曲古抑菌素A对间充质干细胞多能基因的调控研究
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摘要
研究背景
间充质干细胞(Mesenchymal stem cells, MSCs)属于多潜能干细胞。相关研究表明,间充质干细胞对多种疾病具有治疗潜力,例如心肌梗死、神经性疾病和创伤愈合等。依据相关临床前期实验结果,针对多种疾病的临床试验正在逐渐开展中。MSCs分布于多种组织,如骨髓和脂肪组织,但数量极少。在骨髓的有核细胞中,干细胞只占约0.001%-0.01%。有研究证明,间充质干细胞也存在于脐带和胎盘,这增加了获取、使用间充质干细胞的可能性。然而想要获取足够量的间充质干细胞并应用于细胞治疗和组织工程,体外扩增仍然是其中的必需环节。
MSCs长期以来被认为是可以无限传代、扩增的干细胞。近年来的研究表明，在培养、扩增过程中,间充质干细胞会迅速出现衰老现象,细胞形态表现出明显的变化,同时伴有许多旁分泌因子的分泌异常。Oct4, Sox2和Nanog是调控胚胎干细胞自我更新、维持干细胞多向分化潜能的主要转录因子。研究发现Oct4,Sox2和Nanog基因也在MSCs中表达并参与其多能性的调控。在MSCs体外培养、扩增过程中,伴随细胞形态学改变,这些基因也出现表达水平的快速下调。研究表明,MSCs的有限培养、扩增不会引起相关基因的DNA序列改变,多潜能相关基因无显著DNA甲基化现象。然而,MSCs的表观遗传状态在培养、扩增过程中并不稳定。前期研究表明,MSCs的体外培养、扩增会引起多潜能基因启动子区域组蛋白H3-K9、14的去乙酰化,与细胞的老化现象密切相关。
曲古抑菌素A (Trichostatin A, TSA),最初作为抗真菌药物被广泛应用。最近研究发现TSA是一种有效的、特异性组蛋白去乙酰化酶(HDAC)抑制剂。TSA选择性抑制Ⅰ类和Ⅱ类HDAC,但不能作用于Ⅲ类HIDAC。已证实TSA对乳腺癌细胞有明显抑制作用和细胞毒性。根据不同的细胞类型和功能状态,TSA可参与调节多种不同的细胞活动,如细胞分化和细胞增殖等。本研究设计、使用曲古抑菌素A (TSA),作为组蛋白去乙酰化酶(HDAC)抑制剂,抑制MSCs体外培养、扩增引起的组蛋白去乙酰化,从而保持其原始性状。
第一部分人间充质干细胞的体外分离、培养、扩增及多潜能相关基因的检测研究
研究目的：
体外分离、纯化、扩增人胎盘组织来源的间充质干细胞,密切观察MSCs的细胞形态变化,探讨体外扩增对MSCs干细胞形态学特性的影响；检测多潜能相关基因、成骨分化相关基因的mRNA含量,探讨体外扩增过程对MSCs多潜能相关基因、成骨分化相关基因转录水平的影响。
方法：
1.胰酶消化、贴壁培养法体外分离培养并纯化鉴定MSCs:取38W-40W妊娠,健康供者的人胎盘组织,用预冷的磷酸盐缓冲盐水(PBS)洗涤数次,机械剁碎后,37℃水浴下0.25%胰蛋白酶消化30分钟。离心后用含10%胎牛血清和抗生素的DMEM培养基重悬沉淀、种板。37℃、5%CO2培养,隔天换液。待细胞达到80%融合时,0.25%胰蛋白酶/EDTA消化、连续传代、培养；流式细胞仪对MSCs的相关表面标志物进行检测鉴定。
2.体外扩增MSCs、观察细胞形态学变化并绘制扩增曲线：选取体外分离、纯化并鉴定成功的MSCs,当细胞生长到80%融合或以上时,连续传代、培养至10代以上。绘制细胞生长曲线,观察细胞形态学变化。
3. Real-Time PCR检测体外扩增MSCs多潜能相关基因、成骨分化相关基因转录水平：设计、合成多潜能相关基因Oct4、Sox2、Nanog、Rexl和TERT、CD133,成骨分化相关基因ALP和OPN引物,Real-Time PCR检测第1代与连续传代后(第10代)MSCs多潜能相关基因、成骨分化相关基因mRNA相对含量。
结果：
1. MSCs的体外分离、纯化：使用胰酶消化法、贴壁培养法,成功完成MSCs体外分离、纯化并鉴定成功。2. MSCs的体外扩增：采集对数生长期的细胞,进行连续传代,实现MSCs体外扩增。观察细胞形态可见：伴随扩增过程进展,MSCs形态发生显著性改变,表现为细胞增大、不均匀,形态增宽,变扁平。细胞生长曲线提示细胞增殖速度较快,细胞总数呈指数增长。
3.体外扩增MSCs多潜能相关基因、成骨分化相关基因的转录水平改变：应用Real-Time PCR检测体外扩增过程中MSCs多潜能相关基因、成骨分化相关基因的mRNA表达,结果显示：体外扩增的第10代MSCs与第1代相比,多潜能相关基因Oct4、Sox2、Nanog、Rex1和TERT、CD133,转录水平显著降低,成骨分化相关基因ALP和OPN,转录水平显著升高,差异具有统计学意义(P<0.01)。
结论：
1.胰酶消化法、贴壁培养法可以成功分离、纯化人胎盘组织来源的MSCs。
2.传统方法体外扩增MSCs,不能有效维持其干细胞的形态学特征,伴随传代次数增加,细胞形态学变化显著。
3.传统方法体外扩增MSCs,不能有效维持干细胞多向分化潜能,参与维持MSCs多向分化潜能的干细胞多潜能相关基因转录水平下调。
4.传统方法体外扩增MSCs,不能维持干细胞的未分化状态,出现向成骨细胞分化的自主分化趋势,MSCs成骨分化相关基因转录水平上调。
第二部分去乙酰化酶抑制剂曲古抑菌素A(Trichostatin A, TSA)促进MSCs增殖、稳定MSCs形态的功能及机制研究
研究目的：
TSA短期处理MSCs,筛选最佳作用浓度,观察TSA对MSCs细胞形态学和细胞增殖的影响；TSA长期培养MSCs,辅助体外扩增,验证TSA稳定MSCs细胞形态和促进细胞增殖的作用。检测TSA处理对MSCs细胞周期的影响,探讨TSA促进MSCs细胞增殖的作用机制。
方法：
1.梯度浓度TSA短期处理MSCs、观察细胞形态学改变并计数细胞增殖情况：体外分离、纯化的第3代MSCs,分别使用含0、6.25、12.5、25、50、100、200和300nM TSA的培养基,对细胞进行连续培养三天。观察梯度浓度TSA处理组细胞形态差异,计数细胞数目变化。
2.低浓度TSA长期处理MSCs、观察细胞形态学改变并计数细胞增殖情况：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO(TSA溶剂)的完全培养基,对细胞进行连续培养、传代、计数。观察TSA处理组与DMSO对照组细胞形态差异,计数细胞数目变化,绘制生长曲线,检测细胞增殖速度变化。
3.流式细胞仪检测TSA处理对MSCs细胞周期的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO (TSA溶剂)的完全培养基,对细胞进行连续培养。检测完全融合的第10代MSCs,细胞周期各阶段的细胞比例；检测30%融合的第11代MSCs,细胞周期各阶段的细胞比例。
4. Western Blot检测TSA处理对MSCs细胞周期相关蛋白的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO (TSA溶剂)的完全培养基,对细胞进行连续培养。采集TSA处理组与DMSO对照组第6代、第10代MSCs,检测细胞周期相关蛋白Cyclin B1、Cyclin D1和p21的表达。
结果：
1.TSA短期处理对MSCs细胞形态学和细胞增殖的影响：梯度浓度TSA对MSCs短期培养三天,观察MSCs细胞形态学变化及细胞增殖变化。结果显示：较低浓度TSA(6.25nM、12.5nM)培养的MSCs细胞总数显著性增多,差异具有统计学意义(P<0.01)；细胞数目增多的同时,细胞的形态学特征保持良好。较高浓度TSA(200nM、300nM)培养MSCs与低浓度TSA的培养效果相反,引起细胞总数显著性降低(P<0.01)；同时细胞形态异常改变,细胞增大,形态趋于扁平,丧失MSCs的形态学特征
2.TSA长期处理对MSCs细胞形态学和细胞增殖的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO(TSA溶剂)的完全培养基,进行体外扩增至第10代,观察细胞形态学变化和细胞增殖变化。结果显示：TSA处理组与DMSO对照组比较可见：TSA处理组细胞增殖速度、细胞总数较DMSO对照组显著性增加,差异具有统计学意义(P<0.01)；同时TSA处理组细胞的形态学特征保持良好,DMSO细胞形态显著改变；TSA处理组与DMSO对照组的MSCs,细胞的接触抑制均存在,即当细胞生长到充分融合时,细胞增殖就会停止,不会出现局灶性的多层细胞(细胞接触抑制+)
3.TSA处理对MSCs细胞周期的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA.等量DMSO (TSA溶剂)的完全培养基,进行体外扩增。取达到完全融合的第10代MSCs应用流式细胞仪进行细胞周期的检测,结果显示TSA处理组与DMSO对照组相比,处于G1期、S期的细胞比例相似(TSA组：G1期77%、S期17.9%,DMSO组：G1期76.7%、S期17.9%),二者无统计学差异(P>0.05)。将完全融合的第10代MSCs传代培养24h后(第11代,30%融合),流失细胞仪进行细胞周期的检测,结果显示：TSA处理组有较高比例的MSCs从G1期进入S期(G1期23.2%、S期68.7%),与DMSO对照组相比(G1期27.9%、S期62.7%),差异有统计学意义(P<0.05)。
4.TSA处理对MSCs细胞周期相关蛋白的影响：Western Blot检测分别检测TSA处理组与DMSO对照组完全融合的第6代、第10代MSCs的细胞周期相关蛋白表达,结果显示：TSA处理组与DMSO对照组,细胞周期蛋白Cyclin B1、Cyclin D和p21的表达无显著性差异(P>0.05)
结论：
1.低浓度TSA,辅助体外扩增MSCs,可以有效稳定MSCs的干细胞形态学特性。
2.低浓度TSA,辅助体外扩增MSCs,可以显著提高MSCs的细胞增殖速度。
3. MSCs对TSA药物浓度反应敏感。高浓度TSA,反而导致细胞形态异常改变,并且对MSCs的细胞增殖速度有抑制作用。
4.低浓度TSA对MSCs细胞增殖的促进作用,是通过调节MSCs的细胞周期实现的,即通过提高进入S期的细胞比例,显著提高细胞增殖速度。
5.低浓度TSA对MSCs细胞增殖的促进作用,不会引起MSCs相关细胞周期蛋白过度表达,进而不会引起细胞增殖失控、细胞恶性转化。
第三部分去乙酰化酶抑制剂TSA对MSCs多分化潜能的影响及多潜能相关基因的调控研究
研究目的：
低浓度TSA长期培养后,分别诱导MSCs向脂肪细胞、成骨细胞和软骨细胞分化,探讨TSA对MSCs多向分化潜能的影响。检测TSA处理后MSCs多潜能相关基因的转录水平及启动子区域组蛋白乙酰化水平,探讨TSA对MSCs多潜能相关基因的影响及机制,分析TSA维持MSCs细胞形态稳定性及细胞多向分化潜能的原理。
方法：
1.使用脂肪诱导培养基,诱导TSA处理后的MSCs向脂肪细胞分化：体外分离MSCs,分别用含6.25nM TSA、含等量DMSO的完全培养基,连续培养、传代至第6代。更换诱导MSCs向脂肪细胞分化的培养基(含10-6M地塞米松,10μg/ml胰岛素和100μg/ml3-异丁基-L-甲基黄嘌呤),培养3周后,使用油红染色鉴定。
2.使用成骨诱导培养基,诱导TSA处理后的MSCs向成骨细胞分化：体外分离MSCs,分别用含6.25nM TSA、含等量DMSO的完全培养基,连续培养、传代至第6代。更换诱导MSCs向成骨细胞分化的培养基(含10"7M地塞米松,50μg/ml抗坏血酸和10mM β-磷酸甘油),培养3周后,使用茜素红染色鉴定。
3.使用软骨诱导培养,诱导TSA处理后的MSCs向软骨细胞分化：体外分离MSCs,分别用含6.25nM TSA、含等量DMSO的完全培养基,连续培养、传代至第6代,Pellet微球法培养。更换诱导MSCs向软骨细胞分化的培养基(50μg/ml抗坏血酸-2-磷酸,100μg/ml丙酮酸,10ng/ml TGF-β1和50mg/ml ITS Premix),培养3周后,组织固定、切片,HE染色、甲苯胺蓝染色鉴定。
4.Real-Time PCR检测,低浓度TSA辅助扩增,对MSCs多潜能相关基因转录水平的影响：Real-Time PCR检测TSA处理组.DMSO对照组第6代MSCs与第1代MSCs,多潜能相关基因,Oct4、Sox2、Nanog、Rex1和TERT、 CD133mRNA的相对含量。
5.染色质免疫共沉淀(ChIP)法检测,低浓度TSA辅助扩增,对MSCs多潜能相关基因启动子区域组蛋白乙酰化水平的影响：使用特异性抗体,将与目的蛋白结合的DNA片段特异性沉淀、分离出来,Real-Time PCR检测特异性DNA片段的含量,间接反映细胞内与DNA片段结合的目的蛋白水平。检测TSA处理组、DMSO对照组第6代MSCs与第1代MSCs,多潜能相关基因启动子区域组蛋白乙酰化水平变化。
结果：
1.低浓度TSA,辅助体外扩增对MSCs脂肪细胞分化潜能的影响：对向脂肪细胞诱导的TSA处理组与DMSO对照组细胞进行油红染色。结果显示：两组细胞均可见细胞胞浆鲜红色脂肪颗粒着色,表明间充质干细胞已分化为脂肪细胞。
2.低浓度TSA,辅助体外扩增对MSCs成骨细胞分化潜能的影响：对向成骨细胞诱导的TSA处理组与DMSO对照组细胞进行茜素红染色。结果显示：两组细胞均可见染料与钙颗粒结合后显示的红色颗粒,表明间充质干细胞已分化为成骨细胞
3.低浓度TSA,辅助体外扩增对MSCs软骨细胞分化潜能的影响：对向软骨细胞诱导的TSA处理组与DMSO对照组细胞进行组织切片后HE染色染色。结果显示：两组切片均显示细胞变小,胞核变小,胞浆变少,可见胞浆、胞核的空壳,细胞外基质苏木素着色,为透明软骨细胞样外观；组织切片后甲苯胺蓝染色显示胞浆内外紫红色着色。均表明间充质干细胞已分化为软骨细胞。
4.低浓度TSA,辅助体外扩增MSCs对多潜能相关基因转录水平的影响：Real-Time PCR检测MSCs多潜能相关基因mRNA表达。结果显示：DMSO对照组的第6代MSCs与第1代MSCs相比,多潜能相关基因Oct4、Sox2、 Nanog、Rexl和TERT、CD133的转录水平明显下调,差异具有统计学意义(P<0.01)。与DMSO对照组相比,TSA处理组的第6代MSCs,其多潜能相关基因转录水平下调,被显著性抑制(P>0.05)
5.低浓度TSA,辅助体外扩增MSCs对多潜能相关基因启动子区域组蛋白乙酰化水平的影响：染色质免疫共沉淀(ChIP)结果显示, DMSO对照组的第6代MSCs与第1代MSCs相比,多潜能相关基因启动子区域组蛋白H3的乙酰化水平显著性降低,差异具有统计学意义(P<0.01)。与DMSO对照组相比,TSA处理组的第6代MSCs,其多潜能相关基因启动子区域组蛋白H3的乙酰化水平下调,被显著性抑制(P>0.05)。
结论：
1.低浓度TSA,辅助体外扩增MSCs,可以有效逆转多潜能相关基因的转录水平下调。
2.低浓度TSA,辅助体外扩增MSCs,可以有效维持MSCs向脂肪细胞、成骨细胞及软骨细胞的多向分化潜能。
3.低浓度TSA,辅助体外扩增MSCs,是通过抑制基因启动子区域的组蛋白去乙酰化,激活基因转录,抑制多潜能相关基因的转录水平下调,从而有效维持MSCs的多向分化潜能。
Background:
Mesenchymal stem cells (MSCs) are multipotent stem cells. Accumulating evidence suggests that MSCs have profound therapeutic potential for a variety of diseases such as myocardial infarction, neural diseases and wound healing. Due to encouraging preclinical results, a large number of clinical trials for various diseases are underway. MSCs are distributed in a variety of tissues such as the bone marrow and adipose tissue, but represent a rare cell population in tissues. For example, MSCs account only approximately0.001%to0.01%of the nucleated cells in the bone marrow. Recently, it has been demonstrated that MSCs are also present in umbilical cord and placenta. This profoundly increases the availability of MSCs, but ex vivo expansion remains an indispensable procedure to obtain sufficient amounts of MSCs for cell therapies and tissue engineering.
MSCs have long been considered as expandable stem cells. However, recent studies indicate that MSCs age rapidly and undergo considerable changes in cell morphology and production of paracrine factors during culture expansion. Oct4, Sox2and Nanog are main transcription factors that govern embryonic stem cells self-renewal and pluripotency. They are also expressed in MSCs and are involved in their multipotency. Associated with morphological changes, rapid down-regulated expressions of these genes have been detected in MSCs during culture expansion. Previous studies suggest that limited culture expansion of MSCs does not cause alterations in their genetic DNA sequences. However, the epigenetic status of MSCs appears to be unstable in culture. Previous studies indicate that culture expansion of MSCs caused deacetylation of histoneH3-K9and14at promoters of pluripotent genes, which was associated with the appearance of aging signs. Meanwhile, no evident changes in DNA methylation were found in the promoter regions of the pluripotent genes.
Trichostatin A (TSA), which was initially used as an antifungal antibiotic, has recently been found to be a potent and specific inhibitor of HDAC activity. It selectively inhibits the class I and II, but not class III. Previous studies suggest that TSA modulates a wide variety of cellular activities such as cell differentiation and proliferation depending on cell types and their functional states. TSA at concentrations of200～300nM has been found to exhibit pronounced suppressive effect on breast cancer cells with immeasurable toxicities.In this study, we attempted to use a histone deacetylase(HDAC) inhibitor TSA to suppress the reduction of histone acetylation in human MSCs (hMSCs) during culture expansion thus maintaining their primitive properties.
PART1
The down regulated expression of pluripotent genes during the isolation, culture and expansion of MSCs in vitro
Objective:To isolate, culture, expand mesenchymal stem cells. To observe cell morphology changes and detect pluripotent gene expression during cell expansion.
Methods:
1. Briefly, term placentas (38-40weeks' gestation) from healthy donors were harvested. The placental tissue was washed several times with cold phosphate-buffered saline (PBS) and then mechanically minced and enzymatically digested with0.25%trypsin for30minutes at37℃in a water bath. The digest was subsequently pelleted by centrifugation and resuspended in a growth medium consisting of DMEM, supplemented with10%fetal bovine serum and antibiotics. Cells were seeded and incubated in the growth medium at37℃with5%CO2. Medium was replaced every2days. When reaching80%confluence, the cells were lifted by incubating with0.25%trypsin/EDTA and sub-cultured.
2. Real-Time PCR was performed for the expression of Oct4, Sox2, CD133, TERT, REX1, Nanog, alkaline phasphatase (ALP) and osteopontin (OPN).
Result:
1. Successively isolated and cultured mesenchymal stem cells.
2. With successive passages of hMSCs in plastic tissue culture dishes as monolayer, the shape of hMSCs became larger and fatter.
3. In accordance with the morphological changes, Real-Time PCR analysis showed marked decreases of expression levels of pluripotent genes Oct4, Sox2, Nanog, REX1and TERT, CD133, and increased levels of osteogenic genes ALP and OPN in passage10hMSCs compared to hMSCs in passage1.
Conclusion
1. There are some obviously morphological changes of hMSCs that occurred during cell passaging.
2. At the same time, the expression of pluripotent genes decreased according to the cell passaging.
3. Moreover the expression of some osteogenic increased significantly which may related to the auto-differentiation of hMSCs.
PART2
The changes of cell morphology and cell proliferation during the isolation, culture and expansion of MSCs induced by trichostatin A
Objective:
1. To calculate the cell amount and observe cell morphology changes of the isolated, cultured mesenchymal stem cells under short time TSA treatment.
2. To prove that TSA can improve the cell growth speed and stabilize cell morphology of the isolated, cultured and expanded mesenchymal stem cells during long time treatment.
3. To test the cell cycle changes and cell cycle protein changes of the isolated, cultured and expanded mesenchymal stem cells caused by TSA. Analyze the mechanism of TSA induced cell growth accelerate.
Methods:
1. To obtain optimal concentrations of TSA for hMSCs, TSA at concentrations of0,6.25nM,12.5nM,25nM,50nM,100nM,200nM and300nM (dissolved in dimethyl sulfoxide, DMSO) was added to the growth medium. Equal volumes of DMSO alone were used as control. Human MSCs were cultured in24-well plates at a concentration of1x104cells per well in the presence of TSA or DMSO alone and incubated for3days. Then the cells were collected and counted with a hemacytometer.
2.1x105cells per well of passage1hMSCs were seeded in six-well plastic tissue culture plates in triplet wells in the growth medium in the presence of6.25nM TSA or vehicle DMSO and incubated. Cumulative cell numbers from passage2to passage10were calculated.
3. Cells grown to full confluence (passages6and10) and at the first day after passaging (passages7and11in～30%confluence) were harvested for cell cycle analysis and western blotting.
Result:
1. TSA increase the cell amount and stabilize cell morphology of the isolated, cultured mesenchymal stem cells during short time treatment. We proposed that TSA could inhibit the decline of histone acetylation in pluripotent genes and thus retained the primitive properties of hMSCs. To test this, hMSCs were incubated in the growth medium supplemented with TSA at0,6.25,12.5,25,50,100,200and300nM for3days. We found that low concentrations of TSA (6.25nM and12.5nM) increased the cell number by2folds (P＜0.01), and did not cause detectable changes in cell morphology; however,excessive amounts of TSA (200or300nM) decreased hMSC proliferation and lead to significant changes in cell morphology,such as larger and flatter cell body in culture (P＜0.01).
2. TSA improve the cell growth speed and stabilize cell morphology of the isolated, cultured and expanded mesenchymal stem cells during long time treatment. We then analyzed the long term influences of TSA on hMSCs. Human MSCs were" cultured in the presence of TSA (at6.25nM) or an equal amount of DMSO (the dissolvent of TSA) in the growth medium from passage1to passage10. Progressive changes in cell morphology with successive cell passages as described earlier were observed in hMSCs treated with DMSO alone. However, the morphological changes did not occurred in hMSCs cultured in the presence of TSA. Meanwhile, there was a profound increase in the cumulative cell number of hMSCs in culture in the presence of TSA (P＜0.01).
3. TSA changed the cell cycle of the isolated, cultured and expanded mesenchymal stem cells. To investigate whether transformation occurred in TSA-treated cells, we examined cell contact inhibition in cell growth in hMSCs after successive TSA treatment. Similar to DMSO-treated hMSCs, TSA-treated hMSCs stopped proliferating when they reached full confluence and no multi-layer foci were found in the culture. Cell cycle analysis of passage10hMSCs treated with DMSO or TSA showed similar percentages of cells arrested in G1phase (76%versus77%, P＞0.05) when they reached full confluence. However, when cells were passaged to new culture plates (passage11) and incubated in the growth medium for24hours (in～30%confluence), a higher percentage of TSA-treated hMSCs entered S phase compared to DMSO-treated cells (67%versus61%, P＜0.05).
4. We further examined the expression levels of cell cycle proteins in passage6and passage10hMSCs in full confluence by Western blot, and the results showed similar amounts of cyclinD1, cyclin B1and p21in DMSO-and TSA-treated cells.
Conclusion
1. We found that low concentrations of TSA significantly inhibited morphological changes of hMSCs that otherwise occurred during cell passaging.
2. In addition, TSA-treated MSCs grew much faster by increase the cell population in S phase of cell cycle.
3. But TSA treatment did not induce the overexpression of cell cycle protein which may have relationship with cell transformation.
PART3
TSA stabilized the expression of pluripotent genes and their histone H3acetylation levels without affect the pluripotent differentiate ability of MSCs
Objective:To test the pluripotent differentiate ability of the isolated, cultured and expanded mesenchymal stem cells which may be not affected by TSA treatment. Analyze the mechanism of stabilized pluripotent gene expression of the isolated, cultured and expanded mesenchymal stem cells by TSA.
Methods:
1. Passage1hMSCs were grown in the presence of TSA (at6.25nM) or equal amount of vehicle DMSO to passage6. Then the cells were incubated in adipogenic induction media, respectively, for3weeks. The adipogenic induction medium contained10-6M dexamethasone,10μg/ml insulin and100μg/ml3-isobutyl-L-methylxanthine. Cells were finally stained with Oil Red-O to detect lipid.
2. Passage1hMSCs were grown in the presence of TSA (at6.25nM) or equal amount of vehicle DMSO to passage6. Then the cells were incubated in osteogenic induction media, respectively, for3weeks.The osteogenic medium contained10"7M dexamethasone,50μg/ml ascorbic acid and10mM β-glycerophosphate. Cells were finally stained using Alzarin Red for calcium deposition.
3. Passage1hMSCs were grown in the presence of TSA (at6.25nM) or equal amount of vehicle DMSO to passage6. Then the cells were incubated in chondrogenic induction media, respectively, for3weeks. For chondrocyte differentiation, pellet hMSCs were cultured in DMEM (high glucose) containing10-7M dexamethasone,50μg/ml ascorbate-2-phosphate,100μg/ml pyruvate,10ng/ml TGF-β1and50mg/ml ITS Premix. Medium was changed every2days for3weeks. The pellet was fixed, embedded and sectioned for H&E and toluidine blue staining, respectively.
4. Chromatin immunoprecipitation (ChIP) assay was performed using an Acetyl-Histone H3Immunoprecipitation Assay Kit. Histone acetylation was determined using specific antibodies against acetylated histone H3at K9and K14, respectively.
5. Real-Time PCR was performed for the expression of Oct4, Sox2, CD133, TERT, REX1, Nanog, alkaline phasphatase (ALP) and osteopontin (OPN).
Result:
1. TSA may not affect the pluripotent differentiate ability of the isolated, cultured and expanded mesenchymal stem cells. We also examined the multipotent differentiation potential of hMSCs into adipocytes, osteoblasts and chondrocytes, which has been considered as a typical feature of MSCs, and found that similar differentiations into these three cell lineages occurred in TSA-treated hMSCs, compared to DMSO-treated hMSCs.
2. TSA stabilize pluripotent gene expression of the isolated, cultured and expanded mesenchymal stem cells. We examined the expression of pluripotent genes in hMSCs in the above cultures. We found that TSA significantly inhibited the down-expression of Oct4, Sox2, Nanog, REX1and TERT, CD133genes from passage1to passage6hMSCs, which occurred in hMSCs treated with vehicle DMSO alone (P＜0.01).
3. Finally, we examined histone H3acetylation in K9and K14in the promoter regions of TERT, Sox2and Oct4genes in hMSCs. Compared to hMSCs in passage1, hMSCs cultured in the presence of DMSO alone in passage6showed significantly decreased histone H3acetylation levels of the pluripotent genes in K9and K14(P＜0.01). In the presence of TSA (at6.25nM), the acetylation levels of histone H3in K9and K14of these genes in passage6hMSCs showed no significant decreases (P>0.05).
Conclusion
1. TSA-treated MSCs grew much faster, without affect the pluripotent differentiate ability of the isolated, cultured and expanded mesenchymal stem cells.
2. Moreover, TSA stabilized the expression of pluripotent genes and their histone H3acetylation levels in lysine K9and K14in the promoter regions.
间充质干细胞(Mesenchymal stem cells, MSCs)属于多潜能干细胞。相关研究表明,间充质干细胞对多种疾病具有治疗潜力,例如心肌梗死、神经性疾病和创伤愈合等。依据相关临床前期实验结果,针对多种疾病的临床试验正在逐渐开展中。MSCs分布于多种组织,如骨髓和脂肪组织,但数量极少。在骨髓的有核细胞中,干细胞只占约0.001%-0.01%。有研究证明,间充质干细胞也存在于脐带和胎盘,这增加了获取、使用间充质干细胞的可能性。然而想要获取足够量的间充质干细胞并应用于细胞治疗和组织工程,体外扩增仍然是其中的必需环节。
MSCs长期以来被认为是可以无限传代、扩增的干细胞。近年来的研究表明，在培养、扩增过程中,间充质干细胞会迅速出现衰老现象,细胞形态表现出明显的变化,同时伴有许多旁分泌因子的分泌异常。Oct4, Sox2和Nanog是调控胚胎干细胞自我更新、维持干细胞多向分化潜能的主要转录因子。研究发现Oct4,Sox2和Nanog基因也在MSCs中表达并参与其多能性的调控。在MSCs体外培养、扩增过程中,伴随细胞形态学改变,这些基因也出现表达水平的快速下调。研究表明,MSCs的有限培养、扩增不会引起相关基因的DNA序列改变,多潜能相关基因无显著DNA甲基化现象。然而,MSCs的表观遗传状态在培养、扩增过程中并不稳定。前期研究表明,MSCs的体外培养、扩增会引起多潜能基因启动子区域组蛋白H3-K9、14的去乙酰化,与细胞的老化现象密切相关。
曲古抑菌素A (Trichostatin A, TSA),最初作为抗真菌药物被广泛应用。最近研究发现TSA是一种有效的、特异性组蛋白去乙酰化酶(HDAC)抑制剂。TSA选择性抑制Ⅰ类和Ⅱ类HDAC,但不能作用于Ⅲ类HIDAC。已证实TSA对乳腺癌细胞有明显抑制作用和细胞毒性。根据不同的细胞类型和功能状态,TSA可参与调节多种不同的细胞活动,如细胞分化和细胞增殖等。本研究设计、使用曲古抑菌素A (TSA),作为组蛋白去乙酰化酶(HDAC)抑制剂,抑制MSCs体外培养、扩增引起的组蛋白去乙酰化,从而保持其原始性状。
第一部分人间充质干细胞的体外分离、培养、扩增及多潜能相关基因的检测研究
研究目的：
体外分离、纯化、扩增人胎盘组织来源的间充质干细胞,密切观察MSCs的细胞形态变化,探讨体外扩增对MSCs干细胞形态学特性的影响；检测多潜能相关基因、成骨分化相关基因的mRNA含量,探讨体外扩增过程对MSCs多潜能相关基因、成骨分化相关基因转录水平的影响。
方法：
1.胰酶消化、贴壁培养法体外分离培养并纯化鉴定MSCs:取38W-40W妊娠,健康供者的人胎盘组织,用预冷的磷酸盐缓冲盐水(PBS)洗涤数次,机械剁碎后,37℃水浴下0.25%胰蛋白酶消化30分钟。离心后用含10%胎牛血清和抗生素的DMEM培养基重悬沉淀、种板。37℃、5%CO2培养,隔天换液。待细胞达到80%融合时,0.25%胰蛋白酶/EDTA消化、连续传代、培养；流式细胞仪对MSCs的相关表面标志物进行检测鉴定。
2.体外扩增MSCs、观察细胞形态学变化并绘制扩增曲线：选取体外分离、纯化并鉴定成功的MSCs,当细胞生长到80%融合或以上时,连续传代、培养至10代以上。绘制细胞生长曲线,观察细胞形态学变化。
3. Real-Time PCR检测体外扩增MSCs多潜能相关基因、成骨分化相关基因转录水平：设计、合成多潜能相关基因Oct4、Sox2、Nanog、Rexl和TERT、CD133,成骨分化相关基因ALP和OPN引物,Real-Time PCR检测第1代与连续传代后(第10代)MSCs多潜能相关基因、成骨分化相关基因mRNA相对含量。
结果：
1. MSCs的体外分离、纯化：使用胰酶消化法、贴壁培养法,成功完成MSCs体外分离、纯化并鉴定成功。2. MSCs的体外扩增：采集对数生长期的细胞,进行连续传代,实现MSCs体外扩增。观察细胞形态可见：伴随扩增过程进展,MSCs形态发生显著性改变,表现为细胞增大、不均匀,形态增宽,变扁平。细胞生长曲线提示细胞增殖速度较快,细胞总数呈指数增长。
3.体外扩增MSCs多潜能相关基因、成骨分化相关基因的转录水平改变：应用Real-Time PCR检测体外扩增过程中MSCs多潜能相关基因、成骨分化相关基因的mRNA表达,结果显示：体外扩增的第10代MSCs与第1代相比,多潜能相关基因Oct4、Sox2、Nanog、Rex1和TERT、CD133,转录水平显著降低,成骨分化相关基因ALP和OPN,转录水平显著升高,差异具有统计学意义(P<0.01)。
结论：
1.胰酶消化法、贴壁培养法可以成功分离、纯化人胎盘组织来源的MSCs。
2.传统方法体外扩增MSCs,不能有效维持其干细胞的形态学特征,伴随传代次数增加,细胞形态学变化显著。
3.传统方法体外扩增MSCs,不能有效维持干细胞多向分化潜能,参与维持MSCs多向分化潜能的干细胞多潜能相关基因转录水平下调。
4.传统方法体外扩增MSCs,不能维持干细胞的未分化状态,出现向成骨细胞分化的自主分化趋势,MSCs成骨分化相关基因转录水平上调。
第二部分去乙酰化酶抑制剂曲古抑菌素A(Trichostatin A, TSA)促进MSCs增殖、稳定MSCs形态的功能及机制研究
研究目的：
TSA短期处理MSCs,筛选最佳作用浓度,观察TSA对MSCs细胞形态学和细胞增殖的影响；TSA长期培养MSCs,辅助体外扩增,验证TSA稳定MSCs细胞形态和促进细胞增殖的作用。检测TSA处理对MSCs细胞周期的影响,探讨TSA促进MSCs细胞增殖的作用机制。
方法：
1.梯度浓度TSA短期处理MSCs、观察细胞形态学改变并计数细胞增殖情况：体外分离、纯化的第3代MSCs,分别使用含0、6.25、12.5、25、50、100、200和300nM TSA的培养基,对细胞进行连续培养三天。观察梯度浓度TSA处理组细胞形态差异,计数细胞数目变化。
2.低浓度TSA长期处理MSCs、观察细胞形态学改变并计数细胞增殖情况：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO(TSA溶剂)的完全培养基,对细胞进行连续培养、传代、计数。观察TSA处理组与DMSO对照组细胞形态差异,计数细胞数目变化,绘制生长曲线,检测细胞增殖速度变化。
3.流式细胞仪检测TSA处理对MSCs细胞周期的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO (TSA溶剂)的完全培养基,对细胞进行连续培养。检测完全融合的第10代MSCs,细胞周期各阶段的细胞比例；检测30%融合的第11代MSCs,细胞周期各阶段的细胞比例。
4. Western Blot检测TSA处理对MSCs细胞周期相关蛋白的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO (TSA溶剂)的完全培养基,对细胞进行连续培养。采集TSA处理组与DMSO对照组第6代、第10代MSCs,检测细胞周期相关蛋白Cyclin B1、Cyclin D1和p21的表达。
结果：
1.TSA短期处理对MSCs细胞形态学和细胞增殖的影响：梯度浓度TSA对MSCs短期培养三天,观察MSCs细胞形态学变化及细胞增殖变化。结果显示：较低浓度TSA(6.25nM、12.5nM)培养的MSCs细胞总数显著性增多,差异具有统计学意义(P<0.01)；细胞数目增多的同时,细胞的形态学特征保持良好。较高浓度TSA(200nM、300nM)培养MSCs与低浓度TSA的培养效果相反,引起细胞总数显著性降低(P<0.01)；同时细胞形态异常改变,细胞增大,形态趋于扁平,丧失MSCs的形态学特征
2.TSA长期处理对MSCs细胞形态学和细胞增殖的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA、等量DMSO(TSA溶剂)的完全培养基,进行体外扩增至第10代,观察细胞形态学变化和细胞增殖变化。结果显示：TSA处理组与DMSO对照组比较可见：TSA处理组细胞增殖速度、细胞总数较DMSO对照组显著性增加,差异具有统计学意义(P<0.01)；同时TSA处理组细胞的形态学特征保持良好,DMSO细胞形态显著改变；TSA处理组与DMSO对照组的MSCs,细胞的接触抑制均存在,即当细胞生长到充分融合时,细胞增殖就会停止,不会出现局灶性的多层细胞(细胞接触抑制+)
3.TSA处理对MSCs细胞周期的影响：体外分离、纯化的第1代MSCs,分别使用含6.25nM TSA.等量DMSO (TSA溶剂)的完全培养基,进行体外扩增。取达到完全融合的第10代MSCs应用流式细胞仪进行细胞周期的检测,结果显示TSA处理组与DMSO对照组相比,处于G1期、S期的细胞比例相似(TSA组：G1期77%、S期17.9%,DMSO组：G1期76.7%、S期17.9%),二者无统计学差异(P>0.05)。将完全融合的第10代MSCs传代培养24h后(第11代,30%融合),流失细胞仪进行细胞周期的检测,结果显示：TSA处理组有较高比例的MSCs从G1期进入S期(G1期23.2%、S期68.7%),与DMSO对照组相比(G1期27.9%、S期62.7%),差异有统计学意义(P<0.05)。
4.TSA处理对MSCs细胞周期相关蛋白的影响：Western Blot检测分别检测TSA处理组与DMSO对照组完全融合的第6代、第10代MSCs的细胞周期相关蛋白表达,结果显示：TSA处理组与DMSO对照组,细胞周期蛋白Cyclin B1、Cyclin D和p21的表达无显著性差异(P>0.05)
结论：
1.低浓度TSA,辅助体外扩增MSCs,可以有效稳定MSCs的干细胞形态学特性。
2.低浓度TSA,辅助体外扩增MSCs,可以显著提高MSCs的细胞增殖速度。
3. MSCs对TSA药物浓度反应敏感。高浓度TSA,反而导致细胞形态异常改变,并且对MSCs的细胞增殖速度有抑制作用。
4.低浓度TSA对MSCs细胞增殖的促进作用,是通过调节MSCs的细胞周期实现的,即通过提高进入S期的细胞比例,显著提高细胞增殖速度。
5.低浓度TSA对MSCs细胞增殖的促进作用,不会引起MSCs相关细胞周期蛋白过度表达,进而不会引起细胞增殖失控、细胞恶性转化。
第三部分去乙酰化酶抑制剂TSA对MSCs多分化潜能的影响及多潜能相关基因的调控研究
研究目的：
低浓度TSA长期培养后,分别诱导MSCs向脂肪细胞、成骨细胞和软骨细胞分化,探讨TSA对MSCs多向分化潜能的影响。检测TSA处理后MSCs多潜能相关基因的转录水平及启动子区域组蛋白乙酰化水平,探讨TSA对MSCs多潜能相关基因的影响及机制,分析TSA维持MSCs细胞形态稳定性及细胞多向分化潜能的原理。
方法：
1.使用脂肪诱导培养基,诱导TSA处理后的MSCs向脂肪细胞分化：体外分离MSCs,分别用含6.25nM TSA、含等量DMSO的完全培养基,连续培养、传代至第6代。更换诱导MSCs向脂肪细胞分化的培养基(含10-6M地塞米松,10μg/ml胰岛素和100μg/ml3-异丁基-L-甲基黄嘌呤),培养3周后,使用油红染色鉴定。
2.使用成骨诱导培养基,诱导TSA处理后的MSCs向成骨细胞分化：体外分离MSCs,分别用含6.25nM TSA、含等量DMSO的完全培养基,连续培养、传代至第6代。更换诱导MSCs向成骨细胞分化的培养基(含10"7M地塞米松,50μg/ml抗坏血酸和10mM β-磷酸甘油),培养3周后,使用茜素红染色鉴定。
3.使用软骨诱导培养,诱导TSA处理后的MSCs向软骨细胞分化：体外分离MSCs,分别用含6.25nM TSA、含等量DMSO的完全培养基,连续培养、传代至第6代,Pellet微球法培养。更换诱导MSCs向软骨细胞分化的培养基(50μg/ml抗坏血酸-2-磷酸,100μg/ml丙酮酸,10ng/ml TGF-β1和50mg/ml ITS Premix),培养3周后,组织固定、切片,HE染色、甲苯胺蓝染色鉴定。
4.Real-Time PCR检测,低浓度TSA辅助扩增,对MSCs多潜能相关基因转录水平的影响：Real-Time PCR检测TSA处理组.DMSO对照组第6代MSCs与第1代MSCs,多潜能相关基因,Oct4、Sox2、Nanog、Rex1和TERT、 CD133mRNA的相对含量。
5.染色质免疫共沉淀(ChIP)法检测,低浓度TSA辅助扩增,对MSCs多潜能相关基因启动子区域组蛋白乙酰化水平的影响：使用特异性抗体,将与目的蛋白结合的DNA片段特异性沉淀、分离出来,Real-Time PCR检测特异性DNA片段的含量,间接反映细胞内与DNA片段结合的目的蛋白水平。检测TSA处理组、DMSO对照组第6代MSCs与第1代MSCs,多潜能相关基因启动子区域组蛋白乙酰化水平变化。
结果：
1.低浓度TSA,辅助体外扩增对MSCs脂肪细胞分化潜能的影响：对向脂肪细胞诱导的TSA处理组与DMSO对照组细胞进行油红染色。结果显示：两组细胞均可见细胞胞浆鲜红色脂肪颗粒着色,表明间充质干细胞已分化为脂肪细胞。
2.低浓度TSA,辅助体外扩增对MSCs成骨细胞分化潜能的影响：对向成骨细胞诱导的TSA处理组与DMSO对照组细胞进行茜素红染色。结果显示：两组细胞均可见染料与钙颗粒结合后显示的红色颗粒,表明间充质干细胞已分化为成骨细胞
3.低浓度TSA,辅助体外扩增对MSCs软骨细胞分化潜能的影响：对向软骨细胞诱导的TSA处理组与DMSO对照组细胞进行组织切片后HE染色染色。结果显示：两组切片均显示细胞变小,胞核变小,胞浆变少,可见胞浆、胞核的空壳,细胞外基质苏木素着色,为透明软骨细胞样外观；组织切片后甲苯胺蓝染色显示胞浆内外紫红色着色。均表明间充质干细胞已分化为软骨细胞。
4.低浓度TSA,辅助体外扩增MSCs对多潜能相关基因转录水平的影响：Real-Time PCR检测MSCs多潜能相关基因mRNA表达。结果显示：DMSO对照组的第6代MSCs与第1代MSCs相比,多潜能相关基因Oct4、Sox2、 Nanog、Rexl和TERT、CD133的转录水平明显下调,差异具有统计学意义(P<0.01)。与DMSO对照组相比,TSA处理组的第6代MSCs,其多潜能相关基因转录水平下调,被显著性抑制(P>0.05)
5.低浓度TSA,辅助体外扩增MSCs对多潜能相关基因启动子区域组蛋白乙酰化水平的影响：染色质免疫共沉淀(ChIP)结果显示, DMSO对照组的第6代MSCs与第1代MSCs相比,多潜能相关基因启动子区域组蛋白H3的乙酰化水平显著性降低,差异具有统计学意义(P<0.01)。与DMSO对照组相比,TSA处理组的第6代MSCs,其多潜能相关基因启动子区域组蛋白H3的乙酰化水平下调,被显著性抑制(P>0.05)。
结论：
1.低浓度TSA,辅助体外扩增MSCs,可以有效逆转多潜能相关基因的转录水平下调。
2.低浓度TSA,辅助体外扩增MSCs,可以有效维持MSCs向脂肪细胞、成骨细胞及软骨细胞的多向分化潜能。
3.低浓度TSA,辅助体外扩增MSCs,是通过抑制基因启动子区域的组蛋白去乙酰化,激活基因转录,抑制多潜能相关基因的转录水平下调,从而有效维持MSCs的多向分化潜能。
Background:
Mesenchymal stem cells (MSCs) are multipotent stem cells. Accumulating evidence suggests that MSCs have profound therapeutic potential for a variety of diseases such as myocardial infarction, neural diseases and wound healing. Due to encouraging preclinical results, a large number of clinical trials for various diseases are underway. MSCs are distributed in a variety of tissues such as the bone marrow and adipose tissue, but represent a rare cell population in tissues. For example, MSCs account only approximately0.001%to0.01%of the nucleated cells in the bone marrow. Recently, it has been demonstrated that MSCs are also present in umbilical cord and placenta. This profoundly increases the availability of MSCs, but ex vivo expansion remains an indispensable procedure to obtain sufficient amounts of MSCs for cell therapies and tissue engineering.
MSCs have long been considered as expandable stem cells. However, recent studies indicate that MSCs age rapidly and undergo considerable changes in cell morphology and production of paracrine factors during culture expansion. Oct4, Sox2and Nanog are main transcription factors that govern embryonic stem cells self-renewal and pluripotency. They are also expressed in MSCs and are involved in their multipotency. Associated with morphological changes, rapid down-regulated expressions of these genes have been detected in MSCs during culture expansion. Previous studies suggest that limited culture expansion of MSCs does not cause alterations in their genetic DNA sequences. However, the epigenetic status of MSCs appears to be unstable in culture. Previous studies indicate that culture expansion of MSCs caused deacetylation of histoneH3-K9and14at promoters of pluripotent genes, which was associated with the appearance of aging signs. Meanwhile, no evident changes in DNA methylation were found in the promoter regions of the pluripotent genes.
Trichostatin A (TSA), which was initially used as an antifungal antibiotic, has recently been found to be a potent and specific inhibitor of HDAC activity. It selectively inhibits the class I and II, but not class III. Previous studies suggest that TSA modulates a wide variety of cellular activities such as cell differentiation and proliferation depending on cell types and their functional states. TSA at concentrations of200～300nM has been found to exhibit pronounced suppressive effect on breast cancer cells with immeasurable toxicities.In this study, we attempted to use a histone deacetylase(HDAC) inhibitor TSA to suppress the reduction of histone acetylation in human MSCs (hMSCs) during culture expansion thus maintaining their primitive properties.
PART1
The down regulated expression of pluripotent genes during the isolation, culture and expansion of MSCs in vitro
Objective:To isolate, culture, expand mesenchymal stem cells. To observe cell morphology changes and detect pluripotent gene expression during cell expansion.
Methods:
1. Briefly, term placentas (38-40weeks' gestation) from healthy donors were harvested. The placental tissue was washed several times with cold phosphate-buffered saline (PBS) and then mechanically minced and enzymatically digested with0.25%trypsin for30minutes at37℃in a water bath. The digest was subsequently pelleted by centrifugation and resuspended in a growth medium consisting of DMEM, supplemented with10%fetal bovine serum and antibiotics. Cells were seeded and incubated in the growth medium at37℃with5%CO2. Medium was replaced every2days. When reaching80%confluence, the cells were lifted by incubating with0.25%trypsin/EDTA and sub-cultured.
2. Real-Time PCR was performed for the expression of Oct4, Sox2, CD133, TERT, REX1, Nanog, alkaline phasphatase (ALP) and osteopontin (OPN).
Result:
1. Successively isolated and cultured mesenchymal stem cells.
2. With successive passages of hMSCs in plastic tissue culture dishes as monolayer, the shape of hMSCs became larger and fatter.
3. In accordance with the morphological changes, Real-Time PCR analysis showed marked decreases of expression levels of pluripotent genes Oct4, Sox2, Nanog, REX1and TERT, CD133, and increased levels of osteogenic genes ALP and OPN in passage10hMSCs compared to hMSCs in passage1.
Conclusion
1. There are some obviously morphological changes of hMSCs that occurred during cell passaging.
2. At the same time, the expression of pluripotent genes decreased according to the cell passaging.
3. Moreover the expression of some osteogenic increased significantly which may related to the auto-differentiation of hMSCs.
PART2
The changes of cell morphology and cell proliferation during the isolation, culture and expansion of MSCs induced by trichostatin A
Objective:
1. To calculate the cell amount and observe cell morphology changes of the isolated, cultured mesenchymal stem cells under short time TSA treatment.
2. To prove that TSA can improve the cell growth speed and stabilize cell morphology of the isolated, cultured and expanded mesenchymal stem cells during long time treatment.
3. To test the cell cycle changes and cell cycle protein changes of the isolated, cultured and expanded mesenchymal stem cells caused by TSA. Analyze the mechanism of TSA induced cell growth accelerate.
Methods:
1. To obtain optimal concentrations of TSA for hMSCs, TSA at concentrations of0,6.25nM,12.5nM,25nM,50nM,100nM,200nM and300nM (dissolved in dimethyl sulfoxide, DMSO) was added to the growth medium. Equal volumes of DMSO alone were used as control. Human MSCs were cultured in24-well plates at a concentration of1x104cells per well in the presence of TSA or DMSO alone and incubated for3days. Then the cells were collected and counted with a hemacytometer.
2.1x105cells per well of passage1hMSCs were seeded in six-well plastic tissue culture plates in triplet wells in the growth medium in the presence of6.25nM TSA or vehicle DMSO and incubated. Cumulative cell numbers from passage2to passage10were calculated.
3. Cells grown to full confluence (passages6and10) and at the first day after passaging (passages7and11in～30%confluence) were harvested for cell cycle analysis and western blotting.
Result:
1. TSA increase the cell amount and stabilize cell morphology of the isolated, cultured mesenchymal stem cells during short time treatment. We proposed that TSA could inhibit the decline of histone acetylation in pluripotent genes and thus retained the primitive properties of hMSCs. To test this, hMSCs were incubated in the growth medium supplemented with TSA at0,6.25,12.5,25,50,100,200and300nM for3days. We found that low concentrations of TSA (6.25nM and12.5nM) increased the cell number by2folds (P＜0.01), and did not cause detectable changes in cell morphology; however,excessive amounts of TSA (200or300nM) decreased hMSC proliferation and lead to significant changes in cell morphology,such as larger and flatter cell body in culture (P＜0.01).
2. TSA improve the cell growth speed and stabilize cell morphology of the isolated, cultured and expanded mesenchymal stem cells during long time treatment. We then analyzed the long term influences of TSA on hMSCs. Human MSCs were" cultured in the presence of TSA (at6.25nM) or an equal amount of DMSO (the dissolvent of TSA) in the growth medium from passage1to passage10. Progressive changes in cell morphology with successive cell passages as described earlier were observed in hMSCs treated with DMSO alone. However, the morphological changes did not occurred in hMSCs cultured in the presence of TSA. Meanwhile, there was a profound increase in the cumulative cell number of hMSCs in culture in the presence of TSA (P＜0.01).
3. TSA changed the cell cycle of the isolated, cultured and expanded mesenchymal stem cells. To investigate whether transformation occurred in TSA-treated cells, we examined cell contact inhibition in cell growth in hMSCs after successive TSA treatment. Similar to DMSO-treated hMSCs, TSA-treated hMSCs stopped proliferating when they reached full confluence and no multi-layer foci were found in the culture. Cell cycle analysis of passage10hMSCs treated with DMSO or TSA showed similar percentages of cells arrested in G1phase (76%versus77%, P＞0.05) when they reached full confluence. However, when cells were passaged to new culture plates (passage11) and incubated in the growth medium for24hours (in～30%confluence), a higher percentage of TSA-treated hMSCs entered S phase compared to DMSO-treated cells (67%versus61%, P＜0.05).
4. We further examined the expression levels of cell cycle proteins in passage6and passage10hMSCs in full confluence by Western blot, and the results showed similar amounts of cyclinD1, cyclin B1and p21in DMSO-and TSA-treated cells.
Conclusion
1. We found that low concentrations of TSA significantly inhibited morphological changes of hMSCs that otherwise occurred during cell passaging.
2. In addition, TSA-treated MSCs grew much faster by increase the cell population in S phase of cell cycle.
3. But TSA treatment did not induce the overexpression of cell cycle protein which may have relationship with cell transformation.
PART3
TSA stabilized the expression of pluripotent genes and their histone H3acetylation levels without affect the pluripotent differentiate ability of MSCs
Objective:To test the pluripotent differentiate ability of the isolated, cultured and expanded mesenchymal stem cells which may be not affected by TSA treatment. Analyze the mechanism of stabilized pluripotent gene expression of the isolated, cultured and expanded mesenchymal stem cells by TSA.
Methods:
1. Passage1hMSCs were grown in the presence of TSA (at6.25nM) or equal amount of vehicle DMSO to passage6. Then the cells were incubated in adipogenic induction media, respectively, for3weeks. The adipogenic induction medium contained10-6M dexamethasone,10μg/ml insulin and100μg/ml3-isobutyl-L-methylxanthine. Cells were finally stained with Oil Red-O to detect lipid.
2. Passage1hMSCs were grown in the presence of TSA (at6.25nM) or equal amount of vehicle DMSO to passage6. Then the cells were incubated in osteogenic induction media, respectively, for3weeks.The osteogenic medium contained10"7M dexamethasone,50μg/ml ascorbic acid and10mM β-glycerophosphate. Cells were finally stained using Alzarin Red for calcium deposition.
3. Passage1hMSCs were grown in the presence of TSA (at6.25nM) or equal amount of vehicle DMSO to passage6. Then the cells were incubated in chondrogenic induction media, respectively, for3weeks. For chondrocyte differentiation, pellet hMSCs were cultured in DMEM (high glucose) containing10-7M dexamethasone,50μg/ml ascorbate-2-phosphate,100μg/ml pyruvate,10ng/ml TGF-β1and50mg/ml ITS Premix. Medium was changed every2days for3weeks. The pellet was fixed, embedded and sectioned for H&E and toluidine blue staining, respectively.
4. Chromatin immunoprecipitation (ChIP) assay was performed using an Acetyl-Histone H3Immunoprecipitation Assay Kit. Histone acetylation was determined using specific antibodies against acetylated histone H3at K9and K14, respectively.
5. Real-Time PCR was performed for the expression of Oct4, Sox2, CD133, TERT, REX1, Nanog, alkaline phasphatase (ALP) and osteopontin (OPN).
Result:
1. TSA may not affect the pluripotent differentiate ability of the isolated, cultured and expanded mesenchymal stem cells. We also examined the multipotent differentiation potential of hMSCs into adipocytes, osteoblasts and chondrocytes, which has been considered as a typical feature of MSCs, and found that similar differentiations into these three cell lineages occurred in TSA-treated hMSCs, compared to DMSO-treated hMSCs.
2. TSA stabilize pluripotent gene expression of the isolated, cultured and expanded mesenchymal stem cells. We examined the expression of pluripotent genes in hMSCs in the above cultures. We found that TSA significantly inhibited the down-expression of Oct4, Sox2, Nanog, REX1and TERT, CD133genes from passage1to passage6hMSCs, which occurred in hMSCs treated with vehicle DMSO alone (P＜0.01).
3. Finally, we examined histone H3acetylation in K9and K14in the promoter regions of TERT, Sox2and Oct4genes in hMSCs. Compared to hMSCs in passage1, hMSCs cultured in the presence of DMSO alone in passage6showed significantly decreased histone H3acetylation levels of the pluripotent genes in K9and K14(P＜0.01). In the presence of TSA (at6.25nM), the acetylation levels of histone H3in K9and K14of these genes in passage6hMSCs showed no significant decreases (P>0.05).
Conclusion
1. TSA-treated MSCs grew much faster, without affect the pluripotent differentiate ability of the isolated, cultured and expanded mesenchymal stem cells.
2. Moreover, TSA stabilized the expression of pluripotent genes and their histone H3acetylation levels in lysine K9and K14in the promoter regions.
引文
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4. Philippe, B., et al., Culture and Use of Mesenchymal Stromal Cells in Phase I and II Clinical Trials. Stem Cells Int,2010.2010:p.503593.
5. Li, Z., et al., Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation. PLoS One,2011.6(6):p. e20526.
6. Tan, J., et al., The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells. J Genet Genomics,2008.35(10):p.585-93.
7. Noer, A., L.C. Lindeman, and P. Collas, Histone H3 modifications associated with differentiation and long-term culture of mesenchymal adipose stem cells. Stem Cells Dev,2009.18(5):p.725-36.
8. Paranjape, S.M., R.T. Kamakaka, and J.T. Kadonaga, Role of chromatin structure in the regulation of transcription by RNA polymerase Ⅱ. Annu Rev Biochem, 1994.63:p.265-97.
9. Silva, J. and A. Smith, Capturing pluripotency. Cell,2008.132(4):p.532-6.
10. Han, J., et al., Nanog reverses the effects of organismal aging on mesenchymal stem cell proliferation and myogenic differentiation potential. Stem Cells,2012. 30(12):p.2746-59.
11. Zhou, Q., et al., A gene regulatory network in mouse embryonic stem cells. Proc Natl Acad Sci U S A,2007.104(42):p.16438-43.
12. Yannarelli, G., et al., Brief report:The potential role of epigenetics on multipotent cell differentiation capacity of mesenchymal stromal cells. Stem Cells,2013. 31(1):p.215-20.
13. Ju, Z. and K.L. Rudolph, Telomeres and telomerase in stem cells during aging and disease. Genome Dyn,2006.1:p.84-103.
14. Wagner, W., et al., Replicative senescence of mesenchymal stem cells:a continuous and organized process. PLoS One,2008.3(5):p. e2213.
15. Orlando, V., H. Strutt, and R. Paro, Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods,1997.11(2):p.205-14.
16. Kuo, M.H. and C.D. Allis, In vivo cross-linking and immunoprecipitation for studying dynamic Protein.DNA associations in a chromatin environment. Methods,1999.19(3):p.425-33.
17. Mayr, B. and M. Montminy, Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol,2001.2(8):p. 599-609.
18. Van Lente, F., J.F. Jackson, and H. Weintraub, Identification of specific crosslinked histones after treatment of chromatin with formaldehyde. Cell,1975. 5(1):p.45-50.
19. Jackson, V. and R. Chalkley, A new method for the isolation of replicative chromatin:selective deposition of histone on both new and old DNA. Cell,1981. 23(1):p.121-34.
20. Solomon, M.J. and A. Varshavsky, Formaldehyde-mediated DNA-protein cross linking:a probe for in vivo chromatin structures. Proc Natl Acad Sci U S A, 1985.82(19):p.6470-4.
21. Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem cells. Science,1999.284(5411):p.143-7.
22. Prockop, D. J., Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997.276(5309):p.71-4.
23. Phinney, D.G. and D.J. Prockop, Concise review:mesenchymal stem/multipotent stromal cells:the state of transdifferentiation and modes of tissue repair--current views. Stem Cells,2007.25(11):p.2896-902.
24. Wu, Y., et al., Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells,2007.25(10):p.2648-59.
25. Wu, Y., et al., Bone marrow-derived stem cells in wound healing:a review. Wound Repair Regen,2007.15 Suppl 1:p. S18-26.
26. Pittenger, M.F. and B.J. Martin, Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res,2004.95(1):p.9-20.
27. Wu, Y., R.C. Zhao, and E.E. Tredget, Concise review:bone marrow-derived stem/progenitor cells in cutaneous repair and regeneration. Stem Cells,2010. 28(5):p.905-15.
28. Williams, A.R. and J.M. Hare, Mesenchymal stem cells:biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circ Res, 2011.109(8):p.923-40.
29. Javazon, E.H., K.J. Beggs, and A.W. Flake, Mesenchymal stem cells:paradoxes of passaging. Exp Hematol,2004.32(5):p.414-25.
30. Aggarwal, S. and M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood,2005.105(4):p.1815-22.
31. Jiang, X.X., et al., Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood,2005.105(10):p.4120-6.
32. Le Blanc, K., et al., Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet,2004.363(9419):p. 1439-41.
33. Horwitz, E.M., et al., Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A,2002.99(13):p. 8932-7.
34. Prevedouros, K., et al., Seasonal and long-term trends in atmospheric PAH concentrations:evidence and implications. Environ Pollut,2004.128(1-2):p. 17-27.
35. Snykers, S., et al., Chromatin remodeling agent trichostatin A:a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow. BMC Dev Biol,2007.7:p.24.
36. de Boer, J., et al., Inhibition of histone acetylation as a tool in bone tissue engineering. Tissue Eng,2006.12(10):p.2927-37.