Negative effects of Notch1 on the differentiation of muscle-derived stem cells into neuronal-like cells*☆

Xifan Mei, Chang Liu, Zhanpeng Guo, Yajiang Yuan, Shiqiang Fang, Yansong Wang,Yue Guo, Jinhao Zeng

1Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China

2Department of Endocrinology, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China

INTRODUCTION

Due to limited regeneration of spinal cord injuries, it remains difficult to fundamentally change the prognosis of spinal cord injury[1].Muscle-derived stem cells (MDSCs) are a type of stem cell found in muscle tissue with high self-renewal and proliferative capacities,as well as multipotency.MDSCs have been found to differentiate into neuronal-like cells for regeneration of damaged nerves and restoration of neurological function[2].Following spinal cord injury, spinal cord functions can be partially recovered by transplantation of seed cells into the damaged area[3].Because MDSCs can differentiate into neuronal-like cells[4], they are of interest to easily obtain neuronal-like cells for transplantation.The Notch signaling pathways plays an important role in formation of the nervous system.Notch signals spatio-temporally regulate the quantity of nerve cells at a suitable number and the types of neurons and glial cells[5].Notch signaling also regulates the differentiation of precursor nerves, and low level Notch signaling may benefit differentiation into nerve cells[6].TheNotch1gene has been proposed as essential to maintain the level of undifferentiated cells[7]and plays a role in regeneration and differentiation of stem cells[8].Neural precursor cells with inactivatedNotch1genes can differentiate into nerve cells[9].Therefore, the present study determined expression of theNotch1gene, the key node in the Notch signaling pathway, in MDSCs during differentiation into neuronal-like cells.

RESULTS

Morphology of MDSCs after differentiation

Cultured MDSCs were assigned to three groups: control group cultured with complete medium, induced group cultured with differentiation medium and inhibitor group cultured with medium containing gamma-secretase inhibitor, N-(3,5-difluorophenacetyl-L-alanyl)-S- phenylglycine t-butyl ester.

After inducement in differentiation medium for 7 days, cells proliferated well in the induced group, with long processes similar to those of nerve cells.Cell processes were also connected to each other.Morphological changes were progressive and the differentiation rate was high.Cells at this time were not significantly proliferating, and neurospheres were not formed.No morphological changes were observed in control and inhibitor groups (Figure 1).

Neuron-specific enolase (NSE) expression in MDSCs after differentiation for 7 days

NSE is a specific acid protease present in neurons and neuroendocrine cells.In the induced group, cell bodies and processes were strongly NSE-positive, as indicated by brown coloration, after differentiation for 7 days, while control and inhibitor groups were NSE-negative (Figure 2).In addition, the cytoplasm of induced cells was brown, and darkened around the nucleus, but lightened far from the nucleus.Staining of NSE in control and inhibitor group cells was negative.

Figure 1 Morphology of muscle-derived stem cells in the induced group after differentiation for 7 days (× 200).Cell processes appear similar to those of neurons and connections among the cells are observed (arrows).Neurospheres are not observed.

Figure 2 Morphology of neuron-specific enolase(NSE)-positive cells in induced (A) and control (B) groups after differentiation for 7 days (× 200).

Notch1 protein expression in differentiated MDSCs

Notch1 expression was determined using immunofluorescence staining.All groups contained Notch1-positive cells (Figure 3).The control group was strongly Notch1-positive (Figure 3A).The inhibitor group was Notch1-positive (Figure 3C), but weaker compared with that of the control group, and the induced group was weakly Notch1-positive (Figure 3B).

Figure 3 Morphology of Notch 1-positive cells in each group (immunofluorescence staining, × 400).The nucleus is stained blue (Hoechst 33258).Green fluorescence tetraethyl rhodamine isothiocyanate is observed in positive cells.

Notch1 mRNA expression in differentiated MDSCs

Notch1mRNA expression was determined by reverse transcription (RT)-PCR and was expressed in all groups.Moreover,Notch1mRNA of induced group cells was significantly decreased (P＜0.05) compared with that of control and inhibitor group cells.There was no significant difference between control and inhibitor groups (P＞ 0.05;Figure 4).

DISCUSSION

The application of seed cells is a research focus of tissue engineering in recent years.MDSCs are promising seed cells in the field of tissue engineering, particularly for cell transplantation and gene therapy.A large number of animal experiments using MDSCs for gene therapy and tissue engineering have been conducted, particularly for blood disorders such as myelodysplastic syndrome, in which not only the types of seed cells were expanded,but the microenvironment was also regulated[10-11].

Figure 4 Notch1 mRNA expression in each group as detected by reverse transcription-PCR.aP＜0.05, vs.control group.Data were expressed as mean±SD of five samples from each group (two-sample t-test).M: Marker.

In the present study, collagenase and dispase were used for MDSC digestion to prevent the detrimental impact on cellular activity caused by trypsin.Studies show that MDSCs are easily obtained, survive for a long period of time and can differentiate into blood cells, chondrocytes and endothelial cellsin vitro[12-14].Another study found that MDSCs are able to differentiate into neurons and glial cells[15].Therefore, the establishment and improvement of MDSC culture to obtain highly pure cells is of important practical value.

TheNotchgene is a specific mutant form found in drosophila, which can affect the formation of wings, eyes and bristles.Gene mutation can occur in the embryonic cells of drosophila, and its absence can lead to over-differentiation of the nervous system[16].Under physiological conditions, a cell ligand directly connects with an adjacent extracellular subunit of the Notch receptor, triggering the Notch transmembrane subunit lyase region to activate the lyase in the Notch transmembrane subunit.The soluble intracellular domain of Notch forms and transfers to the nucleus.The target geneHes1/5begins transcription, and bHLH proteins,encoded byHes1/5, regulate its downstream target genes.

Some undifferentiated neuroectodermal cells show high expression of Delta, which signals neighboring cellsviaNotch receptors.After cells receive the signal, differentiation into nerve cells is inhibited, and differentiation proceeds into other cell types.A proportion of cells with Delta expression will eventually differentiate into neurons and glial cells[17].Accordingly, stem cells that lack Notch signaling will eventually differentiate into nerve cells.In mammals, various cells control embryonic development and adult homeostasisviathe Notch signaling pathway.In embryonic development, the Notch signaling pathway plays a crucial role in the self-renewal of stem cells and the differentiation of precursor cells[18-20].An analysis of myelodysplastic syndrome found that disruption to Notch signaling during the early development of stem cells decreases the plasticity of MSCs, affecting their differentiation into neurons and lipocytes[21].Notch1gene knockout experiments show the same outcomes.Notch1gene knockout orNotch1and 3 double-knockout leads to apoptosis of nerve cells and a large number of neural precursor cells.

During the development of neural stem cells, the Notch signaling pathway can change the activity of transcription factors and control the transcription of important genes includingMash-1andGaianoby protein interactions[22].Studies have shown that activation of the Notch signaling pathway inhibits differentiation of nerve cells, while blocking Notch signaling reduces differentiation of nerve cells.This observation shows that Notch signaling can be used to control the differentiation of neural stem cells[23-24].As a key node in the Notch signaling pathway, Notch1 is involved in regenerative responses.Activated Notch1 is also an inhibitory factor of leukemia[25].

Notch signaling plays a very important role in the formation and development of the nervous system and the treatment of disease.The present study investigated whether Notch1 regulates stem cell differentiation into neuronal-like cells.To exclude other factors, the inhibitor group was used as a control.DAPT is a type of γ-secretase inhibitor.γ-secretase is a multi-protein complex that hydrolyzes various transmembrane proteins including Notch receptors.γ-secretase inhibitors specifically inactivate γ-secretase resulting in a series of biological effects.After Notch receptor-ligand binding, the combination of Notch and its ligand triggers hydrolysis mediated by tumor necrosis factor-α-converting enzyme and the γ-secretase/presenilin complex to release the active fragment for transfer to the nucleus, thereby activating downstream gene transcription.

In the present study, cells in the induced group resembled nerve cells after differentiation for 7 days, and NSE expression was observed in cells.These results show that MDSCs differentiated into neuronal-like cells.As shown in Figure 3, Notch1 protein was weakly expressed,and the inhibitor group, which was set to exclude other factors, showed a similar result.Inducing factors were not used in the control group, thus, MDSCs did not differentiate into neuronal-like cells, and NSE expression was negative.However, Notch1 protein was strongly positive compared with that of the induced group.RT-PCR and statistical analyses showed that there were significant differences between induced and control groups.These results indicate that during the process of MDSC differentiation into neuronal-like cells, Notch1 gene expression decreases.However, the specific mechanism requires further investigation.

MATERIALS AND METHODS

Design

A comparative observation andin vitrocytology experiment．

Time and setting

Experiments were performed at the Laboratory of Tissue Engineering, First Affiliated Hospital of Liaoning Medical University, China from 2008 to May 2011.

Materials

Ten healthy Sprague-Dawley rats of a clean grade and either gender, aged 3 days and weighing 10±2 g, were provided by the Laboratory Animal Center of Liaoning Medical University (License No.SYXK (Liao) 2003-0011).Experiments were performed in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animalsissued by the Ministry of Science and Technology, China[26].

Methods

Primary culture and identification of MDSCs

Rats were sacrificed and disinfected by soaking in 75%alcohol (3 × 5 minutes).Under aseptic conditions, fat,tendons, muscle, membranes and other connective tissues were removed.Then, skeletal limb muscles were harvested and washed three times with sterile PBS containing 400 mg/L streptomycin and 4 × 105U/L penicillin.Skeletal muscles were cut into pieces with ophthalmic scissors, digested with two times the volume of a digestive enzyme mixture (0.48 g/L dispersion type II enzyme, 0.2 g/L type II collagenase and 0.28 g/L CaCl2)at 37°C with 5% CO2for 1 hour in a humidified cell incubator.The sample was mixed three times for 5 minutes each, incubated with DMEM supplemented with 15%FBS to terminate digestion and filtered through a stainless steel mesh with a 200 μm pore diameter.Cells were centrifuged at 97 ×gfor 10 minutes and the supernatant was discarded.The cell pellet was resuspended in DMEM and transferred to culture flasks for 2 hours and designated as PP1.PP1 cells were passaged into another culture flask and designated as PP2, then incubated overnight.Then, cells were passaged into another culture flask and designated as PP3.Cultures were passaged once every 24 hours until PP6 was obtained.PP5 cells were used for assessment.Cells at 70% confluence were passaged and incubated at 37°C with 5% CO2.MDSCs were digested with an enzyme mixture, followed by Desmin detection[27].Induced group was cultured with differentiation medium (DMEM supplemented with 10% FBS, 20 mg/L NGF, 20 mg/L bFGF and 1% penicillin) and inhibitor group was cultured with medium containing DAPT (Sigma, St.Louis, MO,USA).Morphological changes of the cells were observed under a phase contrast microscope and immunocytochemical staining was performed after 6 days.

Detection of NSE expression in MDSCs using immunocytochemical staining

Coverslips were placed in petri dishes filled with the cell suspension, incubated at 37°C with 5% CO2for 2–3 days.Cells were fixed with paraformaldehyde for 30 minutes,placed in 0.3% Triton X-100 for 5 minutes, PBS washed twice, mixed with 3% H2O2for 30 minutes and placed in blocking buffer for 20 minutes.Cells were then incubated with a mouse anti-rat NSE antibody (1: 100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C,followed by a goat anti-mouse IgG (1: 100; Santa Cruz Biotechnology) for 2 hours and then a streptavidin-biotin complex for 30 minutes.Cells were treated with a diaminobenzidine kit (Wuhan Boster, China) for 30 minute and, stained with hematoxylin for 0.5 minutes.Cells were dehydrated with an 80%, 90%, 95%, 100%, 100% ethanol series (4 minutes), mixed with xylene I and xylene II(10 minutes each) and mounted with glycerin.Five fields of view were randomly selected to quantify 100 cells per field and the number of positive cells was calculated and photographed.

Detection of Notch1 protein expression in MDSCs using immunofluorescence staining

Cells were fixed in 4% polyphosphate for 15 minutes,mixed with 3% BSA for 30 minutes, incubated with the mouse anti-rat Notch1 antibody overnight at 4°C, followed by a goat anti-mouse IgG (ready-to-use; Santa Cruz Biotechnology) for 30 minutes at 37°C.Cells were stained with 30 μL/mL Hoechst 33258 (Sigma) for 8 minutes and observed under a fluorescence microscope(Olympus, Tokyo, Japan).Notch1 protein on the cell surface was labeled with tetraethyl rhodamine isothiocyanate (TRITC, Sigma).

Detection of Notch1 mRNA expression using RT-PCR

Total RNA was isolated from cells using TRIzol reagent(Sigma) according to the manufacturer’s instructions.The quality and quantity of RNA was verified by two discrete electropherogram peaks corresponding to 28S and 18S rRNA at a ratio approaching 2: 1.A Ta-KaRa RNA PCR Kit (TaKaRa Biotechnology, Otsu,Japan) Ver 3.0 was used for reverse transcription according to the manufacturer’s instructions.Based on theNotch1cDNA sequence, primers were designed as follows:

The PCR conditions were 94°C for 5 minutes, followed by 30 cycles of DNA amplification (30 seconds at 95°C,30 seconds at 61°C and 50 seconds at 72°C) and then 10 minutes incubation at 72°C.

PCR products were examined by 2% agarose gel electrophoresis.The absorbance ratio of the target gene to β-actin was calculated for five replicates.

Statistical analysis

Data were expressed as the mean±SD and analyzed using SPSS 13.0 statistical software (SPSS, Chicago, IL,USA).Comparisons among groups were performed using a two-samplet-test.A value ofP＜0.05 was considered statistically significant.

Author contributions:The experiment was designed by

Chang Liu, performed by Xifan Mei, Zhanpeng Guo, Yajiang Yuan, Shiqiang Fang, Yansong Wang, Yue Guo, Jinhao Zeng and evaluated by Xifan Mei.

Conflicts of interest:None declared.

Funding:The study was supported by the Program for Liaoning Innovative Research Team in University (LNIRT), No.2008T113.

Ethical approval:The study was approved by the Animal Ethics Committee of Liaoning Medical University, China.

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