Modulation of hematopoietic and endothelial cell differentiation from mouse embryonic stem cells by different culture conditions

Embryonic stem (ES) cells can differentiate into many different somatic cells in culture. To better correlate hematopoietic and endothelial cell differentiation of ES cells in currently available protocols, we compared fetal liver kinase-1 (Flk-1)-, stem cell leukemia (Scl)-, and vascular endothelial-cadherin (VE-cadherin)-expressing cells generated in embryoid bodies (EBs) and on OP9 cells. We report that the kinetics of Scl and Flk-1 expression were similar in EBs and OP9 cells, although Flk-1 expression was extended on OP9 cells. CD45+ and Ter-119+ cells developed more efficiently in EBs, whereas VE-cadherin+ cells developed largely on OP9 cells. Cell sorting and replating studies showed that Scl+ cells, not Flk-1+ or VE-cadherin+ cells, were enriched for primitive and definitive hematopoietic progenitors. Our studies indicate that optimal hematopoietic and endothelial cell differentiation occur in EBs and on OP9 cells, respectively. Regardless of the culture systems used, Scl is the most relevant marker for enriching primitive and definitive hematopoietic progenitors.     

Selective Imaging of Lung Macrophages Using [11C]PBR28-Based Positron Emission Tomography

Purpose

We tested whether the translocator protein (TSPO)-targeted positron emission tomography (PET) tracer, N-acetyl-N-(2-[¹¹C]methoxybenzyl)-2-phenoxy-5-pyridinamine ([¹¹C]PBR28), could distinguish macrophage dominant from neutrophilic inflammation better than 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) in mouse models of lung inflammation and assessed TSPO association with macrophages in lung tissue from the mouse models and in patients with chronic obstructive pulmonary disease (COPD).

Procedures

MicroPET imaging quantified [¹¹C]PBR28 and [¹⁸F]FDG lung uptake in wild-type (Wt) C57BL/6J or heterozygous transgenic monocyte-deficient Wt/opT mice at 49 days after Sendai virus (SeV) infection, during macrophage-dominant inflammation, and in Wt mice at 3 days after SeV infection or 24 h after endotoxin instillation during neutrophilic inflammation. Immunohistochemical staining for TSPO in macrophages and neutrophils was performed using Mac3 and Ly6G for cell identification in mouse lung sections and CD68 and neutrophil elastase (NE) in human lung sections taken from explanted lungs from patients with COPD undergoing lung transplantation and donor lungs rejected for transplantation. Differences in tracer uptake among SeV-infected, endotoxin-treated, and uninfected/untreated control mice and in TSPO staining between neutrophils and macrophage populations in human lung sections were tested using analysis of variance.

Results

In Wt mice, [¹¹C]PBR28 uptake (% injected dose/ml lung tissue) increased significantly with macrophage-dominant inflammation at 49 days (D49) after SeV infection compared to controls (p = <0.001) but not at 3 days (D49) after SeV infection (p = 0.167). [¹¹C]PBR28 uptake was unchanged at 24 h after endotoxin instillation (p = 0.958). [¹⁸F]FDG uptake increased to a similar degree in D3 and D49 SeV-infected and endotoxin-treated Wt mice compared to controls with no significant difference in the degree of increase among the tested conditions. [¹¹C]PBR28 but not [¹⁸F]FDG lung uptake at D49 post-SeV infection was attenuated in Wt/opT mice compared to Wt mice. TSPO localized predominantly to macrophages in mouse lung tissue by immunostaining, and TSPO staining intensity was significantly higher in CD68+ cells compared to neutrophils in the human lung sections.

Conclusions

PET imaging with [¹¹C]PBR28 can specifically detect macrophages versus neutrophils during lung inflammation and may be a useful biomarker of macrophage accumulation in lung disease.

     

Fructose consumption during pregnancy and lactation induces fatty liver and glucose intolerance in rats

Nutritional insults during pregnancy and lactation are health risks for mother and offspring. Both fructose (FR) and low-protein (LP) diets are linked to hepatic steatosis and insulin resistance in nonpregnant animals. We hypothesized that dietary FR or LP intake during pregnancy may exacerbate the already compromised glucose homeostasis to induce gestational diabetes and fatty liver. Therefore, we investigated and compared the effects of LP or FR intake on hepatic steatosis and insulin resistance in unmated controls (CTs) and pregnant and lactating rats. Sprague-Dawley rats were fed a CT, or a 63% FR, or an 8% LP diet. Glucose tolerance test at day 17 of the study revealed greater (P < .05) blood glucose at 10 (75.6 mg/dL vs 64.0 ± 4.8 mg/dL) minutes and 20 (72.4 mg/dL vs 58.6 ± 4.0 mg/dL) minutes after glucose dose and greater area under the curve (4302.3 mg?dL^− 1? min^− 1 vs 3763.4 ± 263.6 mg?dL^− 1? min^− 1) for FR-fed dams compared with CT-fed dams. The rats were euthanized at 21 days postpartum. Both the FR- and LP-fed dams had enlarged (P < .05) livers (9.3%, 7.1% body weight vs 4.8% ± 0.2% body weight) and elevated (P < .05) liver triacylglycerol (216.0, 130.0 mg/g vs 19.9 ± 12.6 mg/g liver weight) compared with CT-fed dams. Fructose induced fatty liver and glucose intolerance in pregnant and lactating rats, but not unmated CT rats. The data demonstrate a unique physiological status response to diet resulting in the development of gestational diabetes coupled with hepatic steatosis in FR-fed dams, which is more severe than an LP diet.     

Oncogenic potential of the DNA replication licensing protein CDT1

The expression of a gene, designated as Retroviral insertion site (Ris)2, was activated by retroviral DNA integration in an immortalized primitive erythroid cell line, EB-PE. Ris2 was also expressed at high levels in all human tumor cell lines analysed. Consistently, NIH3T3 fibroblasts overexpressing Ris2 formed tumors in Rag2 -/- mice when injected subcutaneously. The putative RIS2 protein shows a high sequence similarity to Xenopus CDT1, Drosophila DUP, and human CDT1, a newly identified DNA replication licensing protein, suggesting that Ris2 is a mouse homologue of CDT1. Cells overexpressing Ris2/Cdt1 exhibited a quicker entry into S phase when released from serum starvation compared to controls. Our results suggest that CDT1, an essential licensing protein for DNA replication, can function as an oncogene in mammals.