Mol. Cells 2016; 39(5): 418-425
Published online April 25, 2016
https://doi.org/10.14348/molcells.2016.2345
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: guanfangxia@126.com (FG); yangbo96@126.com (BY)
Resveratrol (RES) plays a critical role in the fate of cells and longevity of animals via activation of the sirtuins1 (SIRT1) gene. In the present study, we intend to investigate whether RES could promote the self-renewal and neural-lineage differentiation in human umbilical cord derived MSCs (hUC-MSCs)
Keywords human umbilical cord derived mesenchymal stem cells, neural differentiation, resveratrol, self-renewal, SIRT1
Mesenchymal stem cells (MSCs) have been spotlighted in the neural regenerative medicine (Can and Karahuseyinoglu, 2007). However, MSCs become senescent after consecutively passage
Sirt1 gene is a member of the Sirtuins family which possess histone deacetylase activity and plays an important role in regulating the aging of cells and neurodegenerative diseases. However, the protein level of SIRT1 declines with MSCs passage
Since the controversial effects of RES on different cells retard the research progress and limit its clinical application, this study aims to investigate the role of RES in the fate of human umbilical cord derived MSCs (hUC-MSCs)
hUC-MSCs were isolated as previously described (Ma et al., 2014). The cultured cells were harvested at passage 4 (P4) for a typical experiment. The study was approved by the Ethics Committees of the First Affiliated Hospital of Zhengzhou University and consented by the donors of umbilical cord.
hUC-MSCs were harvested and washed with ice-cold PBS. Cells were labeled with the following antibodies: CD29-PE, CD44-FITC, HLA-ABC-FITC, HLA-DR-FITC, CD34-PE, CD45-PE, CD51-FITC, and CD105-PE (BD Bioscience, USA) before analyzed by the FACS Calibur flow cytometer (Becton-Dickinson, USA).
RES 50 μM stock solution (trans-3,4,5-trihydroxystilbene; R5010; Sigma, USA) was suspended in DMSO (Sigma, USA) and diluted to specific concentration before use. The final working concentration of DMSO was less than 0.1% for the
Cell viability was quantitatively determined by a CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the manufacture’s protocol. Briefly, hUC-MSCs at P4 were plated in 96-well plate at a density of 1000 cells/well and cultured in 100 μM DMEM/F12 with RES (0.1, 1, 2.5, 5, 10, 20, 50 and 100 μM) for 1, 3, 6, 8, 10 and 12 days, rinsed with PBS and incubated in DMEM/F12 with 10 μl CCK-8 for each well for 2 h at 37°C and the absorbance (OD) of the solution was measured by a microplate reader (Bio-Rad, USA) at a wavelength of 450 nm.
To examine the effect of RES on the proliferation rate of hUC-MSCs, cells at P4 were seeded in 24-well plate (8000 cells/well) for 24 hours to allow for stabilization and then exposed to RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days before EdU (Ribobio, China) was added. The EdU labeling duration was determined at 24h according to the average cell-doubling time for hUC-MSCs. Images were visualized using fluorescent microscope (Olympus, Japan). The red (EdU-labeled) and blue (Hoechst-labeled) cells were counted.
hUC-MSCs at P4 were plated at similar density in their respective media and cultured for 6 days before senescence-associated β-galactosidase (SA-β-gal) staining (Beyotime, China). Briefly, cells were fixed in 4% (v/v) formaldehyde for 10 min before stained with SA-β-gal-staining solution at pH 6.0 for 12 h. The SA-β-gal-positive cells exhibited blue color. The number of positive cells was counted under a phase-contrast microscope. The experiments were carried out in triplicate.
hUC-MSCs at P4 were incubated with RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days before propidium iodide (PI) staining performed as previously described (Ma et al., 2014).
Cell apoptosis was analyzed by the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) following the manufacturer’s instructions. Briefly, hUC-MSCs at P4 were incubated with RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days and then collected for FITC-Annexin V and PI staining as previously reported (Zhu et al., 2016).
hUC-MSCs at P4 were incubated with RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days before neural differentiation induction, which was modified on the basis of the protocol previously described (Karahuseyinoglu et al., 2007). hUC-MSCs were pre-induced for 24 h in DMEM-LG containing 20% fetal bovine serum (FBS), 10 ng/ml basic fibroblast growth factor (bFGF, Peprotech, USA) was added for an additional 24 h, then incubated in the induction medium for another 24 h: DMEM-LG with 2% DMSO (Sigma, USA), 100 μM butylated hydroxyanisole (Sigma, USA), 25 mM KCl, 10 μM forskolin, 5 μg/ml insulin and 1 μM hydrocortisone, followed by neuro-basal medium, supplemented with 10% FBS, 10 ng/ml epidermal growth factor (Sigma, USA), 10 ng/ml bFGF, 1 × N2 supplement (Gibco, USA), 1 × B-27 supplement (Gibco, USA), and 2 mM L-glutamine (Sigma, USA) for the maintenance of differentiation.
The 1 × 105 cells were plated on 24-well plate and after rinsing with PBS, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After blocking with 10% normal goat serum, cells were incubated overnight at 4°C with specific antibodies against Nestin (1:50, SantaCruz, USA), βIII-tubulin (1:100, Cell Signaling Technology, USA) or Neuron Specific Enolase (NSE, 1:3,000, Abcam, USA). After rinsing in PBS three times, cells were incubated for 1 hour at room temperature in Cy3-conjugated anti-mouse/-rabbit anti IgG (1:1,000, Molecular Probes, USA) for visualization, followed by DAPI staining (Biotech, China). Immunofluorescent images were visualized with fluorescence microscopy. The negative control was incubated with PBS instead of primary antibody, and no immunoreactivity was observed.
Total RNA was isolated using Trizol reagent (Invitrogen) according to the instructions, and mRNA was reverse transcribed using PrimeScript™ RT-PCR Kit (TaKaRa, Japan). qRT-PCR were performed on an ABI 7500 real-time PCR system (Thermo Fisher Scientific, USA) using a SYBR Premix Ex Taq™ (Prefect Real Time, Takara, Japan). Glyceral dehyde 3-phosphate dehydrogenase (GAPDH) was employed as the internal standard. Relative expression levels of different genes were calculated using the 2?ΔΔCt. The sequences of primers for qRT-PCR were shown in Table 1.
Cells were incubated with RES of the indicated concentrations above before Western Blotting, which was performed as previously described (Ma et al., 2014). Primary antibodies (SIRT1, 1:1000; PCNA, 1:1000; p53,1:1000; p21,1:500; p16,1:500, cell signaling technology, USA) were employed.
Data were expressed as mean ± standard deviation (SD). Analysis of Variance (ANOVA) followed by LSD test were employed to determine the significance between different groups.
hUC-MSCs grew out from the Wharton’s jelly 7?10 days after isolation, and displayed a monolayer of bipolar spindle-like or fibroblast-like morphology with a whirlpool-like array. However, the cells underwent morphological changes and became flat, swollen and irregular as the passage number increased (Figs. 1A?1F). Flow cytometry revealed that hUC-MSCs were positive for stromal matrix markers (CD44, CD105), integrin markers (CD29, CD51), and HLA-ABC, while negative for hematopoietic lineage markers (CD34, CD45) and HLA-DR (data not shown).
We found that RES exhibited a dose-dependent promoting effect on the viability of hUC-MSCs at a concentration of 0.1, 1 and 2.5 μM after incubating for 6 days (
The effect of RES on cell proliferation was analyzed by EdU staining at different concentrations and time points. As shown in Figs. 3A and 3B, 0.1, 1 and 2.5 μM RES significantly increased the percentages of EdU-labeling cells, compared with the control. However, the proliferation of hUC-MSCs was significantly inhibited when cells were challenged with 5 and 10 μM RES (
As shown in Figs. 3C and 3D, there were significantly less SA-β-gal positive cells in the 0.1, 1 and 2.5 μM RES treated groups (
Flow cytometry was used to detect the increased percentages of cells in S and G2/M phases (Fig. 3E). There was a reduction in the percentage in G0/G1 phase in hUC-MSCs treated with 0.1, 1 and 2.5 μM RES. However, there was no significant difference when compared with the control (
Flow cytometric detection of Annexin-V-FITC in / Propidium iodide (PI)staining revealed a dose-dependent downward trend in the percentages of apoptotic and necrotic cells among the RES (0.1, 1 and 2.5 μM) treatment groups (Fig. 3F), although there was no significant difference when compared with the control (
hUC-MSCs underwent neuronal differentiation by a multi-step protocol. Most differentiated hUC-MSCs demonstrated neural appearance with retracted cell body, refractive karyon, protruded dendrites and axons after neural induction (Fig. 4A), while hUC-MSCs without induction retained long fusiform shape. RES pre-incubation facilitated the neural differentiation of hUC-MSCs in all concentrations (Fig. 4A). There were more neuronal-shaped cells in RES-pretreated hUC-MSCs than that without pretreatment, especially at the concentration of 2.5, 5 and 10 μM (
The qRT-PCR analysis revealed that after neural differentiation, the expression of Neurogenin1 (Ngn1) was decreased, while Ngn2 and Mash1 were increased (
To explore the molecular mechanism of RES on the cell viability, proliferation, cell cycle, apoptosis and senescence of hUC-MSCs, we examined the expression of critical genes involved in those biological processes by qRT-PCR and Western Blot, including SIRT1, PCNA, p53, p21, and p16. As indicated in Fig. 5A, the expression of SIRT1 and PCNA in hUC-MSCs were significantly increased by 0.1, 1 or 2.5 μM RES (
Regulating the self-renewal and lineage commitment of stem cells is the key factor in regenerative medicine. Studies have shown that RES promotes the survival of adipose-derived mesenchymal stem cells (Pinarli et al., 2013), enhance the proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (Dai et al., 2007) and direct the neuronal differentiation of human bone marrow mesenchymal stem cells (Joe et al., 2015). However, some researchers argued that RES inhibited the proliferation of neural progenitor cells (Park et al., 2012), negatively regulated the neurogenic potential of neural precursors (Saharan et al., 2013). These results suggested that RES might play different or even opposite role in different cell type under specific milieu. Since hUC-MSCs are promising candidate for some intractable diseases, it is essential to gain better understanding of the effect of RES on hUC-MSCs development.
The present study revealed that incubation with 0.1, 1 and 2.5 μM RES for 6 days
In addition, we found that hUC-MSCs expressed low level of Nestin, βIII-tubulin and NSE, indicators of multi-potent neural stem cell, pre-mature neuron and mature neurons respectively (Heng et al., 2014), suggesting the neural differentiation potential of hUC-MSCs. RES could facilitate neural-lineage development of hUC-MSCs in a dose-dependent manner, as evidenced by morphological characters and up-regulated expression of βIII-tubulin and NSE with down-regulated Nestin. Simultaneously, the expression of proneural basic helix loop helix (bHLH) transcription factors, Ngn1, Ngn2 and Mash1 changed. Ngn1 was significantly down-regulated, while Ngn2 and Mash1 were up-regulated in the differentiated hUC-MSCs, and that could be further enhanced by pre-incubation with 2.5, 5 and 10 μM RES. In light with previous studies, Ngn1 is a proneural bHLH transcription factor expressed in newly committed neuronal progenitors and immature neurons and plays an essential role in neurogenesis. It is expressed at a high level in the early stage and decreased in the late stage of neuronal differentiation of MSCs (Cardozo et al., 2012). Ngn2 and Mash1 are activator-type bHLH genes essential for neuronal fate determination (Cardozo et al., 2012; Yu et al., 2013). This study reveals that RES facilitates neuronal differentiation through induction of proneural transcription factor genes in hUC-MSCs, and suggests that RES pretreatment of hUC-MSCs might be beneficial to stem cell based therapy for neural system injury or diseases.
We sought to elucidate the mechanistic basis for these observations and found that RES could regulate the SIRT1 signaling in the hUC-MSCs, which was closely related with cell self-renewal (Liu et al., 2012), senescence (Chen et al., 2014; Rehan et al., 2014), apoptosis (Godoy et al., 2014) and neural differentiation (Hisahara et al., 2008). Consisted with previous studies (Rathbone et al., 2009), the expression of PCNA, a marker of S-phase to detect the DNA duplication and cell proliferation, was closely related with SIRT1. The up-regulated expression of SIRT1 and PCNA after 0.1, 1 and 2.5 μM RES treatment was the underlying mechanism for the cell proliferation boost. Moreover, it was revealed that the activity and half-life of p53 could be reduced by SIRT1, and result in increase in cell survival and decrease in cell apoptosis under various DNA-damaging conditions (Hubbard and Sinclair, 2014). Additionally, in line with the report (Chen et al., 2014) that the level of p16 was up-regulated with decreased SIRT1 expression and contribute to cell senescence and cell cycle arrest (da Luz et al., 2012; Vassallo et al., 2014), the impressed expression of SIRT1 in hUC-MSCs by 5 and 10 μM RES, along with the enhanced p53 and p16 result in cell senescence, cell cycle arrest and apoptosis. Taken together, the effect of RES on the fate of hUC-MSCs is mediated, at least in part, by Sirt1 signaling. However, it is also important to note that, gene expression could be regulated by various factors in a complex way, and the specific roles of SIRT1 may depend on the SIRT1 level, activity, subcellular localization, promoter occupancy and predominant substrates of SIRT1 in the specific microenvironment of different cells (da Luz et al., 2012). Therefore, the controversy in different literatures regarding the effects of RES and SIRT1 could be explained by the response of specific tissues and cells (Chen et al., 2014; Kumazaki et al., 2013; Marambaud et al., 2005; Saharan et al., 2013) and caution should be taken before establishing links between SIRT1 activation and neural protection. Moreover, it is provoking to study the cross-talk between SIRT1 signaling and many other signaling pathways, such as the Wnt/β-catenin, PI3K/AKT (Tsai et al., 2013), ER-ERK1/2 (Dai et al., 2007), ER/NO/cGMP (Song et al., 2006), etc.
Herein, we suggest future studies on the electrophysiology of the neural-like cells, which would further confirm the effects of RES on the function of the neural differentiated hUC-MSCs. Effects of prolonged RES exposure also need to be explored in order to analyze the duration-dependent effect of RES on the biology of hUC-MSCs. Moreover, researches on animals models are warranted to facilitate the clinical application of RES pre-modified hUC-MSCs in treating Alzheimer’ s disease, Parkinson, stroke, trauma induced brain injury and other neuro-degenerative and neural injury disorders.
The present study indicates that RES exerts concentration-dependent effects on the fate of hUC-MSCs
. Sequences of primers for qRT-PCR
Gene | Sequence | bp |
---|---|---|
Ngn1 | 5′-CCAAAGACTTGCTCCACACA-3′ (F) | 164 |
Ngn2 | 5′-CCTGGAAACCATCTCACTTCA-3′ (F) | 81 |
Mash1 | 5′-CCAGTTGTACTTCAGCACC-3′ (F) | 73 |
SIRT1 | 5′-GAGATAACCTTCTGTTCGGTGATGAA-3′ (F) | 194 |
PCNA | 5′-GTAGTAAAGATGCCTTCTGGTG-3′ (F) | 190 |
p53 | 5′-CCGCAGTCAGATCCTAGCG-3′ (F) | 118 |
p21 | 5′-CCTGTCACTGTCTTGTACCCT-3′ (F) | 130 |
p16 | 5′-CTTGCCTGGAAAGATACCG-3′ (F) | 94 |
GAPDH | 5′-ACCCACTCCTCCACCTTTGA-3′ (F) | 125 |
Mol. Cells 2016; 39(5): 418-425
Published online May 31, 2016 https://doi.org/10.14348/molcells.2016.2345
Copyright © The Korean Society for Molecular and Cellular Biology.
Xinxin Wang1,3, Shanshan Ma2,3, Nan Meng1, Ning Yao2, Kun Zhang2, Qinghua Li2, Yanting Zhang2, Qu Xing2, Kang Han2, Jishi Song2, Bo Yang1,*, and Fangxia Guan1,2,*
1The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China, 2School of Life Sciences, Zhengzhou University, Zhengzhou 450001, Henan Province, China, 3These authors contributed equally to this work.
Correspondence to:*Correspondence: guanfangxia@126.com (FG); yangbo96@126.com (BY)
Resveratrol (RES) plays a critical role in the fate of cells and longevity of animals via activation of the sirtuins1 (SIRT1) gene. In the present study, we intend to investigate whether RES could promote the self-renewal and neural-lineage differentiation in human umbilical cord derived MSCs (hUC-MSCs)
Keywords: human umbilical cord derived mesenchymal stem cells, neural differentiation, resveratrol, self-renewal, SIRT1
Mesenchymal stem cells (MSCs) have been spotlighted in the neural regenerative medicine (Can and Karahuseyinoglu, 2007). However, MSCs become senescent after consecutively passage
Sirt1 gene is a member of the Sirtuins family which possess histone deacetylase activity and plays an important role in regulating the aging of cells and neurodegenerative diseases. However, the protein level of SIRT1 declines with MSCs passage
Since the controversial effects of RES on different cells retard the research progress and limit its clinical application, this study aims to investigate the role of RES in the fate of human umbilical cord derived MSCs (hUC-MSCs)
hUC-MSCs were isolated as previously described (Ma et al., 2014). The cultured cells were harvested at passage 4 (P4) for a typical experiment. The study was approved by the Ethics Committees of the First Affiliated Hospital of Zhengzhou University and consented by the donors of umbilical cord.
hUC-MSCs were harvested and washed with ice-cold PBS. Cells were labeled with the following antibodies: CD29-PE, CD44-FITC, HLA-ABC-FITC, HLA-DR-FITC, CD34-PE, CD45-PE, CD51-FITC, and CD105-PE (BD Bioscience, USA) before analyzed by the FACS Calibur flow cytometer (Becton-Dickinson, USA).
RES 50 μM stock solution (trans-3,4,5-trihydroxystilbene; R5010; Sigma, USA) was suspended in DMSO (Sigma, USA) and diluted to specific concentration before use. The final working concentration of DMSO was less than 0.1% for the
Cell viability was quantitatively determined by a CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the manufacture’s protocol. Briefly, hUC-MSCs at P4 were plated in 96-well plate at a density of 1000 cells/well and cultured in 100 μM DMEM/F12 with RES (0.1, 1, 2.5, 5, 10, 20, 50 and 100 μM) for 1, 3, 6, 8, 10 and 12 days, rinsed with PBS and incubated in DMEM/F12 with 10 μl CCK-8 for each well for 2 h at 37°C and the absorbance (OD) of the solution was measured by a microplate reader (Bio-Rad, USA) at a wavelength of 450 nm.
To examine the effect of RES on the proliferation rate of hUC-MSCs, cells at P4 were seeded in 24-well plate (8000 cells/well) for 24 hours to allow for stabilization and then exposed to RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days before EdU (Ribobio, China) was added. The EdU labeling duration was determined at 24h according to the average cell-doubling time for hUC-MSCs. Images were visualized using fluorescent microscope (Olympus, Japan). The red (EdU-labeled) and blue (Hoechst-labeled) cells were counted.
hUC-MSCs at P4 were plated at similar density in their respective media and cultured for 6 days before senescence-associated β-galactosidase (SA-β-gal) staining (Beyotime, China). Briefly, cells were fixed in 4% (v/v) formaldehyde for 10 min before stained with SA-β-gal-staining solution at pH 6.0 for 12 h. The SA-β-gal-positive cells exhibited blue color. The number of positive cells was counted under a phase-contrast microscope. The experiments were carried out in triplicate.
hUC-MSCs at P4 were incubated with RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days before propidium iodide (PI) staining performed as previously described (Ma et al., 2014).
Cell apoptosis was analyzed by the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) following the manufacturer’s instructions. Briefly, hUC-MSCs at P4 were incubated with RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days and then collected for FITC-Annexin V and PI staining as previously reported (Zhu et al., 2016).
hUC-MSCs at P4 were incubated with RES (0, 0.1, 1, 2.5, 5 and 10 μM) for 6 days before neural differentiation induction, which was modified on the basis of the protocol previously described (Karahuseyinoglu et al., 2007). hUC-MSCs were pre-induced for 24 h in DMEM-LG containing 20% fetal bovine serum (FBS), 10 ng/ml basic fibroblast growth factor (bFGF, Peprotech, USA) was added for an additional 24 h, then incubated in the induction medium for another 24 h: DMEM-LG with 2% DMSO (Sigma, USA), 100 μM butylated hydroxyanisole (Sigma, USA), 25 mM KCl, 10 μM forskolin, 5 μg/ml insulin and 1 μM hydrocortisone, followed by neuro-basal medium, supplemented with 10% FBS, 10 ng/ml epidermal growth factor (Sigma, USA), 10 ng/ml bFGF, 1 × N2 supplement (Gibco, USA), 1 × B-27 supplement (Gibco, USA), and 2 mM L-glutamine (Sigma, USA) for the maintenance of differentiation.
The 1 × 105 cells were plated on 24-well plate and after rinsing with PBS, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After blocking with 10% normal goat serum, cells were incubated overnight at 4°C with specific antibodies against Nestin (1:50, SantaCruz, USA), βIII-tubulin (1:100, Cell Signaling Technology, USA) or Neuron Specific Enolase (NSE, 1:3,000, Abcam, USA). After rinsing in PBS three times, cells were incubated for 1 hour at room temperature in Cy3-conjugated anti-mouse/-rabbit anti IgG (1:1,000, Molecular Probes, USA) for visualization, followed by DAPI staining (Biotech, China). Immunofluorescent images were visualized with fluorescence microscopy. The negative control was incubated with PBS instead of primary antibody, and no immunoreactivity was observed.
Total RNA was isolated using Trizol reagent (Invitrogen) according to the instructions, and mRNA was reverse transcribed using PrimeScript™ RT-PCR Kit (TaKaRa, Japan). qRT-PCR were performed on an ABI 7500 real-time PCR system (Thermo Fisher Scientific, USA) using a SYBR Premix Ex Taq™ (Prefect Real Time, Takara, Japan). Glyceral dehyde 3-phosphate dehydrogenase (GAPDH) was employed as the internal standard. Relative expression levels of different genes were calculated using the 2?ΔΔCt. The sequences of primers for qRT-PCR were shown in Table 1.
Cells were incubated with RES of the indicated concentrations above before Western Blotting, which was performed as previously described (Ma et al., 2014). Primary antibodies (SIRT1, 1:1000; PCNA, 1:1000; p53,1:1000; p21,1:500; p16,1:500, cell signaling technology, USA) were employed.
Data were expressed as mean ± standard deviation (SD). Analysis of Variance (ANOVA) followed by LSD test were employed to determine the significance between different groups.
hUC-MSCs grew out from the Wharton’s jelly 7?10 days after isolation, and displayed a monolayer of bipolar spindle-like or fibroblast-like morphology with a whirlpool-like array. However, the cells underwent morphological changes and became flat, swollen and irregular as the passage number increased (Figs. 1A?1F). Flow cytometry revealed that hUC-MSCs were positive for stromal matrix markers (CD44, CD105), integrin markers (CD29, CD51), and HLA-ABC, while negative for hematopoietic lineage markers (CD34, CD45) and HLA-DR (data not shown).
We found that RES exhibited a dose-dependent promoting effect on the viability of hUC-MSCs at a concentration of 0.1, 1 and 2.5 μM after incubating for 6 days (
The effect of RES on cell proliferation was analyzed by EdU staining at different concentrations and time points. As shown in Figs. 3A and 3B, 0.1, 1 and 2.5 μM RES significantly increased the percentages of EdU-labeling cells, compared with the control. However, the proliferation of hUC-MSCs was significantly inhibited when cells were challenged with 5 and 10 μM RES (
As shown in Figs. 3C and 3D, there were significantly less SA-β-gal positive cells in the 0.1, 1 and 2.5 μM RES treated groups (
Flow cytometry was used to detect the increased percentages of cells in S and G2/M phases (Fig. 3E). There was a reduction in the percentage in G0/G1 phase in hUC-MSCs treated with 0.1, 1 and 2.5 μM RES. However, there was no significant difference when compared with the control (
Flow cytometric detection of Annexin-V-FITC in / Propidium iodide (PI)staining revealed a dose-dependent downward trend in the percentages of apoptotic and necrotic cells among the RES (0.1, 1 and 2.5 μM) treatment groups (Fig. 3F), although there was no significant difference when compared with the control (
hUC-MSCs underwent neuronal differentiation by a multi-step protocol. Most differentiated hUC-MSCs demonstrated neural appearance with retracted cell body, refractive karyon, protruded dendrites and axons after neural induction (Fig. 4A), while hUC-MSCs without induction retained long fusiform shape. RES pre-incubation facilitated the neural differentiation of hUC-MSCs in all concentrations (Fig. 4A). There were more neuronal-shaped cells in RES-pretreated hUC-MSCs than that without pretreatment, especially at the concentration of 2.5, 5 and 10 μM (
The qRT-PCR analysis revealed that after neural differentiation, the expression of Neurogenin1 (Ngn1) was decreased, while Ngn2 and Mash1 were increased (
To explore the molecular mechanism of RES on the cell viability, proliferation, cell cycle, apoptosis and senescence of hUC-MSCs, we examined the expression of critical genes involved in those biological processes by qRT-PCR and Western Blot, including SIRT1, PCNA, p53, p21, and p16. As indicated in Fig. 5A, the expression of SIRT1 and PCNA in hUC-MSCs were significantly increased by 0.1, 1 or 2.5 μM RES (
Regulating the self-renewal and lineage commitment of stem cells is the key factor in regenerative medicine. Studies have shown that RES promotes the survival of adipose-derived mesenchymal stem cells (Pinarli et al., 2013), enhance the proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (Dai et al., 2007) and direct the neuronal differentiation of human bone marrow mesenchymal stem cells (Joe et al., 2015). However, some researchers argued that RES inhibited the proliferation of neural progenitor cells (Park et al., 2012), negatively regulated the neurogenic potential of neural precursors (Saharan et al., 2013). These results suggested that RES might play different or even opposite role in different cell type under specific milieu. Since hUC-MSCs are promising candidate for some intractable diseases, it is essential to gain better understanding of the effect of RES on hUC-MSCs development.
The present study revealed that incubation with 0.1, 1 and 2.5 μM RES for 6 days
In addition, we found that hUC-MSCs expressed low level of Nestin, βIII-tubulin and NSE, indicators of multi-potent neural stem cell, pre-mature neuron and mature neurons respectively (Heng et al., 2014), suggesting the neural differentiation potential of hUC-MSCs. RES could facilitate neural-lineage development of hUC-MSCs in a dose-dependent manner, as evidenced by morphological characters and up-regulated expression of βIII-tubulin and NSE with down-regulated Nestin. Simultaneously, the expression of proneural basic helix loop helix (bHLH) transcription factors, Ngn1, Ngn2 and Mash1 changed. Ngn1 was significantly down-regulated, while Ngn2 and Mash1 were up-regulated in the differentiated hUC-MSCs, and that could be further enhanced by pre-incubation with 2.5, 5 and 10 μM RES. In light with previous studies, Ngn1 is a proneural bHLH transcription factor expressed in newly committed neuronal progenitors and immature neurons and plays an essential role in neurogenesis. It is expressed at a high level in the early stage and decreased in the late stage of neuronal differentiation of MSCs (Cardozo et al., 2012). Ngn2 and Mash1 are activator-type bHLH genes essential for neuronal fate determination (Cardozo et al., 2012; Yu et al., 2013). This study reveals that RES facilitates neuronal differentiation through induction of proneural transcription factor genes in hUC-MSCs, and suggests that RES pretreatment of hUC-MSCs might be beneficial to stem cell based therapy for neural system injury or diseases.
We sought to elucidate the mechanistic basis for these observations and found that RES could regulate the SIRT1 signaling in the hUC-MSCs, which was closely related with cell self-renewal (Liu et al., 2012), senescence (Chen et al., 2014; Rehan et al., 2014), apoptosis (Godoy et al., 2014) and neural differentiation (Hisahara et al., 2008). Consisted with previous studies (Rathbone et al., 2009), the expression of PCNA, a marker of S-phase to detect the DNA duplication and cell proliferation, was closely related with SIRT1. The up-regulated expression of SIRT1 and PCNA after 0.1, 1 and 2.5 μM RES treatment was the underlying mechanism for the cell proliferation boost. Moreover, it was revealed that the activity and half-life of p53 could be reduced by SIRT1, and result in increase in cell survival and decrease in cell apoptosis under various DNA-damaging conditions (Hubbard and Sinclair, 2014). Additionally, in line with the report (Chen et al., 2014) that the level of p16 was up-regulated with decreased SIRT1 expression and contribute to cell senescence and cell cycle arrest (da Luz et al., 2012; Vassallo et al., 2014), the impressed expression of SIRT1 in hUC-MSCs by 5 and 10 μM RES, along with the enhanced p53 and p16 result in cell senescence, cell cycle arrest and apoptosis. Taken together, the effect of RES on the fate of hUC-MSCs is mediated, at least in part, by Sirt1 signaling. However, it is also important to note that, gene expression could be regulated by various factors in a complex way, and the specific roles of SIRT1 may depend on the SIRT1 level, activity, subcellular localization, promoter occupancy and predominant substrates of SIRT1 in the specific microenvironment of different cells (da Luz et al., 2012). Therefore, the controversy in different literatures regarding the effects of RES and SIRT1 could be explained by the response of specific tissues and cells (Chen et al., 2014; Kumazaki et al., 2013; Marambaud et al., 2005; Saharan et al., 2013) and caution should be taken before establishing links between SIRT1 activation and neural protection. Moreover, it is provoking to study the cross-talk between SIRT1 signaling and many other signaling pathways, such as the Wnt/β-catenin, PI3K/AKT (Tsai et al., 2013), ER-ERK1/2 (Dai et al., 2007), ER/NO/cGMP (Song et al., 2006), etc.
Herein, we suggest future studies on the electrophysiology of the neural-like cells, which would further confirm the effects of RES on the function of the neural differentiated hUC-MSCs. Effects of prolonged RES exposure also need to be explored in order to analyze the duration-dependent effect of RES on the biology of hUC-MSCs. Moreover, researches on animals models are warranted to facilitate the clinical application of RES pre-modified hUC-MSCs in treating Alzheimer’ s disease, Parkinson, stroke, trauma induced brain injury and other neuro-degenerative and neural injury disorders.
The present study indicates that RES exerts concentration-dependent effects on the fate of hUC-MSCs
. Sequences of primers for qRT-PCR.
Gene | Sequence | bp |
---|---|---|
Ngn1 | 5′-CCAAAGACTTGCTCCACACA-3′ (F) | 164 |
Ngn2 | 5′-CCTGGAAACCATCTCACTTCA-3′ (F) | 81 |
Mash1 | 5′-CCAGTTGTACTTCAGCACC-3′ (F) | 73 |
SIRT1 | 5′-GAGATAACCTTCTGTTCGGTGATGAA-3′ (F) | 194 |
PCNA | 5′-GTAGTAAAGATGCCTTCTGGTG-3′ (F) | 190 |
p53 | 5′-CCGCAGTCAGATCCTAGCG-3′ (F) | 118 |
p21 | 5′-CCTGTCACTGTCTTGTACCCT-3′ (F) | 130 |
p16 | 5′-CTTGCCTGGAAAGATACCG-3′ (F) | 94 |
GAPDH | 5′-ACCCACTCCTCCACCTTTGA-3′ (F) | 125 |
Jeong-Woo Park, Jihyeon Jeong, and Young-Seuk Bae
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