Accepted Article
Accepted Date: 14-May-2014 Article Type: Original Article
Senescence of Bone Marrow Mesenchymal Stromal Cells is accompanied by Activation of p53/p21 Pathway in Myelodysplastic Syndromes Fei Chengming, Zhao Youshan, Guo Juan, Gu Shucheng, Li Xiao, Chang Chunkang Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China
Corresponding author: Chang Chunkang, MD, PhD, Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital Mail address: No.600, Yi Shan Road, Shanghai, China Postcode: 200233 Tel: 86-21-24058336 Fax: 86-21-64088983 E-mail:[email protected]
Funding: This study was supported in part by the National Nature Science Foundation of China (NNSFC81170463)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejh.12385 This article is protected by copyright. All rights reserved.
Accepted Article
The contribution of bone marrow mesenchymal stromal cells (BMMSCs) to the pathogenesis of myelodysplastic syndrome (MDS) has created controversies. In this study, we confirmed that BMMSCs from MDS patients showed prominent features of senescence, which were characterized by increased cell size, decreased proliferation and colony forming potential, alteration of cytoskeleton and increased senescence-associated β-galactosidase (SA-β-Gal) activity. Interestingly, the apoptosis assay results showed that the percentage of apoptosis cells was very low and the difference was not significant between MDS patients and normal controls. Moreover, the osteogenic differentiation potential of BMMSCs from lower-risk but not higher-risk MDS was impaired, indicated by cytochemical stainings and reduced expressions of RUNX2. In addtion，BMMSCs from MDS patients had impaired hematopoietic supporting function . Furthermore, the expression of p53 and p21 which played an important role in regulating the senescence progress of BMMSCs was significantly increased, whereas levels of p16 and pRb expression were not change in the BMMSCs from MDS patients. Taken together, our comprehensive analysis shows that BMMSCs from MDS patients exhibited senescent behavior and activation of p53/p21 pathway probably played an important role in the senescence process.
Key words: bone marrow mesenchymal stromal cells, senescence, p53/p21 pathway, microenvironment, myelodysplastic syndromes
Introduction Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal diseases characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of transformation to acute myeloid leukemia（AML）. The development of MDS
This article is protected by copyright. All rights reserved.
Accepted Article
is a complex and multistep process, it has been clearly recognized that the abnormality of bone marrow microenvironment that seems contribute to hematopoietic failure is one of important reasons related to MDS [1-3]. Bone marrow mesenchymal stromal cells (BMMSCs) have been suggested to be the precursor cells in the bone marrow marrow and their further differentiated progeny constitute the functional components of bone marrow microenvironment that support haematopoiesis [4-6]. Thus, it is reasonable to assume that a primary BMMSCs defect might exist in MDS. However, knowledge is very limited for the role of BMMSCs in the pathogenesis of MDS, previous studies have shown conflicting results with regard to the biological behavior of BMMSCs in MDS, such as proliferative potential, phenotype, differentiation capacity, cytogenetic profile, immunosuppressive properties and in vitro hematopoiesis supporting capacity [7-10]. Our previous study has observed cytogenetic aberrations in BMMSCs from MDS patients [11]. The reported cytogenetic abnormalities in MDS-derived BMMSCs are quite variable, reflecting the predisposition to genetic instability [12, 13]. This genetically unstable stroma may facilitate the growth of malignant clonal cells leading to a permissive milieu [14, 15].
It was also reported that cellular senescence might represent a mechanism responsible for the impaired growth of BMMSCs observed in MDS [16]. Cellular senescence, a state of permanent cell cycle arrest, is linked to organism aging and disease development [17]. Senescent BMMSCs were characterized by increased cell size, decreased proliferation and colony forming potential, and increased senescence-associated β-galactosidase (SA-β-Gal) activity [18]. The senescence growth arrest is established and maintained by the p53/p21 and p16/pRb pathways, these pathways interact but can independently regulate the cell senescence progress [19, 20].
This article is protected by copyright. All rights reserved.
Accepted Article
In the current study, we further investigate the constitutive differences between MDS-derived BMMSCs and normal controls BMMSCs. Besides the general characteristics of BMMSCs, such as proliferation, colony forming , osteogenic differentiation potential and hematopoiesis supporting capacity, we also evaluated cytoskeleton and SA-β-Gal activity of BMMSCs. Furthermore, p53/p21 and p16/pRb pathways, which were involved in the cellular senescence, were demonstrated in this study.
Materials and methods Patients Thirty-two patients with MDS (aged from37 to 80) and seven (aged from 41 to 69) normal controls were investigated in this study. None of patients had received treatment during this period of time, other than supportive therapy. MDS was diagnosed in accordance with the minimum diagnostic criteria establishhed by the Conference on MDS (Vienna, 2006) [21]. The classification and prognostic risk scoring of MDS were performed according to the WHO criteria [22] and the International Prognostic Scoring System (IPSS) [23]. All patients were classified for the study as “lower-risk” (IPSS-low/int-1), and as “higher-risk” (IPSS-int-2/high). Detailed information about these MDS patients is shown in Table 1. All subjects provided informed consent. The research was approved by the ethics committee of the Sixth Hospital affiliated with Shanghai Jiao Tong University, and all patient relevant research strictly abided by the Declaration of Helsinki. Isolation and culture of BMMSCs Mononuclear cells (MNCs) were isolated from fresh BM aspirates and separated by a Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden). MNCs were seeded at an initial This article is protected by copyright. All rights reserved.
Accepted Article
concentration of 1X106 cells/ml and cultured in Human Mesenchymal Stem Cell Growth Medium (Cyagen Biosciences Inc, Guangzhou, China) supplemented with 10% fetal bovine serum (FBS), glutamine, 100 U/ml Penicillin-Streptomycin at 37 with 5% CO2 in fully humidified atmosphere. After 72 h, the culture medium was replaced and non-adherent cells were removed，thereafter medium was replaced every 3-4 days. Upon reaching more than 80-90% confluency, cells were detached with 0.25% trypsin–EDTA (Gibico), At the third passage (P3), adherent BMMSCs were harvested and utilized for experiment analyses. Isolation of CD34+ cells CD34+ cells were isolated by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) from BMMNCs. CD34+ cells purity was evaluated with FACS (BD Biosciences, NJ, USA) and was >90%. Cell growth assay BMMSCs were seeded at 2000 cells/well in 96-well plate for 1-7 days. At different time points the cell number was measured using Cell-Counting Kit-8 (CCK8) proliferation assay kit (Beyotime, China). BMMSCs were mixed with 10 μl of CCK-8 solution/well and incubated for further 2 hours at 37°C. The amount of formazan dye generated by cellular dehydrogenase activity was measured for absorbance at 450 nm with a microplate reader. The optical density (OD) values of each well represented the survival/proliferation of BMMSCs. Colony-forming unit-fibroblast (CFU-F) assay BMMSCs were seeded seeded at 100 cells/ well in 6-well plate. After 15 days of culture with medium replaced every 3 days, the cultures were fixed and stained with crystal violet staining solution in methanol (Beyotime, China) for 20 min at RT. The wells were washed with water and allowed to dry. Colonies were counted macroscopically, and data were reported as colony numbers per well.
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Accepted Article
Cell cycle assay BMMSCs were fixed with 70% (w/v) ice-cold ethanol for 24h at 4 , washed with PBS, then treated with 50μg/ml RNase for 30 min at 37 followed by 10μg/ml propidium iodine for 30min in the dark at 4 . DNA content was analyzed by FACS Calibur system (BD, Mountain View, Calif, USA) using the Cellquest software. Apoptosis assay The number of apoptotic cells was quantified using the Annexin-V-FITC Apoptosis Detection Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Early apoptotic cells were defined as Annexin-V-positive, 7AAD-negative cells. The analyses were performed on an FACS flow cytometer (Becton-Dickinson, Sunnyvale, CA). Cytoskeleton assay BMMSCs were washed with PBS and fixed in 4 % paraformaldehyde for 30 min, then incubated with 0.01mg/ml fluorescein isothiocyanate(FITC)-conjugated phalloidin(Cytoskeleton, Inc. Denver, USA)overnight at 4 , the nuclei were counterstained with DAPI, then mounting to the slides with Fluoromount (Fisher). The stained cells were viewed at 100X magnification using a fluorescent microscopy (Olympus, Tokyo, Japan), and the cell morphology was monitored. To quantify the fluorescence and to measure cells area, we performed image analysis using Image-Pro Plus 6.0 software. One hundred cells were randomly analyzed for each sample, and the optical density of the signals was quantitated.. Single images were digitized for image analysis at 256 gray levels. The single cells were randomly selected by the operators by using the cursor, and then positive areas were automatically estimated. Constant optical threshold and filter combination were used. SA-β-Gal assay BMMSCs cultured on plates were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After rinsing with PBS, cells were This article is protected by copyright. All rights reserved.
Accepted Article
incubated with a freshly prepared SA-β-Gal staining solution (Beyotime, China) for 16 h at 37°C. Under light microscopy, the number of blue cells (SA-β-Gal positive cells) out of at least 500 cells in 10 randomly chosen fields was used to calculate the percentage of senescent cells. Osteogenic differentiation assay BMMSCs were seeded at 3×104 cells/well in 6-well plate pre-coated with gelatin solution in Human Mesenchymal Stem Cell Osteogenic Differentiation Medium( Cyagen Biosciences Inc，Guangzhou,China) containing 10% FBS , 100 U/ml Penicillin-Streptomycin, 0.2mM Ascorbate, 10mM β-Glycerophosphate, 10-7M Dexamethasone , then the medium was replaced every 3-4 days. After 3 weeks differentiation, cells can be fixed and stained with Alizarin red, visualized using light microscopy. After photography, the bound staining was eluted with 10% (wt/vol) cetylpyridinium chloride (sigma), and alizarin red-S in samples was quantified by measuring absorbance at 572 nm. Reverse transcriptase–polymerase chain reaction (RT- PCR) Total RNA was extracted from BMMSCs using the RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. cDNA was prepared using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada) following the manufacturer’s protocol. To perform real-time PCR, each 20μl RT-PCR mix contained 10μl of RealMasterMix (Takara, Dalian, China), 0.8μl of each prime, 2μL cDNA, and distilled water. PCR was performed on an ABI 7500 real-time PCR machine (Applied Biosystems). Conditions were as follows: hold stage was 95 °C for 30 s, cycling was 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. The primer sequences of p21, p53, p16, pRb and RUNX2 are listed in Table 2. The threshold cycle (Ct) value was subsequently determined, and the relative quantification of mRNA expression was calculated using the comparative Ct method. The relative quantification value of the target, which was normalized to that of an
This article is protected by copyright. All rights reserved.
Accepted Article
endogenous control (GAPDH gene) and relative to that of a calibrator (the mean expression level of normal controls), was expressed as 2−△△Ct(fold difference), where△ Ct = Ct of the target gene − Ct of the endogenous control gene (GAPDH), and
△ △ Ct =△ Ct of the samples for the target gene − △ Ct of the calibrator for the target gene. Western blot analysis Whole cell lysates were obtained from BMMSCs and equal quantities of protein were separated by 12% SDS-PAGE and blotted onto a PVDF membrane. The PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% skimmed milk powder for 1 h, incubated with p21
(1:100; Abcam
rabbit
p53
(1:1000; Abcam Inc.),
rabbit
Inc.) and mouse GAPDH (1:1000; Abcam Inc) as loading
control. Membranes were incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Specific bands were visualized by using ECL Western Blotting Detection Reagents (Amersham Biosciences).
Hematopoietic assay 1.2×106 BMMSCs were cultivated on 96-well plates until at least 80% confluence was reached and then irradiated with 30 Gray. Afterwards，1×104 CD34+ cells were plated on these BMMSCs feeder layers and cultivated in long term bone marrow culture (LTBMC) medium consisting of MyeloCult H5100 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 10-6mol/L hydrocortisone (Sigma) for 5 weeks with weekly changes of culture medium. After 5 weeks, the medium was replaced by clonogenic methylcellulose medium consisting of 50 ng/mL of SCF, 10 ng/mL of IL-3 (Sigma), 20 ng/mL of GM-CSF (Sigma), and 4 U/mL of erythropoietin (Sigma). After 2 weeks, hematopoietic colonies greater than 50 cells were counted.
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Accepted Article
Statistical analysis The data were presented as mean± SD. All statistical analyses were performed using the SPSS 17.0 System. Multiple pairwise comparisons were made using one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.
Results BMMSCs from MDS patients
exhibited abnormal cellular morphology and
cytoskeleton We first assessed cell morphology and found that normal cells displayed fibroblast-like morphology whereas BMMSCs from MDS patients were larger and appeared disorganized (Fig. 1A). This phenotypic change correlates with alteration of cytoskeleton content, immunostaining for filamentous actin (F-actin) revealed deformation of actin filaments running through the cell body of MDS-derived BMMSCs when compared to the control ( Fig. 1B). The quantification of cell size showed that BMMSCs from MDS patients were larger than normal cells(Fig.1C, p
Accepted Date: 14-May-2014 Article Type: Original Article
Senescence of Bone Marrow Mesenchymal Stromal Cells is accompanied by Activation of p53/p21 Pathway in Myelodysplastic Syndromes Fei Chengming, Zhao Youshan, Guo Juan, Gu Shucheng, Li Xiao, Chang Chunkang Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China
Corresponding author: Chang Chunkang, MD, PhD, Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital Mail address: No.600, Yi Shan Road, Shanghai, China Postcode: 200233 Tel: 86-21-24058336 Fax: 86-21-64088983 E-mail:[email protected]
Funding: This study was supported in part by the National Nature Science Foundation of China (NNSFC81170463)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejh.12385 This article is protected by copyright. All rights reserved.
Accepted Article
The contribution of bone marrow mesenchymal stromal cells (BMMSCs) to the pathogenesis of myelodysplastic syndrome (MDS) has created controversies. In this study, we confirmed that BMMSCs from MDS patients showed prominent features of senescence, which were characterized by increased cell size, decreased proliferation and colony forming potential, alteration of cytoskeleton and increased senescence-associated β-galactosidase (SA-β-Gal) activity. Interestingly, the apoptosis assay results showed that the percentage of apoptosis cells was very low and the difference was not significant between MDS patients and normal controls. Moreover, the osteogenic differentiation potential of BMMSCs from lower-risk but not higher-risk MDS was impaired, indicated by cytochemical stainings and reduced expressions of RUNX2. In addtion，BMMSCs from MDS patients had impaired hematopoietic supporting function . Furthermore, the expression of p53 and p21 which played an important role in regulating the senescence progress of BMMSCs was significantly increased, whereas levels of p16 and pRb expression were not change in the BMMSCs from MDS patients. Taken together, our comprehensive analysis shows that BMMSCs from MDS patients exhibited senescent behavior and activation of p53/p21 pathway probably played an important role in the senescence process.
Key words: bone marrow mesenchymal stromal cells, senescence, p53/p21 pathway, microenvironment, myelodysplastic syndromes
Introduction Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal diseases characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of transformation to acute myeloid leukemia（AML）. The development of MDS
This article is protected by copyright. All rights reserved.
Accepted Article
is a complex and multistep process, it has been clearly recognized that the abnormality of bone marrow microenvironment that seems contribute to hematopoietic failure is one of important reasons related to MDS [1-3]. Bone marrow mesenchymal stromal cells (BMMSCs) have been suggested to be the precursor cells in the bone marrow marrow and their further differentiated progeny constitute the functional components of bone marrow microenvironment that support haematopoiesis [4-6]. Thus, it is reasonable to assume that a primary BMMSCs defect might exist in MDS. However, knowledge is very limited for the role of BMMSCs in the pathogenesis of MDS, previous studies have shown conflicting results with regard to the biological behavior of BMMSCs in MDS, such as proliferative potential, phenotype, differentiation capacity, cytogenetic profile, immunosuppressive properties and in vitro hematopoiesis supporting capacity [7-10]. Our previous study has observed cytogenetic aberrations in BMMSCs from MDS patients [11]. The reported cytogenetic abnormalities in MDS-derived BMMSCs are quite variable, reflecting the predisposition to genetic instability [12, 13]. This genetically unstable stroma may facilitate the growth of malignant clonal cells leading to a permissive milieu [14, 15].
It was also reported that cellular senescence might represent a mechanism responsible for the impaired growth of BMMSCs observed in MDS [16]. Cellular senescence, a state of permanent cell cycle arrest, is linked to organism aging and disease development [17]. Senescent BMMSCs were characterized by increased cell size, decreased proliferation and colony forming potential, and increased senescence-associated β-galactosidase (SA-β-Gal) activity [18]. The senescence growth arrest is established and maintained by the p53/p21 and p16/pRb pathways, these pathways interact but can independently regulate the cell senescence progress [19, 20].
This article is protected by copyright. All rights reserved.
Accepted Article
In the current study, we further investigate the constitutive differences between MDS-derived BMMSCs and normal controls BMMSCs. Besides the general characteristics of BMMSCs, such as proliferation, colony forming , osteogenic differentiation potential and hematopoiesis supporting capacity, we also evaluated cytoskeleton and SA-β-Gal activity of BMMSCs. Furthermore, p53/p21 and p16/pRb pathways, which were involved in the cellular senescence, were demonstrated in this study.
Materials and methods Patients Thirty-two patients with MDS (aged from37 to 80) and seven (aged from 41 to 69) normal controls were investigated in this study. None of patients had received treatment during this period of time, other than supportive therapy. MDS was diagnosed in accordance with the minimum diagnostic criteria establishhed by the Conference on MDS (Vienna, 2006) [21]. The classification and prognostic risk scoring of MDS were performed according to the WHO criteria [22] and the International Prognostic Scoring System (IPSS) [23]. All patients were classified for the study as “lower-risk” (IPSS-low/int-1), and as “higher-risk” (IPSS-int-2/high). Detailed information about these MDS patients is shown in Table 1. All subjects provided informed consent. The research was approved by the ethics committee of the Sixth Hospital affiliated with Shanghai Jiao Tong University, and all patient relevant research strictly abided by the Declaration of Helsinki. Isolation and culture of BMMSCs Mononuclear cells (MNCs) were isolated from fresh BM aspirates and separated by a Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden). MNCs were seeded at an initial This article is protected by copyright. All rights reserved.
Accepted Article
concentration of 1X106 cells/ml and cultured in Human Mesenchymal Stem Cell Growth Medium (Cyagen Biosciences Inc, Guangzhou, China) supplemented with 10% fetal bovine serum (FBS), glutamine, 100 U/ml Penicillin-Streptomycin at 37 with 5% CO2 in fully humidified atmosphere. After 72 h, the culture medium was replaced and non-adherent cells were removed，thereafter medium was replaced every 3-4 days. Upon reaching more than 80-90% confluency, cells were detached with 0.25% trypsin–EDTA (Gibico), At the third passage (P3), adherent BMMSCs were harvested and utilized for experiment analyses. Isolation of CD34+ cells CD34+ cells were isolated by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) from BMMNCs. CD34+ cells purity was evaluated with FACS (BD Biosciences, NJ, USA) and was >90%. Cell growth assay BMMSCs were seeded at 2000 cells/well in 96-well plate for 1-7 days. At different time points the cell number was measured using Cell-Counting Kit-8 (CCK8) proliferation assay kit (Beyotime, China). BMMSCs were mixed with 10 μl of CCK-8 solution/well and incubated for further 2 hours at 37°C. The amount of formazan dye generated by cellular dehydrogenase activity was measured for absorbance at 450 nm with a microplate reader. The optical density (OD) values of each well represented the survival/proliferation of BMMSCs. Colony-forming unit-fibroblast (CFU-F) assay BMMSCs were seeded seeded at 100 cells/ well in 6-well plate. After 15 days of culture with medium replaced every 3 days, the cultures were fixed and stained with crystal violet staining solution in methanol (Beyotime, China) for 20 min at RT. The wells were washed with water and allowed to dry. Colonies were counted macroscopically, and data were reported as colony numbers per well.
This article is protected by copyright. All rights reserved.
Accepted Article
Cell cycle assay BMMSCs were fixed with 70% (w/v) ice-cold ethanol for 24h at 4 , washed with PBS, then treated with 50μg/ml RNase for 30 min at 37 followed by 10μg/ml propidium iodine for 30min in the dark at 4 . DNA content was analyzed by FACS Calibur system (BD, Mountain View, Calif, USA) using the Cellquest software. Apoptosis assay The number of apoptotic cells was quantified using the Annexin-V-FITC Apoptosis Detection Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Early apoptotic cells were defined as Annexin-V-positive, 7AAD-negative cells. The analyses were performed on an FACS flow cytometer (Becton-Dickinson, Sunnyvale, CA). Cytoskeleton assay BMMSCs were washed with PBS and fixed in 4 % paraformaldehyde for 30 min, then incubated with 0.01mg/ml fluorescein isothiocyanate(FITC)-conjugated phalloidin(Cytoskeleton, Inc. Denver, USA)overnight at 4 , the nuclei were counterstained with DAPI, then mounting to the slides with Fluoromount (Fisher). The stained cells were viewed at 100X magnification using a fluorescent microscopy (Olympus, Tokyo, Japan), and the cell morphology was monitored. To quantify the fluorescence and to measure cells area, we performed image analysis using Image-Pro Plus 6.0 software. One hundred cells were randomly analyzed for each sample, and the optical density of the signals was quantitated.. Single images were digitized for image analysis at 256 gray levels. The single cells were randomly selected by the operators by using the cursor, and then positive areas were automatically estimated. Constant optical threshold and filter combination were used. SA-β-Gal assay BMMSCs cultured on plates were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After rinsing with PBS, cells were This article is protected by copyright. All rights reserved.
Accepted Article
incubated with a freshly prepared SA-β-Gal staining solution (Beyotime, China) for 16 h at 37°C. Under light microscopy, the number of blue cells (SA-β-Gal positive cells) out of at least 500 cells in 10 randomly chosen fields was used to calculate the percentage of senescent cells. Osteogenic differentiation assay BMMSCs were seeded at 3×104 cells/well in 6-well plate pre-coated with gelatin solution in Human Mesenchymal Stem Cell Osteogenic Differentiation Medium( Cyagen Biosciences Inc，Guangzhou,China) containing 10% FBS , 100 U/ml Penicillin-Streptomycin, 0.2mM Ascorbate, 10mM β-Glycerophosphate, 10-7M Dexamethasone , then the medium was replaced every 3-4 days. After 3 weeks differentiation, cells can be fixed and stained with Alizarin red, visualized using light microscopy. After photography, the bound staining was eluted with 10% (wt/vol) cetylpyridinium chloride (sigma), and alizarin red-S in samples was quantified by measuring absorbance at 572 nm. Reverse transcriptase–polymerase chain reaction (RT- PCR) Total RNA was extracted from BMMSCs using the RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. cDNA was prepared using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada) following the manufacturer’s protocol. To perform real-time PCR, each 20μl RT-PCR mix contained 10μl of RealMasterMix (Takara, Dalian, China), 0.8μl of each prime, 2μL cDNA, and distilled water. PCR was performed on an ABI 7500 real-time PCR machine (Applied Biosystems). Conditions were as follows: hold stage was 95 °C for 30 s, cycling was 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. The primer sequences of p21, p53, p16, pRb and RUNX2 are listed in Table 2. The threshold cycle (Ct) value was subsequently determined, and the relative quantification of mRNA expression was calculated using the comparative Ct method. The relative quantification value of the target, which was normalized to that of an
This article is protected by copyright. All rights reserved.
Accepted Article
endogenous control (GAPDH gene) and relative to that of a calibrator (the mean expression level of normal controls), was expressed as 2−△△Ct(fold difference), where△ Ct = Ct of the target gene − Ct of the endogenous control gene (GAPDH), and
△ △ Ct =△ Ct of the samples for the target gene − △ Ct of the calibrator for the target gene. Western blot analysis Whole cell lysates were obtained from BMMSCs and equal quantities of protein were separated by 12% SDS-PAGE and blotted onto a PVDF membrane. The PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% skimmed milk powder for 1 h, incubated with p21
(1:100; Abcam
rabbit
p53
(1:1000; Abcam Inc.),
rabbit
Inc.) and mouse GAPDH (1:1000; Abcam Inc) as loading
control. Membranes were incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Specific bands were visualized by using ECL Western Blotting Detection Reagents (Amersham Biosciences).
Hematopoietic assay 1.2×106 BMMSCs were cultivated on 96-well plates until at least 80% confluence was reached and then irradiated with 30 Gray. Afterwards，1×104 CD34+ cells were plated on these BMMSCs feeder layers and cultivated in long term bone marrow culture (LTBMC) medium consisting of MyeloCult H5100 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 10-6mol/L hydrocortisone (Sigma) for 5 weeks with weekly changes of culture medium. After 5 weeks, the medium was replaced by clonogenic methylcellulose medium consisting of 50 ng/mL of SCF, 10 ng/mL of IL-3 (Sigma), 20 ng/mL of GM-CSF (Sigma), and 4 U/mL of erythropoietin (Sigma). After 2 weeks, hematopoietic colonies greater than 50 cells were counted.
This article is protected by copyright. All rights reserved.
Accepted Article
Statistical analysis The data were presented as mean± SD. All statistical analyses were performed using the SPSS 17.0 System. Multiple pairwise comparisons were made using one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.
Results BMMSCs from MDS patients
exhibited abnormal cellular morphology and
cytoskeleton We first assessed cell morphology and found that normal cells displayed fibroblast-like morphology whereas BMMSCs from MDS patients were larger and appeared disorganized (Fig. 1A). This phenotypic change correlates with alteration of cytoskeleton content, immunostaining for filamentous actin (F-actin) revealed deformation of actin filaments running through the cell body of MDS-derived BMMSCs when compared to the control ( Fig. 1B). The quantification of cell size showed that BMMSCs from MDS patients were larger than normal cells(Fig.1C, p
![[Myelodysplastic syndromes].](/assets/img/hold.png)
