Embryonic Regulation of the Mouse Hematopoietic Niche

Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called “niche cells” surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells

Specific Inhibition of Tumor Cells by Oncogenic <i>EGFR</i> Specific Silencing by RNA interference

<div><p>Anticancer agents that have minimal effects on normal cells and tissues are ideal cancer drugs. Here, we show specific inhibition of human cancer cells carrying oncogenic mutations in the epidermal growth factor receptor (EGFR) gene by means of oncogenic allele-specific RNA interference (RNAi), both <i>in vivo</i> and <i>in vitro</i>. The allele-specific RNAi (ASP-RNAi) treatment did not affect normal cells or tissues that had no target oncogenic allele, whereas the suppression of a normal <i>EGFR</i> allele by a conventional <i>in vivo</i> RNAi caused adverse effects, i.e., normal EGFR is vital. Taken together, our current findings suggest that specific inhibition of oncogenic <i>EGFR</i> alleles without affecting the normal <i>EGFR</i> allele may provide a safe treatment approach for cancer patients and that ASP-RNAi treatment may be capable of becoming a safe and effective, anticancer treatment method. </p> </div

Adverse effects of knockdown of normal <i>Egfr</i> in normal ICR mice.

<p>(<b>A</b>) Plasma analyses. siEgfr siRNA targeting the wild-type mouse <i>Egfr</i> gene, another siRNA (as indicated), or delivery vehicle was administered 3 times, on Days 1, 3, and 5, to 10-week-old ICR mice via the lateral tail vein. The day after administration, measurement of body weight was carried out (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone.0073214.s019" target="_blank">Table S5</a>). Two days after the last administration, the mice were subjected to hematological analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone.0073214.s020" target="_blank">Table S6</a>) followed by separation of blood plasma. The plasma specimens were examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone-0073214-g002" target="_blank">Figure 2</a>. Examined biochemical parameters are indicated (n=4 mice/group; mean ± SDs; * <i>P</i><0.05 by Tukey-Kramer test; n.s., no statistical significance). (<b>B</b>) TUNEL assay. Cryosections of intestinal tissue were prepared from ICR mice that had been treated with the indicated siRNAs; cryosections were examined using a TUNEL assay. A marked increase in intestinal apoptosis was detected in the siEgfr-treated ICR mice.</p

Effects of ASP-RNAi on tumor growth.

<p>(<b>A</b>) Schematic drawing of the experimental plan. s.c.: subcutaneous. (<b>B</b>) <i>In vivo</i> luminescent imaging. Subcutaneous tumor models were established with PC-3/luc cells and siRNAs at indicated doses were administered according to the experimental plan (A). Tumors were monitored by an IVIS imaging system. (<b>C</b>) Tumor growth. Luminescent intensities of the tumors treated with siRNAs were quantified and analyzed (mean ± SDs, n = 5 mice/group). (<b>D</b>) Wet weight of tumors treated with siRNAs. Three weeks after treatment (Day 28), the treated tumors were isolated and their wet weight was measured [mean ± SDs; n = 5 mice/group; *<i>P</i> < 0.05 by Dunnett’s test (vs. siControl)]. (<b>E</b>) Western blot analysis. Subcutaneous tumors were treated with 2.0 mg/kg b.w. of si747/49_3D8 (red box) or siControl (open box). Three days after treatment (Day 10), the treated tumors were isolated and examined by Western blotting using indicated antibodies. (<b>F</b>) Plasma biochemical parameters in mice treated with siRNAs. Plasma specimens were prepared from treated mice at Day 28, and subjected to plasma biochemical analyses to examine alkaline phosphatase (ALP), total protein (TP), GPT, GOT, and total (T-), direct (D-) and indirect (I-) bilirubin (BIL). Significant difference in each parameter was examined by Dunnett’s test as in D. n.s., no significant difference. (<b>G</b>) Body weight. Body weight of the treated mice was measured at Day 28. Statistical analysis was carried out as in F.</p

Effects of gefitinib on tumor growth.

<p>(<b>A</b>) Schematic drawing of the experimental plan. p.o.: per os (oral administration). (<b>B</b>) <i>In vivo</i> luminescent imaging. Subcutaneous tumor models were established with PC-3/luc cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone-0073214-g002" target="_blank">Figure 2</a>, and gefitinib was administered at the indicated doses. Photographic images of luminescent signals at Day 7 and 28 after the inoculation of PC-3/luc cells are shown. (<b>C</b>) Tumor growth. Luminescent intensities of the tumors treated with gefitinib were quantified and analyzed (n = 5 tumor models/group). Error bars represent SDs. (<b>D</b>) Wet weight of the tumors treated with gefitinib. Three weeks after treatment (Day 28), the treated tumors were isolated and wet weight was measured. A significant difference against the vehicle control group (open bar) is indicated by an asterisk [*<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed)]. Error bars represent SDs. (<b>E</b>) Examination of plasma biochemical parameters in the treated mice. Plasma specimens were prepared from the mice at Day 28, and subjected to plasma biochemical analyses as in Figures 2 and 4 [n = 5 mice/group; mean ± SDs; <i>P</i> < 0.05 by Dunnett’s test (vs. siControl); n.s., no significant difference]. (<b>F</b>) Body weight. Body weight of the treated mice was measured at Day28 [n = 5 mice/group; mean ± SDs; *<i>P</i> < 0.05 by Dunnett’s test (vs. siControl)]. (<b>G</b>) TUNEL assay. Cryosections of intestinal tissues were prepared from model mice treated with gefitinib (0, 50, 100 mg/kg b.w.) at Day 28 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073214#pone-0073214-g004" target="_blank">Figure 4</a>, and the cryosections were examined by a TUNEL assay.</p

Effects of systemic siRNA administration on tumor growth in lung cancer models.

<p>(<b>A</b>) <i>In vivo</i> luminescent imaging. PC-3/luc cells were intravenously administered to nude mice and examined using an IVIS imaging system 5 days after cell injection. PC-3/luc-positive mice were randomly divided into two groups, and subjected to systemic administration of indicated siRNAs twice (Day 5 and 7) via the lateral tail vein. The treated mice were examined using the IVIS imaging system; photographic images of luminescent signals at Day 5 and 10 (after cell injection) are shown (left panels). Box plots represent the luminescent intensities of the treated mice at Day 5 and 10 (right panels) [n = 5 mice/group; *<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed); n.s., no significant difference]. (<b>B</b>) Lung tissues isolated from siRNA-treated mice. Lung tissues were isolated from PC-3-bearing mice treated with the indicated siRNAs, and the wet weights were measured [mean ± SDs; n=5 mice/group; *<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed)]. (<b>C</b>) Histological analysis of lung tissues. Cryosections of lung tissue were prepared from PC3-bearing mice treated with the indicated siRNAs; cryosections were stained with conventional hematoxylin and eosin solution. (<b>D</b>) <i>In vivo</i> imaging of active caspase. Six hours after administration of the indicated siRNAs on Day 5, VivoGlo Caspase 3/7 substrate (Promega) was administrated intraperitoneally to the treated mice; <i>in vivo</i> imaging and subsequent imaging analysis were carried out. Box plots represent luminescent intensities [n = 6 mice/group; *<i>P</i> < 0.05 by Student’s <i>t</i>-test (two-tailed)].</p