Trichostatin A Stabilizes the Expression of Pluripotent Genes in Human Mesenchymal Stem Cells during Ex Vivo Expansion

Background

Mesenchymal stem cells (MSCs) have been considered as ideal cells for the treatment of a variety of diseases. However, aging and spontaneous differentiation of MSCs during culture expansion dampen their effectiveness. Previous studies suggest that ex vivo aging of MSCs is largely caused by epigenetic changes particularly a decline of histone H3 acetylation levels in promoter regions of pluripotent genes due to inappropriate growth environment.

Methodology/Principal Findings

In this study, we examined whether histone deacetylase inhibitor trichostatin A (TSA) could suppress the histone H3 deacetylation thus maintaining the primitive property of MSCs. We found that in regular adherent culture, human MSCs became flatter and larger upon successive passaging, while the expression of pluripotent genes such as Oct4, Sox2, Nanog, Rex-1, CD133 and TERT decreased markedly. Administration of low concentrations of TSA in culture significantly suppressed the morphological changes in MSCs otherwise occurred during culture expansion, increased their proliferation while retaining their cell contact growth inhibition property and multipotent differentiation ability. Moreover, TSA stabilized the expression of the above pluripotent genes and histone H3 acetylation levels in K9 and K14 in promoter regions of Oct4, Sox2 and TERT.

Conclusions/Significance

Our results suggest that TSA may serve as an effective culture additive to maintain the primitive feature of MSCs during culture expansion.     

Epigenetic changes of mesenchymal stem cells in three‐dimensional (3D) spheroids

Mesenchymal stem cells (MSCs) hold profound promise in tissue repair/regeneration. However, MSCs undergo remarkable spontaneous differentiation and aging during monolayer culture expansion. In this study, we found that 2–3 days of three‐dimensional (3D) spheroid culture of human MSCs (hMSCs) that had been expanded in monolayer for six passages increased their clonogenicity and differentiation potency to neuronal cells. Moreover, in accordance with these changes, the expression levels of miRNA which were involved in stem cell potency were changed and levels of histone H3 acetylation in K9 in promoter regions of Oct4, Sox2 and Nanog were elevated. Our results indicate that spheroid culture increases their multi‐potency and changes the epigenetic status of pluripotent genes in hMSCs.     

Knockdown of p21(Cip1/Waf1) enhances proliferation, the expression of stemness markers, and osteogenic potential in human mesenchymal stem cells

Mammalian aging of many tissues is associated with a decline in the replicative and functional capacity of somatic stem cells. Understanding the basis of this decline is a major goal of aging research. Human bone marrow-derived multipotent stromal cells (MSCs) have been applied in the treatment of fracture nonunion. Clinical application of MSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often at the expense of stemness and differentiation potentiality. We first demonstrated that late-passage MSCs exhibited decreased proliferation capacity, reduced expression of stemness markers such as Oct-4 and Nanog, and deterioration of osteogenic potential. Further, late-passage MSCs showed increased expression of p21(Cip1/Waf1) (p21), an inhibitor of the cyclin-dependent kinase. Knockdown of p21 by lentivirus-mediated shRNAs against p21 in late-passage MSCs increased the proliferation capacity, the expression of Oct-4 and Nanog, and osteogenic potential compared with cells transduced with control shRNA. More importantly, reduction in p21 expression in MSCs enhanced the bone repair capacity of MSCs in a rodent calvarial defect model. Knockdown of p21 in MSCs also increased the telomerase activity and telomere length, and did not show chromosomal abnormalities or acquire transformation ability. Therefore, these data successfully demonstrate the involvement of senescence gene in the expression of stemness markers and osteogenic potential of MSCs.     

Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation

Background

Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood.

Methodology/Principal Findings

Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes.

Conclusions/Significance

Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.     

Ascorbic acid induces in vitro proliferation of human subcutaneous adipose tissue derived mesenchymal stem cells with upregulation of embryonic stem cell pluripotency markers Oct4 and SOX 2

Mesenchymal stem cells (MSCs) have immense therapeutic potential because of their ability to self-renew and differentiate into various connective tissue lineages. The in vitro proliferation and expansion of these cells is necessary for their use in stem cell therapy. Recently our group has developed and characterized mesenchymal stem cells from subcutaneous and visceral adipose tissue. We observed that these cells show a slower growth rate at higher passages and therefore decided to develop a supplemented medium, which will induce proliferation. Choi et al. have recently shown that the use of ascorbic acid enhances the proliferation of bone marrow derived MSCs. We therefore studied the effect of ascorbic acid on the proliferation of MSCs and characterized their phenotypes using stem cell specific molecular markers. It was observed that the use of 250 μM ascorbic acid promoted the significant growth of MSCs without loss of phenotype and differentiation potential. There was no considerable change in gene expression of cell surface markers CD105, CD13, Nanog, leukemia inhibitory factor (LIF) and Keratin 18. Moreover, the MSCs maintained in the medium supplemented with ascorbic acid for a period of 4 weeks showed increase in pluripotency markers Oct4 and SOX 2. Also cells in the experimental group retained the typical spindle shaped morphology. Thus, this study emphasizes the development of suitable growth medium for expansion of MSCs and maintenance of their undifferentiated state for further therapeutic use.     

Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities

Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.     

二甲双胍调控糖尿病大鼠骨髓间充质干细胞的增殖和分化

虽然二甲双胍广泛用于治疗2型糖尿病,但是其对骨骼的潜在影响知之甚少。因此,本研究评估了二甲双胍对培养的大鼠骨髓间充质干细胞(MSCs)和脂肪细胞两者的分化以及增殖的影响。首先随机组形成对照实验,其中对照组为在不经二甲双胍处理培养基中培养MSCs细胞21 d,而二甲双胍组则在用100μmol/L二甲双胍处理培养基中培养MSCs 21 d。结果表明,二甲双胍增强了大鼠MSCs的成骨细胞分化细胞中ALP的活性,抑制了培养中MSCs脂肪形成分化的过程,但是增强了MSCs细胞的增殖能力。     

胎儿肺组织来源间充质干细胞的分离、培养、鉴定及重编程初探

该文主要研究将进行胎儿肺部组织来源的间充质干细胞（mesenchymal stem cells derived from fetal lung,FL-MSCs）转变成为诱导多能干细胞（induced pluripotent stem cells,iPS细胞）。首先使用酶消化法对胎儿肺部组织进行分离,然后采用常规方法进行培养并成功获得成纤维细胞样细胞。使用共聚焦技术检测获得的细胞,发现角蛋白表达呈阴性;共聚焦技术检测c-Myc、Oct4、Nanog以及Nestin四个干性相关因子,发现它们呈阳性;检测成纤维细胞样细胞的免疫表型,符合间充质干细胞的表型判断标准;然后进行诱导分化实验,发现这些细胞可以向成脂、成骨细胞分化,经过以上实验鉴定获得的成纤维细胞为FL-MSCs。使用Yamanaka四因子体系对FL-MSCs进行诱导,可以形成类似人胚胎干细胞（human embryonic stem cells,hES细胞）的克隆,采用核型分析、STR检测分析以及畸胎瘤形成实验初步验证获得的克隆为iPS。     

Cell growth arrest and apoptosis induced by Oct4 or Nanog knockdown in mouse embryonic stem cells: a possible role of Trp53

It has been clear that both Oct4 and Nanog play essential roles in maintaining embryonic stem cells (ESCs) undifferentiation. However, the roles of Oct4 and Nanog in ESCs growth and apoptosis have been much less explored. In this study, we systematically examined the effects of Oct4 or Nanog knockdown on mouse ESCs (mESCs) growth and apoptosis as well as potential mechanisms. Our results show that Oct4 or Nanog knockdown induces growth arrest and apoptosis in mESCs, indicating that the two genes also play important roles in mESCs survival and growth. Moreover, upregulation in Trp53 and its downstream genes expression was detected in Oct4 or Nanog knockdown mESCs, suggesting a possible role of Trp53 in Oct4 or Nanog knockdown induced mESCs growth arrest and apoptosis.     

人类胚胎干细胞系H9培养和分化中细胞异质性的发现

目的:在人类胚胎干细胞系H9培养和分化过程中探讨该细胞系的异质性。方法:对人类胚胎干细胞系H9进行体外未分化培养和诱导分化,鉴定其多潜能性和分化状态;在诱导其向拟胚体细胞的分化过程中,检测多潜能相关基因及分化特异基因的表达情况。结果:发现多潜能相关基因(Oct4、SOX2和Nanog)和种系特异性基因(Cdx2、Bachurary、SOX1、Fgf5和AFP)并不限于分别在未分化细胞和分化细胞中表达。结论:提示H9细胞系在培养过程中的非基因异质性现象,为进一步认识胚胎干细胞的自我更新和多潜能性提供了有意义的参考。     

Depletion of HOXA5 inhibits the osteogenic differentiation and proliferation potential of stem cells from the apical papilla

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self‐renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real‐time RT‐PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16^INK4A and p18^INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16^INK4A, p18^INK4C, and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental‐tissue‐derived MSCs.     

Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus-diseased gestation

Human mesenchymal stem cells isolated from amniotic fluid (AF-MSCs) demonstrate the potency for self-renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF-MSCs from two cell sources and the expression of the cell surface-specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes’ expression specific for adipogenic (peroxisome proliferator-activated receptor-ɣ, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage-specific differentiation was related to differential expression of miR-17, miR-21, miR-34a, and miR-146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF-MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF-MSCs differentiation in fetus-diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.