Antinociceptive activity of doliroside B.

CONTEXT: Dolichos trilobus Linn (Leguminosae) is often used in Yi ethnic medicine to treat pain, fracture, and rheumatism. OBJECTIVE: To explore the therapeutic potential of doliroside B (DB) from D. trilobus and its disodium salt (DBDS) and the underlying mechanism in pain. MATERIALS AND METHODS: In the writhing test, Kunming mice were orally treated with DB and DBDS at doses of 0.31, 0.62, 1.25, 2.5, and 5 mg/kg. Vehicle, morphine, indomethacin, and acetylsalicylic acid were used as negative and positive control on the nociception-induced models, respectively. In the hot plate test, mice were orally treated with DB and DBDS at doses of 2.5, 5, 10, and 20 mg/kg. In the formalin test, mice were orally treated with DB and DBDS at doses of 2.5, 5, 10, and 20 mg/kg. In the meanwhile, lipopolysaccharide-induced inflammatory model in RAW264.7 macrophages was adopted to study the mechanism of pain alleviation for DBDS. RESULTS: DBDS (5 mg/kg) inhibited the writhing number by 80.2%, which exhibited the highest antinociceptive activity in pain models. DBDS could selectively inhibite the activity of COX-1. Meanwhile, it also reduced the production of NO, iNOS, and IL-6 by 55.8%, 69.0%, and 49.9% inhibition, respectively. It was found that DBDS also positively modulated the function of GABAA1 receptor. DISCUSSION AND CONCLUSIONS: DBDS displayed antinociceptive activity by acting on both the peripheral and central nervous systems, which may act on multitargets. Further work is warranted for developing DBDS into a potential drug for the treatment of pain.

Phenylpropanoid and Iridoid Glucosides from the Whole Plant of Hemiphragma heterophyllum and Their alpha-Glucosidase Inhibitory Activities.

Three phenylpropanoid glucosides (1:  - 3: ) and one iridoid glucoside (11: ), together with eleven known glucosides, were isolated from the ethanol extract of the whole plant of Hemiphragma heterophyllum. Their structures were elucidated by means of 1D and 2D NMR spectroscopy, HRMS, and chemical methods. All compounds except 11: and 13:  - 15: showed varying degrees of α-glucosidase inhibitory activity. Compounds 5, 9: , and 12: were marginally active in the bioassay, while compounds 1:  - 4: , 6:  - 8: , and 10: exhibited appreciable inhibitory activity with an IC50 value of 33.6 ~ 83.1 µM, which was much lower than that of the positive control acarbose (IC50 = 310.8 µM).

Interleukin-7 augments CD8+ T cells function and promotes viral clearance in chronic hepatitis C virus infection.

Interleukin (IL)-7 is a potent proliferation, activation, and survival cytokine for CD8+ T cells to improve viral and tumor specific CD8+ T cell responses. However, the role of IL-7 in regulation of dysfunctional hepatitis C virus (HCV)-specific CD8+ T cells was not fully elucidated. Thus, a total of 53 patients with chronic hepatitis C and 24 healthy individuals were enrolled in the current study. Serum IL-7 and its receptor α chain CD127 expression was measured. The modulatory function of IL-7 to CD8+ T cells was investigated in both direct and indirect contact co-culture with HCVcc-infected Huh7.5 cells. Both serum IL-7 and CD127 expression on CD8+ T cells was significantly reduced in chronic HCV-infected patients, which was negatively correlated with HCV RNA. Stimulation of IL-7 promoted both cytotoxicity and cytokines (interferon-γ, tumor necrosis factor-α, and IL-2) production of CD8+ T cells from patients with chronic hepatitis C. Moreover, IL-7 increased proliferation of CD8+ T cells, while downregulated a critical repressor of cytokine signaling, suppressor of cytokine signaling 3 (SOCS3). The IL-7-mediated enhancement effects to CD8+ T cells were dependent on IL-6 production. The current data suggested that IL-7 induced both cytolytic and noncytolytic functions of CD8+ T cells probably via repression of SOCS3. IL-7 might be considered as one of the therapeutic candidates for treatment of chronic HCV infection.

Response of microbial community composition and function to land use in mining soils of Xikuang Mountain in Hunan.

Nine land types in the northern mining area (BKQ) (mining land, smelting land, living area), the old mining area (LKQ) (whole-ore heap, wasteland, grassland), and southern mining area (NKQ) (grassland, shrubs, farmland) of Xikuang Mountain were chosen to explore the composition and functions of soil bacterial communities under different habitats around mining areas. The composition and functions of soil bacterial communities were compared among the sampling sites using 16S rRNA high-throughput sequencing and metagenomic sequencing. α diversity analysis showed the soil bacterial diversity and abundance in the old mining area were significantly higher than those in the northern mining area. ß diversity analysis demonstrated that the soil bacterial community composition was highly similar among different vegetation coverages in the southern mining area. Microbial community function analysis showed the annotated KEGG function pathways and eggNOG function composition were consistent between the grassland of the old mining area and the grassland of the southern mining area. This study uncovers the soil bacterial community composition and functions among different habitats in the mining areas of Xikuang Mountain and will underlie soil ecosystem restoration in different habitats under heavy metal pollution around the mining areas there.

Proteomic and metabolomic analyses illustrate the mechanisms of expression of the O6 -methylguanine-DNA methyltransferase gene in glioblastoma.

AIM: Glioblastoma (GBM) has been reported to be the most common high-grade primary malignant brain tumor in clinical practice and has a poor prognosis. O6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation has been related to prolonged overall survival (OS) in GBM patients after temozolomide treatment. METHODS: Proteomics and metabolomics were combined to explore the dysregulated metabolites and possible protein expression alterations in white matter (control group), MGMT promoter unmethylated GBM (GBM group) or MGMT promoter methylation positive GBM (MGMT group). RESULTS: In total, 2745 upregulated and 969 downregulated proteins were identified in the GBM group compared to the control group, and 131 upregulated and 299 downregulated proteins were identified in the MGMT group compared to the GBM group. Furthermore, 131 upregulated and 299 downregulated metabolites were identified in the GBM group compared to the control group, and 187 upregulated and 147 downregulated metabolites were identified in the MGMT group compared to the GBM group. The results showed that 94 upregulated and 19 downregulated proteins and 20 upregulated and 16 downregulated metabolites in the MGMT group were associated with DNA repair. KEGG pathway enrichment analysis illustrated that the dysregulated proteins and metabolites were involved in multiple metabolic pathways, including the synthesis and degradation of ketone bodies, amino sugar and nucleotide sugar metabolism. Moreover, integrated metabolomics and proteomics analysis was performed, and six key proteins were identified in the MGMT group and GBM group. Three key pathways were recognized as potential biomarkers for recognizing MGMT promoter unmethylated GBM and MGMT promoter methylation positive GBM from GBM patient samples, with areas under the curve of 0.7895, 0.7326 and 0.7026, respectively. CONCLUSION: This study provides novel mechanisms to understand methylation in GBM and identifies some biomarkers for the prognosis of two different GBM types, MGMT promoter unmethylated or methylated GBM, by using metabolomics and proteomics analyses.

Identification of ubiquitin-specific protease 32 as an oncogene in glioblastoma and the underlying mechanisms.

Glioblastoma (GBM) patients present poor prognosis. Deubiquitination by deubiquitinating enzymes (DUBs) is a critical process in cancer progression. Ubiquitin-specific proteases (USPs) constitute the largest sub-family of DUBs. Evaluate the role of USP32 in GBM progression and provide a potential target for GBM treatment. Clinical significance of USP32 was investigated using Gene Expression Omnibus databases. Effects of USP32 on cell growth and metastasis were studied in vitro and in vivo. Differentially expressive genes between USP32-knockdown U-87 MG cells and negative control cells were detected using RNA sequencing and used for Gene Ontology and Kyoto Encyclopedia of Genes and Genomic pathway enrichment analyses. Finally, RT-qPCR was used to validate the divergent expression of genes involved in the enriched pathways. USP32 was upregulated in GBM patients, being correlated to poor prognosis. USP32 downregulation inhibited cell growth and metastasis in vitro. Furthermore, USP32 knockdown inhibited tumorigenesis in vivo. In addition, UPS32 was identified as a crucial regulator in different pathways including cell cycle, cellular senescence, DNA replication, base excision repair, and mismatch repair pathways. USP32 acts as an oncogene in GBM through regulating several biological processes/pathways. It could be a potential target for GBM treatment.

Secondary metabolites isolated from Agrimonia pilosa Ledeb.

A pilot study on the ethanol extracts of Agrimonia pilosa found to have anti-α-glucosidase and anti-inflammatory activities. Subsequent chemical study afforded a new phenylethyl isocoumarin glycoside (1) and eight known compounds (2-9). The structure of 1 was elucidated by comprehensive spectroscopic analysis and chemical transformations. All compounds showed modest α-glucosidase inhibitory activity (IC50 values ranging from 36.8 to 210.7 µM), which was lower than that of the positive control acarbose (IC50=301.9 µM). Those compounds except inactive compounds 3 and 6 showed weak anti-inflammatory activity.[Formula: see text].

Association Between Body Mass Index and Intracranial Aneurysm Rupture: A Multicenter Retrospective Study.

Background and Aims: It has recently emerged the concept of "obesity paradox," a term used to describe an inverse association between obesity and clinical outcomes in cardiovascular diseases and stroke. The purpose of this study was to investigate the association between body mass index (BMI) and the risk of intracranial aneurysm rupture. Methods: In this study, we conducted a retrospective analysis of a prospectively maintained database of patients with intracranial aneurysms from 21 medical centers in China. A total of 3,965 patients with 4,632 saccular intracranial aneurysms were enrolled. Patients were separated into unruptured (n = 1,977) and ruptured groups (n = 1,988). Univariable and multivariable logistic regression analyses were performed to determine the association between BMI and intracranial aneurysm rupture. Results: Compared to the patients with normal BMI (18.5 to < 24.0 kg/m2), the odds of intracranial aneurysm rupture were significantly lower in patients with BMI 24.0 to < 28.0 kg/m2 (OR = 0.745, 95% CI = 0.638-0.868, P = 0.000) and patients with BMI ≥ 28.0 kg/m2 (OR = 0.628, 95% CI = 0.443-0.890, P = 0.009). Low BMI (<18.0 kg/m2) was not associated with intracranial aneurysm rupture (OR = 0.894, 95% CI = 0.483-1.657, P = 0.505). For males, both the BMI 24.0 to < 28.0 kg/m2 (OR = 0.606, 95% CI = 0.469-0.784, P = 0.000) and the BMI ≥ 28.0 kg/m2 (OR = 0.384, 95% CI = 0.224-0.658, P = 0.001) were associated with a lower rupture risk, whereas the inverse association was not observed in females. Both the BMI 24.0 to < 28.0 kg/m2 (OR = 0.722 for aged 50-60y, 95% CI = 0.554-0.938, P = 0.015; OR = 0.737 for aged >60y, 95% CI = 0.586-0.928, P = 0.009) and the BMI ≥ 28.0 kg/m2 (OR = 0.517 for aged 50-60y, 95% CI = 0.281-0.950, P = 0.0034; OR = 0.535 for aged >60y, 95% CI = 0.318-0.899, P = 0.0018) was associated with a lower rupture risk in patients aged ≥50 years, whereas the association was not significant in patients aged <50 years. Conclusions: Increased BMI is significantly and inversely associated with saccular intracranial aneurysm rupture in males and patients aged ≥50 years.

Medicinal dietary plants of the Yi in Mile, Yunnan, China.

BACKGROUND: The Yi is the largest ethnic group in Yunnan Province (China), with a population of five million. The Yi people tend to live in mountainous areas, and their culture includes a unique dietary system for treating and protecting people against illnesses. Medicinal plants occupy an essential place in the Yi diet because they play a key role in health and the prevention and treatment of diseases. However, few studies have addressed these medicinal dietary plants and their importance in the Yi's daily lives. The aim of this study was to (1) investigate the medicinal dietary plants used by the Yi in Mile City, (2) document the traditional knowledge held about these plants, (3) identify species with important cultural significance to the Yi in Mile City, and (4) analyze the special preparation methods and consumption habits of these plants. METHODS: Field investigations were performed in six villages in Mile City, Honghe Hani and Yi Autonomous Prefecture, Yunnan, from July 2017 to May 2018. Information was collected using direct observation, semi-structured interviews, key informant interviews, individual discussions, and focus group discussions. The use value (UV) and frequency of utilization index (FUI) of these plants were analyzed. Plant samples and voucher specimens were collected for taxonomic identification. RESULTS: This study documented 124 species belonging to 62 families and 102 genera. These plants included angiosperms (117 spp.), gymnosperms (3), pteridophytes (2), lichen (1), and fungus (1). The 20 species with the highest UV were noted as being particularly important to the Yi people's daily life in Mile City. The primary medicinal preparation method for plants recorded in the study was decoction. The most commonly used plant parts were fruits and roots. The most frequently used edible parts were fruits, and the most frequently used medicinal parts were roots. The medicinal parts were used to treat diseases such as rheumatism, edemas, kidney deficiency, spleen deficiency, gastritis, parasites, and so on. CONCLUSION: A wide variety of medicinal dietary plants are used by the Yi people in Mile City. Those plants, which have both rich nutritional and medicinal value, occupy an essential part of the Yi dietary and medicine culture. Ethnobotanical surveys of medicinal dietary plants provide a theoretical reference for the conservation and sustainable use of the plant resources and could contribute to the protection of the Yi food culture and traditional medicine in Mile City. In addition, this information provides a sound basis for developing and using Yi ethnic medicine and health products.

Room temperature N-heterocyclic carbene manganese catalyzed selective N-alkylation of anilines with alcohols.

The first example of room temperature non-noble metal homogeneous system catalyzed selective N-alkylation of anilines with alcohols by a bis-NHC manganese complex is presented. This system was applied to a large range of alcohols and anilines, including biologically relevant motifs and challenging methanol. Experimental and computational studies suggest an outer-sphere mechanism for this NHC-Mn system.

Effect of FIGF overexpression on liver cells transforming to insulin-producing cells.

Limitation in the number of insulin-producing pancreatic ß-cells is a typical feature of diabetes. It has been indicated that activating pancreatic transcription factors can promote the transformation of hepatocytes into insulin-secreting ß-like cells, indicating that direct hepatocyte differentiation seems promising as a treatment for diabetes. Nevertheless, the reprogramming efficiency still remains low. Our previous study found that the expression of c-fos-induced growth factor (FIGF) was increased in the pancreatic tissues in partial pancreatectomy mice compared to that in normal mice. Here, we observed that treatment with Ad-FIGF was found to enhance MafA and Ngn3-induced reprogramming of BNL CL.2 cells to ß-like cells with the ability of secreting insulin. And FIGF overexpression increased the levels of histone H3/H4 acetylation at MafA and Ngn3 promoter regions in BNL CL.2 cells. Importantly, in vivo study further confirmed that forced expression of FIGF facilitated the insulin expression and decreased the blood glucose levels in STZ mice. These results strengthen the possibility of developing cell-based therapies for diabetes through utilizing ß-like cells derived from non-insulin-secreting cells.

High Expression of Long Noncoding RNA HOTAIRM1 is Associated with the Proliferation and Migration in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable malignancy. Long noncoding RNA (LncRNA) HOTAIRM1 (HOX antisense intergenic RNA myeloid 1) has been shown to play important roles in the progression of several type cancers. However, the exact role of HOTAIRM1 in PDAC development remains largely unknown. This study aims to evaluate the potential function of HOTAIRM1 in the development and progress of PDAC. HOTAIRM1 expression was measured by RT-qPCR in forty seven paired human PDAC tissues and five PDAC cell lines. SW1990 and PANC-1 cells were transfected with siHOTAIRM1 to achieve HOTAIRM1 silence. MTT assay and colony formation assay were used to detect the effect of HOTAIRM1 knockdown on cell proliferation. The impact of HOTAIRM1 silence on cell cycle and apoptosis was assessed by flow cytometry assay. Transwell migration assay was performed to explore the influence of HOTAIRM1 downregulation on the migratory potential of PDAC cells. Western blot assay was applied to determine the expression changes of cell cycle, apoptosis, and migration-related genes before and after downregulating HOTAIRM1. HOTAIRM1 expression was abnormally upregulated in PDAC tissues and cells when compared with the control samples, and was positively associated with the expression of KRAS gene mutation. In vitro functional experiments, HOTAIRM1 expression was significantly downregulated by transfection with siHOTAIRM1 in SW1990 and PANC cell lines. HOTAIRM1 knockdown attenuated cell proliferation by inducing cell cycle arrest at G0/G1 phase, promoted cell apoptosis, and inhibited cell migration in PDAC cells by regulating related-genes expression. In conclusion, HOTAIRM1 plays a critical role in PDAC progression, which may be a novel diagnostic and rational therapeutic target for the treatment of pancreatic ductal adenocarcinoma.

Novel oligodeoxynucleotide agonists of TLR9 containing N3-Me-dC or N1-Me-dG modifications.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N3-Me-cytosine or N1-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs). IMOs containing N-Me-cytosine or N-Me-guanine in C- or G-position, respectively, of the CpG dinucleotide showed activation of HEK293 cells expressing TLR9, but not TLR3, 7 or 8. IMOs containing N-Me-cytosine or N-Me-guanine modification showed activity in mouse spleen cell cultures, in vivo in mice, and in human cell cultures. In addition, IMOs containing N-Me-substitutions reversed antigen-induced Th2 immune responses towards a Th1-type in OVA-sensitized mouse spleen cell cultures. These studies suggest that TLR9 tolerates a methyl group at N1-position of G and a methyl group at N3-position of C may interfere with TLR9 activation to some extent. These are the first studies elucidating the role of N3-position of cytosine and N1-position of guanine in a CpG motif for TLR9 activation and immune stimulation.

Antioxidant and alpha-glucosidase inhibitory activities of isoflavonoids from the rhizomes of Ficus tikoua Bur.

A new unique isoflavone derivatives with a cyclic-monoterpene-substituent, ficusin C (1), together with five known compounds (2-6), were isolated from the rhizomes of Ficus tikoua. Their structures were elucidated on the basis of spectroscopic data interpretation, mass spectrometric analysis and comparison with literature data of related compounds. Antioxidant and α-glucosidase inhibitory activities of these compounds were evaluated by 1,1-diphenyl-2-picryhydrazyl (DPPH) radical-scavenging assay and α-glucosidase inhibitory experiment, respectively.

Agonists of Toll-like receptor 9 containing synthetic dinucleotide motifs.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends. Agonists of TLR9 containing cytosine or guanine modification showed activity in HEK293 cells expressing TLR9, mouse spleen, and human cell-based assays and in vivo in mice. The results presented here provide insight into which specific chemical modifications at C or G of the CpG motif are recognized by TLR9 and the ability to modulate immune responses substituting natural C or G in immune modulatory oligonucleotides.

Polyol monoterpenes isolated from Chenopodium ambrosioides.

Phytochemical study on the 95% ethyl alcohol extract of stems of Chenopodium ambrosioides resulted in the isolation of two new polyol monoterpenes, 4-hydroxy-4(α or ß)-isopropyl-2-methyl-2-cyclohexen-1-one (1) and 1-methyl-4ß- isopropyl-1-cyclohexene-4α,5α,6α-triol (2), together with five known compounds, (1S,2S,3R,4S)-1-methyl-4-(propan-2-yl)cyclohexane-1,2,3,4-tetrol (3), (1R,2S,3S,4S)- 1,2,3,4-tetrahydroxy-p-menthane (4), (1R,2S)-3-p-menthen-1,2-diol (5), (1R,4S)-p- menth-2-en-1-ol (6) and 1,4-dihydroxy-p-menth-2-ene (7). The structures of the new compounds were established on the basis of detailed spectroscopic evidence including extensive 1D and 2D NMR techniques. Compounds 1-7 were evaluated for their anti-inflammatory activity, and compound 1 showed moderate ability to inhibit NO production of LPS-stimulated RAW 264.7 macrophages with an IC50 value of 16.83 µM.

Effects of Different Ratios of Sewage Sludge and Cattle Manure on Growth and Propagation of Eisenia Fetida.

Domestic sewage sludge and cattle manure are rich in nutrition elements, but without proper disposal, are harmful to the environment. Here with an indoor culture method, we used Eisenia fetida to dispose different ratios of sewage sludge and cattle manure, and thereby investigated the effects and acting rules of these sludge-manure mixtures on the growth and reproduction of E. fetida. We find these mixtures are food sources for E. fetida, and their physiochemical properties are significantly changed after disposal by earthworms. Paired samples t-test shows the average change after different treatments is -20.37% for total organic carbon, 85.71% for total Kjeldahl N, -6.67% for total P, 8.33% for pH, -24.78% for EC (ms·cm-1), and -57.10% for C/N ratio. The average growth rate after treatment CD-70 is 9.20 mg·worm-1·day-1; the average growth rates of E. fetida on day 0-28, day 29-56, and day 57-91 are 9.33, 11.90 and 6.95 mg·worm-1·day-1, respectively, indicating a trend of "rapid-rapidest-slow" growth. Other treatments all show this trend. Though all earthworms developed reproductive rings during the test periods, the appearing time and the cocoon production time both differed among these treatments. The cocoon production amount is maximized to 233 after treatment CD-70. The cocoon production rates are significantly different among these treatments, and the maximum and mean are 0.32 and 0.17-0.32, cocoons·worm-1· day-1, respectively. E. fetida can modestly enrich Cd, but is not very effective over Sb or other heavy metals. E. fetida can remove a part of heavy metals from sewage sludge and cattle manure. Generally, the mixtures of sewage sludge and cattle manure can largely affect the growth and propagation of E. fetida in a ratio-dependent way.

Oligodeoxynucleotides containing synthetic immunostimulatory motifs augment potent Th1 immune responses to HBsAg in mice.

Toll-like receptor 9 (TLR9) modulators have potent Th1-adjuvant activity. We recently reported the development of immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) or R'pG (R'=1-(2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs. IMOs activate TLR9 pathways, resulting in cytokine secretion profiles different from those induced by CpG DNA. In the present study we evaluated the adjuvant activity of IMOs containing CpG, CpR, or R'pG motifs in combination with hepatitis B surface antigen (HBsAg) in a mouse model. Mice immunized with HBsAg plus IMO produced higher levels of IgG2a and lower levels of IgG1 than did mice immunized with HBsAg alone or with alum. High IgG2a responses were found at week 4 and remained high until 14 weeks after immunization. Adoptive transfer of splenocytes from HBsAg/IMO-immunized mice to naïve mice resulted in strong IgG2a production in response to antigen boost. Splenocytes of mice immunized with HBsAg/IMO produced high levels of IFN-gamma, but not Th2 cytokines IL-4 and IL-5, in antigen-recall experiments in vitro. The use of IMOs as adjuvants to HBsAg resulted in the production of strong anti-HBsAg antibodies at antigen doses as low as 0.2 microg. These data demonstrate that IMOs enhance the immunogenicity of HBsAg through potent Th1 immune responses, which may allow lower doses of antigen in vaccination.

Alteration of histone acetylation pattern during long-term serum-free culture conditions of human fetal placental mesenchymal stem cells.

Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.

Placental mesenchymal stem cells of fetal origin deposit epigenetic alterations during long-term culture under serum-free condition.

OBJECTIVE: Fetal placental mesenchymal stem cells (fPMSCs) have shown promising cell therapy potentials. However, their genetic and epigenetic stability during in vitro propagation has not been well studied. We thus interrogated the methylation alterations and tumorigenicity of fPMSCs after in vitro expansion using serum-free medium. RESEARCH DESIGN AND METHODS: The properties of fPMSCs cultured in a serum-free medium at passage 3 and passage 8 were ascertained by determining their MSC markers, proliferative capacity, chromosomal stability, activity of global DNA methyltransferases and methylation profile. Their potential of malignant transformation was also evaluated in a severe combined immunodeficiency (SCID) murine model. RESULTS: The fPMSCs could maintain their MSC characteristics but quickly reached a senescent state of proliferation during in vitro expansion. 246 genes with differential DNA methylation of promoters were identified, along with a significantly downregulated global DNA methyltransferase activity. The genes associated with aging and tumorigenesis had a significantly demethylated tendency over in vitro propagation. However, the deposition of epigenetic alterations did not translate into malignant transformation in SCID mice. CONCLUSION: The fPMSCs cultured in serum-free medium have a tendency to deposit methylation modifications over in vitro expansion, therefore the detection of genetic and/or epigenetic alterations is necessary for fPMSCs before they are employed for clinical uses.