MiR-24 is required for hematopoietic differentiation of mouse embryonic stem cells

Overexpression of miRNA, miR-24, in mouse hematopoietic progenitors increases monocytic/ granulocytic differentiation and inhibits B cell development. To determine if endogenous miR-24 is required for hematopoiesis, we antagonized miR-24 in mouse embryonic stem cells (ESCs) and performed in vitro differentiations. Suppression of miR-24 resulted in an inability to produce blood and hematopoietic progenitors (HPCs) from ESCs. The phenotype is not a general defect in mesoderm production since we observe production of nascent mesoderm as well as mesoderm derived cardiac muscle and endothelial cells. Results from blast colony forming cell (BL-CFC) assays demonstrate that miR-24 is not required for generation of the hemangioblast, the mesoderm progenitor that gives rise to blood and endothelial cells. However, expression of the transcription factors Runx1 and Scl is greatly reduced, suggesting an impaired ability of the hemangioblast to differentiate. Lastly, we observed that known miR-24 target, Trib3, is upregulated in the miR-24 antagonized embryoid bodies (EBs). Overexpression of Trib3 alone in ESCs was able to decrease HPC production, though not as great as seen with miR-24 knockdown. These results demonstrate an essential role for miR-24 in the hematopoietic differentiation of ESCs. Although many miRNAs have been implicated in regulation of hematopoiesis, this is the first miRNA observed to be required for the specification of mammalian blood progenitors from early mesoderm

Retroperitoneal Hematoma as an Atypical Presentation of Choriocarcinoma: A Case Report

A 38-year-old female with an etonogestrel implant in place and history of previous ectopic pregnancy presented with acute abdominal pain and vaginal bleeding. She was found to have a beta-hCG of \u3e12,000 mIU/mL and free fluid noted on a focused assessment with sonography in trauma exam. She underwent an emergent diagnostic laparoscopy due to the suspicion of a ruptured ectopic pregnancy. Findings at the time of surgery included a normal-appearing uterus and left fallopian tube, a surgically absent right fallopian tube and large volume hemoperitoneum with a rapidly expanding left retroperitoneal hematoma. A postoperative computerized tomography (CT) angiogram suggested active bleeding from a pseudoaneurysm of the left renal artery which was successfully embolized by interventional radiology. Biopsy confirmed gestational trophoblastic neoplasia (GTN) after metastases to the brain. In this report, we describe the details of this case of GTN with an atypical presentation

Minimal asbestos exposure in germline BAP1 heterozygous mice is associated with deregulated inflammatory response and increased risk of mesothelioma

Germline BAP1 mutations predispose to several cancers, in particular malignant mesothelioma. Mesothelioma is an aggressive malignancy generally associated to professional exposure to asbestos. However, to date we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. We hypothesized that germline BAP1 mutations might influence the asbestos-induced inflammatory response that is linked to asbestos carcinogenesis, thereby increasing the risk of developing mesothelioma after minimal exposure. Using a BAP1+/- mouse model, we found that, compared to their wild type littermates, BAP1+/- mice exposed to low-dose asbestos fibers showed significant alterations of the peritoneal inflammatory response, including significantly higher levels of pro-tumorigenic alternatively polarized M2 macrophages, and lower levels of several chemokines and cytokines. Consistent with these data, BAP1+/- mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos, doses that rarely induced mesothelioma in wild type mice. Our findings suggest that minimal exposure to carcinogenic fibers may significantly increase the risk of malignant mesothelioma in genetically predisposed individuals carrying germline BAP1 mutations, possibly via alterations of the inflammatory response

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived

Expression of miR-24 target Trib3 impairs hematopoietic development of ESCs.

<p><b>A)</b> Trib3 gene expression in d4 EBs derived from the indicated ESC clones. The increase in expression observed in the miArrest-24 clones compared to uninfected and miArrest-SCR infected RW4 cells was significant with a P value of <0.00002 as determined by unpaired t-test. <b>B)</b> Relative Trib3 mRNA expression as determined by Q-RT-PCR of RNA isolated from d4 EBs fractionated as describe in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004959#pgen.1004959.g007" target="_blank">Fig. 7</a>. <b>C)</b> ESCs were taken out of LIF and spin-infected with either MigR1 or MigR1-Trib3 virus for 1.5h. Cells were then cultured as hanging drops for 48h to form EBs. EBs were then transferred to liquid culture and grown and additional 6d before flow cytometry analysis. Surface expression of the HPC markers CD41 and cKit were examined on GFP+ cells. <b>D)</b> Three independent infections were performed and the average percent of CD41+ HPCs observed is shown. The decrease in CD41+ cells induced by Trib3 expression is significant with a P value of <0.001.</p

BL-CFCs develop from EBs with antagonized miR-24.

<p>RW4 parental cells, miArrest-Scr clones, and miArrest-24 clones were differentiated in liquid culture for <b>A)</b> 2.75d or <b>B)</b> 3.0d. EBs were dissociated into single cell suspensions and plated in methylcellulose for growth of BL-CFCs. 4d later BL-CFCs were counted. 2 independent miArrest-Scr clones were examined for d2.75 and d3 differentiations. 4 miArrest-24 clones for d2.75 and 7 miArrest-24 clones for d3 were examined. For d2.75, RW4 n = 4, miArrest Scr n = 6, and miArrest-24 n = 8. For d3.0, RW4 n = 8, miArrest Scr n = 8, and miArrest-24 n = 16. <b>C)</b> RNA was isolated from d3, d4, and d5 EBs derived from the indicated ESC clones. Expression of the transcription factor Etv2 was examined. The decrease in expression observed in the miArrest-24 clones compared to uninfected and miArrest-SCR infected RW4 cells was significant at d3 (P<0.006), d4 (P<0.002), and d5 (P<0.006) as determined with by unpaired t-test. <b>D)</b> Twenty BL-CFC colonies from individual cultures were isolated and used to generate cDNA. Quantitative-RT-PCR was performed with the indicated gene specific primers. Data was averaged from results obtained from the differentiation of 3 scrambled clones, and 3 mir-24 KD clones. The decreases in Gata1, Runx1, and Scl expression are significant with respective P values of P<0.001, P< 10^-4, and P<10^-7.</p

MiR-24 is required for blood development from mouse embryonic stem cells.

<p>ESC clones were generated from RW4 cells infected with either miArrest-24 (ShRNA targeting miR-24) or miArrest-SCR (Scrambled shRNA) lentivirus. <b>A)</b> RW4 wildtype, SCR (miArrest-SCR), and <b>B)</b> miArrest-24 clones (24–1, 24–6B, and 24–14B) were differentiated into EBs for 14d in methylcellulose media. Top 3 panels are 25X magnification, and bottom panels are 50X. <b>C)</b> Quantitative RT-PCR analysis of expression of the hematopoietic transcription factors Gata1, and Sfpi1 from RNA obtained from the indicated ESC clones differentiated into EBs in liquid culture for 6d. The decrease in expression observed in the miArrest-24 clones compared to uninfected and miArrest-SCR infected RW4 clones was significant for Gata1, and Sfpi1 with respective P values of <0.0005, and <0.0008 as determined by unpaired t-test. Error bars represent the standard error of the mean (SEM).</p

MiR-24 is required for HPC generation.

<p>ESC clones infected with control miArrest-SCR, or miArrest-24 lentivirus were differentiated into EBs for 6d in liquid culture and analyzed by flow cytometry and quantitative RT-PCR. <b>A)</b> Flow cytometry analysis of CD41 and cKit (CD117) cell surface expression on single cells isolated from EBs generated from the indicated ESC clones. CD41+cKit- population contains primitive HPCs and CD41+cKit+ contains primitive and definitive HPCs. <b>B, C)</b> Expression of the transcription factors <b>B)</b> Scl, and <b>C)</b> Runx1, which are required for the development and/or function of hemogenic endothelium. RNA was isolated from d4 and d6 EBs. The decrease in expression observed in the miArrest-24 clones compared to uninfected and miArrest-SCR infected RW4 cells was significant at d4 for Scl, and Runx1 with respective P values of <0.007, and <0.002, as well as at d6 with respective P values of <0.003, and <0.008. P values were determined by unpaired t-test. Error bars represent the SEM.</p

MiR-24 is not required for the differentiation of embryonic stem cells into embryoid bodies.

<p>Two independently derived miArrest-SCR, and miArrest-24 ESC clones were differentiated into EBs. RNA was collected at d3 and d4 of differentiation, and used to analyze gene expression by quantitative RT-PCR. <b>A)</b> Formation of the germ layers was analyzed by assaying expression of Pax6 (Ectoderm), FoxA2 (Endoderm), and T (Mesoderm). No significant differences were observed in gene expression comparing scrambled shRNA clones to miR-24 shRNA clones. <b>B)</b> To further analyze mesoderm differentiation the expression of lateral plate mesoderm gene Twist, and paraxial plate mesoderm gene Tbx6 were assayed. No significant difference was observed in Tbx6 expression, however the decrease in Twist expression was significant at d3 and d4 with respective P values of <0.002, and <10^-8 respectively. Error bars in panels A and B represent the SEM. <b>C</b>. The indicated ESC clones were differentiated in liquid culture for 4d. Development of paraxial and lateral plate mesoderm was analyzed by flow cytometry. Lateral plate mesoderm is FLK+PDGFRα-, paraxial mesoderm is FLK1-PDGFRα+, and nascent uncommitted mesoderm is FLK1+PDGFRα+.</p