Trichostatin A Regulating The Expression Of Pluripotent Genes In Mesenchymal Stem Cells

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.PART1The down regulated expression of pluripotent genes during the isolation, culture and expansion of MSCs in vitroObjective: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.Conclusion1. 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 AObjective: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.Conclusion1. 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. PART3TSA stabilized the expression of pluripotent genes and their histone H3acetylation levels without affect the pluripotent differentiate ability of MSCsObjective: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).Conclusion1. 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.