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Endotoxin-Induced Silencing of Mesoderm Induction and Functional Differentiation: Role of HMGB1 in Pluripotency and Infection

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Endotoxin-Induced Silencing of Mesoderm Induction and Functional Differentiation: Role of HMGB1 in Pluripotency and Infection For reprint orders, please contact: ESEARCH RTICLE [email protected] R A Endotoxin-induced silencing of mesoderm induction and functional differentiation: role of HMGB1 in pluripotency and infection Kavitha Objectives: Mechanisms underpinning Gram-negative bacterial vaginosis-induced birth Sivasubramaniyan1, anomalies are obscure. Ethical issues limit such studies on peri-implantation-stage human Rajesh Reddy Atluri1, Kanchan Sarda1, embryos. Here we have used embryoid bodies (EBs) as an in vitro model to examine the Milan Arvind1, effect of Gram-negative bacterial endotoxins/lipopolysaccharides (LPS) on the faithful Vishnu Balaji1 & induction of germ lineages during embryogenesis. The role of LPS-inducible cytokine and Kaushik Dilip Deb1† pluripotency-related DNA-binding protein HMGB1 was also studied in these EBs. †Author for correspondence Methods: EBs derived from the human embryonic stem cell line HUES9 were exposed to 1Manipal University, 12.5 pg/ml of LPS for 48 h. The expression profile of the ectoderm, endoderm, mesoderm Embryonic Stem Cell and and trophectoderm lineage markers, such as βIII-tubulin, GATA4, BMP2, Brachury and Developmental Biology Program, Manipal Institute β-hCG, were studied, by RT-PCR and immunofluorescence. Inhibition of mesoderm of Regenerative Medicine, induction was confirmed by RT-PCR analysis for hANP, cTnT, ABCG2, GATA2, BMP4 and #10 Service Road, Domlur HAND1. Osteoblast differentiation was induced in the EBs, and confirmed by von Kosa and Layout, Bangalore 560071, India Alizarin red staining. A comet assay was also carried out to assess the degree of apoptosis Tel.:+91 802 535 6660; in these EBs. Results and conclusions: We found that the LPS-treated EBs were Fax: +91 802 535 6662; selectively silenced for mesoderm markers and failed to differentiate into functional E-mail: kaushik.deb@ manipalhospital.org osteoblasts. HMGB1 expression was absent in the normal EBs and was found to be localized in the cytoplasm of the LPS-treated EBs. Overall, our data indicate that endotoxin-induced HMGB1 expression in the peri-implantation-stage embryos can bring about severe birth defects of, for example, the bone and heart. This study also indicates that HMGB1 could be involved in maintenance of pluripotency in the human embryonic stem cells by impeding their differentiation. Human embryonic stem cells (hESCs) have Gram-negative bacterial infections of the been widely used to understand the molecular maternal genital tract, known as bacterial vagi- mechanisms underpinning human develop- nosis, can lead to the formation of poor-quality ment. These pluripotent cells provide a reliable embryos, which fail to implant [6]. Subclinical source for studying differentiation to all the germ or silent infections of Gram-negative bacteria layer lineages, namely ectoderm, endoderm, mes- such as Chlamydia trachomatis can also cause oderm and trophectoderm lineages [1,2]. HESCs birth defects with poorly developed tissues and have been successfully directed toward the for- organs of the fetus [7]. Ethical issues limit stud- mation of different tissues of various lineages [3]. ies on the molecular mechanisms underlying These cells can also be used to produce preim- such pathogenesis in human embryos. Endo- plantation embryo- or blastocyst-like entities, toxin lipopolysaccaharides (LPS) is the main known as embryoid bodies (EBs), which con- antigenic component of Gram-negative bacte- sist of a differentiated population of cells repre- rial cell wall and is regularly shed in the sur- senting all the germ layers. These EBs, rounding environment. LPS is known to cause therefore, closely mimic a growing embryo, various perinatal complications [8]. In previous which consists of the placental precursors (tro- studies we have established the role of various phectoderm) and the cells of the embryo proinflamatory and other LPS-inducible proper (ectoderm, endoderm and mesoderm) cytokines and growth factors, such as IL-1α, Keywords: embryoid bodies, [4]. It is known that ectoderm forms the skin IL-1β, TNF-α and CSF1 during embryo human embryonic stem cell, lipopolysaccharides, and the nervous system, the mesoderm forms implantation and in subsequent pregnancy mesoderm tissues such as the cardiomyocytes, bone and loss [9–11]. However, the molecular events blood, and the endoderm forms, for example, underlying poor fetal development and birth part of the liver, lungs and intestine of the developing defects during silent infections are not known. embryo [5]. We hypothesize that the presence of LPS in the 10.2217/17460751.3.1.23 © 2008 Future Medicine Ltd ISSN 1746-0751 Regen. Med. (2008) 3(1), 23–31 23 RESEARCH ARTICLE – Sivasubramaniyan, Atluri, Sarda, Arvind, Balaji & Deb environment of the developing fetus may selec- factor (βFGF), and penicillin–streptomycin tively inhibit the induction of one or more of 50 U/ml (all from Invitrogen, CA, USA). For the lineages during early pregnancy. induction of EB formation, the hESCs were In this study we have used EBs as an in vitro seeded on a low-adherent 60-mm plate (BD model to examine the effect of LPS on the dif- Biosciences, CA, USA) containing ES media ferentiation and faithful induction of the germ without FGF2. Human ESCs from three con- lineages during peri-implantation embryonic fluent 35-mm dishes were collected after development. The expression of LPS-inducible trypsinization and used for inducing EBs in and pluripotency-related gene high-mobility each l60-mm low-adherent dish. The EBs group box 1 (HMGB1) was studied to assess its formed were screened for their typical round possible involvement in the aberrant differenti- morphology under a binocular microscope ation of the LPS-treated EBs [12,13]. HMGB1 is (Nikon). On day 2.5 they were collected and explicitly expressed by the cells of the inner cell washed three times in the culture media and mass and is absent in the trophectoderm cells of finally transferred to fresh media. This step was the blastocyst [14]. HMGB1 is also known as a carried out to dispose of the dying MEFs and DNA-binding protein that can regulate expres- also to select the healthy-looking EBs. sion of genes [12]. Owing to its versatile roles both during development and in response to Selection of time points & dose of LPS endotoxins, we hypothesized that HMGB1 The induction of all the germ lineages in the may be a key player in mediating LPS-induced EBs and effect of LPS was tested at two different developmental defects. time points (day 4.5 and 9.5) and by using dif- In previous studies we have shown that LPS ferent doses of LPS. Early EBs at day 2.5 were exposure can render the preimplantation washed in the culture media and were divided embryo or 5-day-old blastocyst inefficient for into groups of approximately 30 each. Each implantation [15]. We therefore used early-stage group was then exposed to a dose of 5, 10, 15 or 5-day-old EBs to closely mimic the peri- 20 pg/ml LPS. A control group of EBs unex- implantation stage of embryonic development posed to LPS was also taken. Following 48 h of (day 4–5). We found that LPS exposure for incubation the EBs were harvested on day 4.5 or 48 h inhibited functional mesoderm formation 9.5. The RNA was isolated from each group and in these EBs. LPS-induced HMGB1 expression RT-PCR-based screening was carried out for the in these EBs also indicates its possible role in expression of the ectoderm, endoderm, meso- silencing mesoderm induction. These findings derm and trophectoderm lineage markers. All indicate for the first time that the presence of the doses (ranging from 5–20 pg LPS/ml) and endotoxins in the maternal environment can both the time points studied exerted similar lead to predictable mesoderm tissue-specific effects on the expression pattern of the lineage birth defects, such as malformation of bones. markers. The median-dose 12.5 pg LPS/ml and This study also indicates that HMGB1 is the earlier time point of complete lineage induc- related to pluripotency in hESCs and that its tion, for example, day 4.5 was chosen as a repre- expression silences mesoderm-specific genes sentative dose and time for this study. All and differentiation. subsequent studies and analyses were carried out using this dose and time point. Material & methods Culture of hESCs & production of EBs Exposure of EBs to LPS hESC line HUES-9 was obtained from Harvard EBs at day 2.5 were exposed to 12.5 pg/ml of University (MA, USA) and was used after insti- endotoxin/LPS (Sigma) for 48 h supplemented tutional ethics committee approval. It was in culture medium. The normal and the endo- maintained on mouse embryonic feeder (MEF) toxin-treated EBs were harvested on day 4.5. cells. HUES-9 was maintained in embryonic Postexposure, the control and endotoxin- stem cell medium (ES medium) consisting of treated EBs were divided into two groups. One 80% KnockOut Dulbecco's Modified Eagle's group (n = 30) was lysed in TRIZOL for RNA Medium (DMEM) and 20% KnockOut serum isolation and the other group (n = 90) was fixed replacement (KSR), supplemented with 2 mM in 4% parafomaldehyde for immunofluores- L-glutamine, 1% nonessential amino acid solu- cence. The expression profile of the ectoderm, tion, 0.1 mM β-mercaptoethanol, 4 ng/ml endoderm, mesoderm and trophectoderm human recombinant basic fibroblast growth lineage markers, such as βIII-tubulin, GATA4, 24 Regen. Med. (2008) 3(1) futurefuture sciencescience groupgroup Role of HMGB1 in pluripotency and infection – RESEARCH ARTICLE BMP2, Brachury and β-hCG, were studied by as a result of LPS treatment. The results were RT-PCR and immunofluorescence. The expres- then compared with the control cells (non-LPS- sion of the LPS-inducible and pluripotency- treated EBs). To count the number of cells per related DNA-binding protein HMGB1 was also EB, the number of DAPI-stained nuclei were studied in both the control and treated EBs.
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