6 (IL-6) family of cytokines that comprises leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M, IL-11, cardiotrophin-like cytokine (CLC), and neuropoietin/cardiotrophin-2 (NP/CT-2) in addition to IL-6 (Garcia-cenador et al. 2013a, b). CT-1 is an endogenous cytokine displaying protective properties in the heart and liver (Schillaci et al. 2013; Aguilar-Melero et al. 2013) and promotes the survival of dopaminergic neurons by blocking apoptotic cell death (Lopez-Yoldi et al. 2015; Peng et al. 2010). In our previous study, CT-1 was found to promote the survival and neural differentiation of hUCB-MSCs in vitro. Furthermore, hUCB-MSCs cultured in neural induction medium (NIM) and treated with both CT-1 generated a high level of neural-like cells (Peng et al. 2017). To gain a better understanding of the mechanisms underlying the neural differentiation of hUCB-MSCs, this study aimed to evaluate hUCB-MSC neural differentiation after transfection with recombinant adenovirus expressing enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) and to investigate the possible mechanisms involved.

Materials and methods

Isolation, culture and identification of hUCB-MSCs

Umbilical cord blood (UCB) was obtained from mothers with consent and was separated and maintained according to our previous study (Peng et al. 2016). The separated cells were with mouse anti-human CD44-fluorescein-isothiocyanate (FITC), anti-CD105 (endoglin)-phycoerythrin (PE), anti-CD29-PE, and anti-CD34-Peridinin-Chlorophyll-Protein Complex (PerCP) and nonspecific mouse IgG1-FITC or IgG1-PE (BD Biosciences, Franklin Lakes, USA). The labeled-cell pellet was resuspended in 0.3 ml phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) for 30 min at 4 °C and then washed twice with PBS. The sample was analyzed by flow cytometry using a FACSCalibur (Becton–Dickinson, USA).

Transfection

hUCB-MSCs were grown in 24-well plates at a density of 1 × 105 cells for 1 day and then transfected with Adv-EGFP-CT-1 and recombinant adenovirus expressing enhanced green fluorescence protein (Adv-EGFP) at different multiplicities of infection (MOIs) (50, 100, 200 and 300 PFU/cell), respectively. The transduction efficiency was assessed by fluorescence microscopy at 24 h, 48 h and 72 h after transfection with Adv-EGFP-CT-1 or Adv-EGFP (Sainuo, Beijing, China), respectively. The best transduction efficiency of Adv-EGFP-CT-1 was determined by cell immunofluorescence at 24 h, 48 h and 72 h after transfection. The transduction efficiency was highest when cells were exposed to the transduction medium for 72 h at an MOI of 100 PFU/cell. Therefore, cells were transfected with Adv-EGFP-CT-1 or Adv-EGFP at an MOI of 100 PFU/cell for 72 h and used for assays.

Neural differentiation

Neural differentiation induction was performed with modifications to the procedure described by Lim (Lim et al. 2008). Cells were divided into four groups: control group (untreated cells); EGFP + NIM group (MSCs after transfection with Adv-EGFP expressing green fluorescence protein and NIM treatment); CT-1 + NIM group (MSCs after transfection with Adv-EGFP-CT-1 and NIM treatment; and NIM group (NIM-treated uninfected cells). MSCs were detached enzymatically and mechanically and plated in 6-well plates at 5 × 105 cells/ml. The next day, the cells in the EGFP + NIM and CT-1 + NIM groups were transfected with Adv-EGFP or Adv-EGFP-CT-1 (MOI of 100 PFU/cell), respectively. At 48 h after the initial plating, the medium (except for control cells) was replaced with preinduction medium composed of DMED/F12, 10% fetal bovine serum (FBS) and 10 ng/ml basic fibroblast growth factor (bFGF, Sigma, USA). At 72 h after the initial plating, the cells (except for control cells) were induced by replacing the pretreatment medium with NIM containing 100 µM butylated hydroxyanisole, 2% dimethylsulfoxide, 25 mM KCl, 5 U/ml heparin, 20 ng/ml bFGF, 5 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 100 µM putrescine, 30 µM sodium selenite, and 0.5 µM all-trans retinoic acid. The cells in each group were incubated at 37 °C under 5% CO2 in a humid environment for 6 h and 4 days respectively after NIM incubation, and the specific neural markers Nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) were assessed by immunofluorescence. Cell morphological changes were observed by microscopy.

Detection of cellular biomarkers by immunofluorescence

After induction, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized using PBS containing 0.1% Triton X-100 at room temperature for 30 min and blocked with 3% BSA in PBS. The cells were incubated overnight at 4 °C with the following primary antibodies:rabbit polyclonal anti-NeuN (1:1000, Abcam, Cambridge, USA), rabbit polyclonal anti-Nestin (1:1000, Abcam), and rabbit polyclonal anti-GFAP (1:1000, Abcam). After washing with PBS, the cells were incubated with the following secondary antibody: anti-rabbit CY3 (1:100, Bioss, Beijing, China). For quantitative analysis, the cells were incubated with 4,6-diamidino-2-phenylindole (DAPI, 2 µg/ml) for 3 min at room temperature. The preparations were examined by fluorescence microscopy (Leica, Wetzlar, Germany) and laser scanning confocal microscopy (Nikon, Japan). Cells positive for each antigen were counted as percentages of the total DAPI-stained cell population. For quantitative assessment of cell differentiation in different groups, the relative numbers of cells expressing different markers (Nestin, NeuN and GFAP) were counted as percentages of the total DAPI-stained cell population. ImageJ software was used to merge images.

Western blotting

Cells were plated in 24-well plates for 1 day, transfected with Adv-EGFP-CT-1 (MOI of 100PFU/cell), and cultured in NIM for 4 days according the procedure by Lim et al. (2008). Expression of CT-1 receptor glycoprotein 130 (gp130)/leukemia inhibitory factor receptor β (LiFRβ), Raf-1, phosphorylated Raf-1 (p-Raf-1), extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were detected at different times (24 h, 48 h and 72 h) after transfection with Adv-EGFP-CT-1. Furthermore, we treated the differentiated cells with the extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 (Beyotime, Shanghai, China). The expression of p-ERK1/2, GFAP, Nestin and NeuN was measured in differentiated cells followed by treatment with 10 µM PD98059 or 100 µM PD98059 for 30 min. Differentiated cells in the absence of PD98059 were used as the control group. Cell extracts were prepared in buffer containing 1% Triton X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), and the protein concentration was measured by the BCA protein assay (Beyotime, Shanghai, China). Cell extracts were mixed with 5× loading buffer and boiled for 5 min, and the proteins were separated for 1.5 h by 12% sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad, USA). The resolved proteins were transferred to a polyvinylidene fluoride membrane (PVDF) using the wet transfer method (25 mM Tris, 150 mM glycine, 20% methanol) for 45–50 min. Each membrane was blocked with 5% milk in TBS for 1 h at room temperature and incubated with rabbit anti-human gp130 (1:500, Santa Cruz, San Juan Ranch, USA), anti-LiFRβ (1:500, Santa Cruz), p-Raf-1 (1:500,Santa Cruz), p-ERK1/2 (1:500, Santa Cruz) and ERK1/2 (1:500, Santa Cruz), anti-GFAP (1:1000, Abcam, Cambridge, USA), anti-NeuN (1:1000, Abcam), anti-Nestin (1:1000, Abcam), anti-β-actin (1:1000, Beyotime, Beijing, China) or anti-β-tubulin (1:1000, Beyotime) antibodies overnight at 4 °C. The membranes were washed three times with 0.1% Tween-20 TBST, exposed to fluorescence-conjugated goat anti-rabbit IgG as a secondary antibody for 1 h, and detected using an Odyssey infrared imaging system (LI-COR, USA).

Statistical analysis

Results are presented as the mean ± standard deviation (SD). Neural differentiation data were analyzed using one-way analysis of variance (ANOVA) (SPSS 17.0, SPSS Inc, Chicago, IL). One-way ANOVA was performed for each group. Differences were considered statistically significant at P < 0.05.

Results

Isolation and characterization of hUCB-MSCs

Osteoclast-like and mesenchymal-like cells were observed in the initial stage. Floating cells (osteoclast-like cells) were removed from the half-changed medium, and attached cells were subsequently passaged and observed at 5–7 days after the initial plating. According to flow cytometry analysis of MSC-specific surface markers, the percentages of hUCB-MSCs positive for CD105, CD29 and CD44 were 89.31%, 98.41% and 99.2%, respectively. The cells were negative for CD34, a hematopoietic stem cell lineage marker (data not shown).

hUCB-MSCs transfection with Adv-EGFP-CT-1

The transduction efficiency was highest (87.60 ± 1.36%) when cells were exposed to Adv-EGFP-CT-1 for 72 h at an MOI of 100 PFU/cell. Moreover, hUCB-MSCs transfected with Adv-EGFP-CT-1 sustained expression of CT-1 (Fig. 1). At 72 h after transfection with an MOI of 100 PFU/cell, cell morphology and growth characteristics were the same as those of uninfected cells. Cells transfected with Adv-EGFP-CT-1 at an MOI of 100 PFU/cell for 72 h were used for assays.

Fig. 1.

Fig. 1

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Expression of CT-1 after human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) transfected with cardiotrophin-1 (CT-1) were measured at different time. a Immunofluorescence analysis of CT-1 expression in hUCB-MSCs is shown in red, and 4,6-diamino-2-phenyl indole (DAPI) staining is shown in blue. The last vertical column represents CT-1 incorporation (red) and DAPI (blue) in hUCB-MSCs. Transduction efficiency was the highest when the cells were exposed to recombinant adenovirus with enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) at a multiplicity of infection (MOI) of 100 PFU/cell for 72 h (Scale bar = 100 μm). b Histograms show transduction efficiency in hUCB-MSCs at different times after transfection with CT-1 at a MOI of 100 PFU/cell. (Color figure online)

Morphological changes in the neural differentiation of hUCB-MSCs induced by Adv-EGFP-CT-1

We observed morphological changes occurring in hUCB-MSCs during neural differentiation. In Adv-EGFP-CT-1-transfected hUCB-MSCs at 6 h after NIM induction (CT-1 + NIM), the cytoplasm had retracted toward the nucleus, and the process became thinner because of continuous shrinkage of the cell body. In the following 4 days, some cells gradually developed neuron-like cell reticular processes (Fig. 2b). In contrast, these morphological changes were weak in the other three groups. After NIM treatment, most of the cells exhibited pyramidal or spherical cell bodies with multiple processes at 6 h and 4 days (Fig. 2a), whereas the cells treated with Adv-EGFP and NIM exhibited a bipolar or multipolar morphology (Fig. 2c). Untreated hUCB-MSCs displayed a fibroblast-like appearance (Fig. 2d).

Fig. 2.

Fig. 2

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The morphological changes in human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) at 4 days after neurogenic differentiation in vitro. a The cells were pyramidal or spherical cell bodies with bipolar processes after NIM treatment. b The cytoplasm in hUCB-MSCs retracted toward the nucleus and the process become thinner because of continuous shrinkage of the cell body. Most of the cells exhibited a network-like structure (arrow indicate neural-like cells) in transfected with recombinant adenovirus with enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) and incubated in neural induction medium (NIM). c Cells treated with Adv-EGFP and NIM exhibited a bipolar or multipolar morphology. d The untreated hUCB-MSCs exhibited a spindle-like shape and maintained a fibroblast-like morphology (×100) (scale bar = 100 µm)

Effect of Adv-EGFP-CT-1 on neural differentiation in hUCB-MSCs

We next determined whether CT-1 expression induces expression of neuronal markers Nestin, GFAP and NeuN by performing immunofluorescence in the four groups at 6 h and 4 days after induction. Interestingly, treatment with Adv-EGFP-CT-1 and NIM (CT-1 + NIM group) resulted in greater expression of the neural progenitor marker Nestin than in other groups at 6 h after induction. However, Nestin expression decreased dramatically at 4 days after induction (Fig. 3a, b). NeuN levels were highest at 4 days after induction in the CT-1 + NIM group compared with the other three groups (P < 0.05) (Fig. 3a, d). Furthermore, GFAP expression increased in the CT-1 + NIM group at 4 days after induction (Fig. 3a, c). Conversely, only a few cells in the NIM and EGFP + NIM groups expressed GFAP and NeuN at 4 d after induction, and most cells in the control group were negative for NeuN and GFAP.

Fig. 3.

Fig. 3

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Expression of neural markers in human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). a Immunofluorescence analysis of neural-specific markers Nestin, GFAP and NeuN in uninduced cells (Control), neural induction medium (NIM)-treated uninfected cells, recombinant adenovirus expressing enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1)-infected NIM-treated cells (CT-1 + NIM) and Adv-EGFP-infected cells further cultured in NIM (EGFP + NIM) at 6 h and 4 days after induction. Each vertical column represents Nestin (red), GFAP (red) and NeuN (red); 4,6-diamidino-2-phenylindole (DAPI) staining is shown in blue. Arrows indicate Nestin+/DAPI+, GFAP+/DAPI+, and NeuN+/DAPI+ cells. Scale bar = 100 µm. b–d Quantitative analysis of relative Nestin+/DAPI cell numbers (b), GFAP+/DAPI cell numbers (c) and NeuN+/DAPI cell numbers (d) in uninduced cells (control), NIM-treated uninfected cells, Adv-EGFP-CT-1-infected NIM-induced cells (CT-1 + NIM) and Adv-EGFP-infected cells further cultured in NIM (EGFP + NIM) at 6 h and 4 days after induction. Twenty-five to 100 cells per group were analyzed in randomly chosen fields. The bars represent the mean ± SD of at least three independent experiments. Significance was determined by one-way ANOVA. *P < 0.05 compared with CT-1 + NIM group. (Color figure online)

Furthermore, we used confocal microscopy to observe expression of neural markers in hUCB-MSCs after induction. Adv-EGFP-CT-1-transfected cells cultured in NIM expressed EGFP (green), stained with DAPI (blue), and stained with Nestin (red) (Fig. 4a), GFAP (red) (Fig. 4b) and NeuN (data not shown) at 4 days after induction, 75 of 156 DAPI+ cells were NeuN+ (data not shown), 20 of 156 DAPI+ cells were Nestin+, and 85 of 156 DAPI+ cells were GFAP+.

Fig. 4.

Fig. 4

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The expression of Nestin and glial fibrillary acidic protein (GFAP) markers following treatments with Adv-EGFP-CT-1 and neural induction medium (NIM) at 4 days after induction by confocal microscopy. Confocal microscopy analysis of neural induction medium (NIM)-treated transfected recombinant adenovirus with enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) cells expressing EGFP (green), stained with 4,6-diamidino-2-phenylindole (DAPI) (blue), and stained with anti-Nestin (red) (a) and anti-GFAP (red) (b) antibodies, respectively. After 4 days of incubation with NIM in transfected Adv-EGFP-CT-1 cells, 75 of 156 DAPI+ cells were NeuN+ (data not shown), 20 of 156 DAPI+ cells were Nestin+ and 85 of 156 DAPI+ cells were GFAP+. At least 3 slices were analyzed (×400). Scale bar = 100 µm. (Color figure online)

Expression of CT-1 receptors and signaling molecules

To investigate the possible mechanisms by which CT-1 mediates neural differentiation of hUCB-MSCs, we used Western blotting to examine expression of ERK1/2, p-ERK1/2, Raf-1, p-Raf-1, and gp130/LiFRβ in cells at 24 h, 48 h and 72 h after transfection with Adv-EGFP-CT-1. The results revealed a time-dependent increase in p-ERK-1/2 and p-Raf-1 levels within 72 h in Adv-EGFP-CT-1-transfected cells. However, expression of gp130/LiFRβ, ERK1/2 and Raf-1 no longer increased in the cells at 48 h after transfection. Moreover, there were no significant differences in total ERK, Raf-1 and gp130/LiFRβ levels between 48 h and 72 h after transfection (Fig. 5a, c). To better define the involvement of ERK signaling in Adv-EGFP-CT-1-stimulated neural differentiation, we treated differentiated cells with PD98059 for 30 min and found that the expression of Nestin, GFAP and NeuN were dramatically downregulated by 100 µm but not by 10 µm PD98059 (Fig. 5b, d). CT-1 stimulated p-Raf-1 and p-ERK1/2 upregulation, and the MEK inhibitor PD98059 dramatically reduced CT-1-induced neural differentiation.

Fig. 5.

Fig. 5

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Activation of the ERK pathway in hUCB-MSCs by transfection with Adv-EGFP-CT-1. a, c Western blot analysis of gp130/LiFRβ and Raf-1, phosphorylated Raf-1 (p-Raf-1), ERK, and p-ERK1/2 levels in human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) at 24 h, 48 h and 72 h after transfected recombinant adenovirus expressing enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1). Cell lysates were subjected to SDS-poly-acrylamide gel electrophoresis. The blots are representative of at least two independent experiments (*P < 0.05 compared with 24 h, **P < 0.05 compared with 48 h). b, d Western blot analysis of phospho-ERK1/2, Nestin, GFAP and NeuN levels. Infected Adv-EGFP-CT-1 cells were treated with NIM for 4 days and then with 10 µmol/l or 100 µmol/l MEK inhibitor PD98059 for 30 min, after which the levels of p-ERK1/2, Nestin, GFAP and NeuN were determined by Western blot analysis (*P < 0.05 compared with the 100 µM PD98059, **P < 0.05 compared with the 10 µM PD98059 group. ERK1/2: extracellular signal-regulated kinase 1/2; GFAP: glial fibrillary acidic protein; NeuN: neuronal nuclei)

Discussion

Previous studies have shown that CT-1 has cytoprotective effects on several tissues, such as cardiac and vascular tissues, and renal fibrosis (Garcia-Cenador et al. 2013a, b; López-Andrés et al. 2012). Additionally, CT-1 is reported to act as a trophic factor for a few types of neurons, such as sensory, dopaminergic, motor and cortical neurons (Lopez-Yoldi et al. 2015). According to previous studies, CT-1 delays degenerative disease progression in motor neuron disease (Wang et al. 2015). CT-1 may also play a critical role in obesity and metabolic syndrome, and it attenuates cognitive impairment in the mouse brain by inhibiting GSK-3β activity (Wang et al. 2013).

We previously demonstrated that transplantation of neural stem cells (NSCs) overexpressing CT-1 can significantly reduce brain lesions and inhibit the formation of hippocampal mossy fiber spouting (MFS) (Shu et al. 2011). Here, we report the effect of CT-1 on the neural differentiation of hUCB-MSCs following neural induction. After culture in NIM, only a few cells exhibited a neuron-like morphology and expressed GFAP and NeuN. However, following transfection of hUCB-MSCs with adenovirus containing CT-1, the cells effectively expressed CT-1. Furthermore, the transfected cells showed a significant increase in expression of the neuronal markers Nestin, GFAP and NeuN after treatment with NIM, which contains inducing factors such as all-trans retinoic acid and bFGF. Nestin is a neurofilament protein that is one of the most specific markers of multipotent NSCs; NeuN is a well-known specific marker for surviving neurons, and GFAP is typically observed in astrocytes. According to our results, CT-1 may induce neuronal differentiation of hUCB-MSCs. Our previous results also suggested that CT-1 may act as an inhibitor of apoptotic cell death during hUCB-MSC neural differentiation (Peng et al. 2017). Moreover, CT-1 gene transfer was found to improve animal survival and liver graft function and to activate cell survival signaling pathways through Akt, ERK and signal transducer and activator of transcription (STAT-3) phosphorylation (Song et al. 2008).

The possible mechanisms of neural differentiation in hUCB-MCs transfected with CT-1 are currently not well understood. Gp130, a CT-1 receptor, is barely activated by natural cytokines in the absence of other receptor subunits (Rose-John 2018), and Gp130 signaling plays a vital role in the maintenance of neural stem and progenitor cells during animal ontogenesis (Kotasová et al. 2014). According to some studies, CT-1 binds to gp130/LIFRβ in addition to LIF; furthermore, the receptor subunits associate with each other, gp130 combined with LiFRβ to form a heterodimer, which is shared by LIF and CNTF; subsequently, the heterodimer activates downstream signaling molecules, such as Raf-1, ERK1/2, and Janus kinase (JAK). Signal transduction via gp130 involves three major downstream pathways: the JAK/STAT pathway, the Ras/Raf MAPK (MAPK, MEK/ERK) signaling cascade, and the phosphatidylinositol 3-kinase-dependent (PI3K)/protein kinase B (AKT or PKB) pathway. Furthermore, the Ras/Raf/ERK signaling cascade plays an essential role in cell proliferation and differentiation during neuronal development (Zhong 2016).

In this study, the relative level of gp130/LiFRβ peaked in CT-1 + NIM transfected cells, with p-ERK1/2 and p-raf-1 gradually increasing up to 72 h after induction. Nonetheless, it is unclear whether other molecules were activated during this process. Our results indicate that CT-1 activates ERK1/2 and Raf-1 phosphorylation through gp130/LiFRβ. Subsequently, we assessed levels of p-ERK1/2, Nestin, GFAP and NeuN after treatment with different concentrations of PD98059 and found that inhibition of MEK with PD98059 in differentiated cells effectively blocked expression of neural markers such as GFAP, NeuN and Nestin. Thus, the ERK signaling pathway may be involved in the neural differentiation of hUCB-MSCs transfected with CT-1. Consistent with earlier studies, these results suggest that MAPK/ERK and PI3K/Akt-dependent signaling pathways are involved in the survival of differentiated cells treated with BDNF and NIM (Lim et al. 2008). In addition, our previous study demonstrated that the PI3K/Akt pathway contributes to cell survival and neuronal differentiation in hUCB-MSCs treated with CT-1 and NIM (Peng et al. 2017). Alternatively, some studies have emphasized the importance of anti-apoptotic MAPK and ERK1/2 activation as a cardioprotective signaling pathway downstream of CT-1 (Brar et al. 2001). The precise consequences of each signaling pathway differ among cell types. In several embryonic stem cell lines, Ras/MAPK signaling upregulates genes involved in proliferation and self-renewal (Davis and Pennypacker 2018). Although the activated PI3K/Akt pathway is also involved in ERK1/2 inhibition, some studies have indicated that ERK1/2 activation is PI3K/Akt dependent. The PI3K/Akt pathway also regulates the ERK1/2 cascade by triggering Raf-1, B-Raf, or MEK to regulate cell survival, proliferation and differentiation (Romano et al. 2006).

In summary, the results of this study indicate that CT-1 stimulates neural differentiation in hUCB-MSCs through the ERK pathway. Although activation of ERK signaling may contribute to the neural differentiation of hUCB-MSCs treated with CT-1, it is insufficient to trigger this process alone. One possible explanation is that other signaling pathways, such as JAK/STAT, may be involved in hUCB-MSC neural differentiation mediated by Adv-EGFP-CT-1. However, crosstalk among these pathways is not fully understood and needs to be further investigated.

Conclusion

The findings suggest that hUCB-MSCs can differentiate into neurons and glial cells after transfection with Adv-EGFP-CT-1 and treatment with NIM. The ERK pathway may play an important role in CT-1-mediated neural differentiation of hUCB-MSCs.

Acknowledgements

This study was funded by a grant from the National Natural Science Foundation of China (No. 31260286). We are grateful to the doctors and nurses of the Obstetrical Department of Maternity and child health care hospital in Zunyi for the collection of cord blood.

Footnotes

Publisher's Note

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