Maternal environment and DNA methylation patterns

Data collected in the field for 26 Eastern bluebird nests and the one randomly selected nestling from each of the 25 successfully hatched nests. Nest data include yolk testosterone, breeding density, clutch size, and brood size. Nestling data include sex, mass, growth rate, and methylation patterns

Induction of Macrophage-Like Immunosuppressive Cells from Mouse ES Cells That Contribute to Prolong Allogeneic Graft Survival

<div><p>Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.</p></div

ES-SCs prolonged the survival of allogeneic graft.

<p>Host 129X1 or C3H mice were intraperitoneally injected with phosphate buffered saline (PBS, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111826#pone-0111826-g003" target="_blank">Figure 3A, B, D, E, G and H</a>) or 35 Gy-irradiated ES-SCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111826#pone-0111826-g003" target="_blank">Figure 3C, F and I</a>) 14 days before transplantation. (A–C) Hematoxylin and eosin staining of E14 EB grafts transplanted into renal subcapsule at day 14 after graft transplantation. Data are shown as representative of two grafts. Scale bars, 1.0 mm. (D–F) Immunohistological staining for CD3 antigen (indicating T cell infiltration) of ESCs-derived cardiomyocytes transplanted into renal subcapsule at day 7 after graft transplantation. (G–I) Hematoxylin and eosin staining of ESCs-derived cardiomyocytes transplanted into renal subcapsule at day 7 after graft transplantation. Data are shown as representative of two grafts. Scale bars, 100 µm.</p

Graft (ESCs-derived cardiomyocytes) survival.

<p>Administration of ES-SCs prolonged allogeneic ESCs-derived cardiomyocytes survival <i>in vivo</i><b><i>.</i></b> Recipient mice were pretreated by PBS, ES-SCs or ES-DCs as described in Materials and Methods. Thereafter, mice received transplantation of cardiomyocytes generated from 129-derived ESCs, and graft survival was monitored by direct inspection. C3H (PBS-injected) vs. C3H (ES-SCs-treated), p<0.01; Generalized Wilcoxon’s test.</p>a<p>Several recipient mice were sacrificed for histological examination.</p><p>Graft (ESCs-derived cardiomyocytes) survival.</p

Graft (ESCs) survival.

<p>Administration of ES-SCs prolonged allogeneic ESCs-derived embryoid body-like sphere survival <i>in vivo</i>. Recipient mice were pretreated with PBS, ES-SCs or ES-DCs as described in Materials and Methods. Thereafter, mice received transplantation of embryo body-like sphere generated from 129-derived ESCs, and graft survival was monitored by direct inspection. C3H (PBS-injected) vs. C3H (ES-SCs-treated), p<0.001; Generalized Wilcoxon’s test.</p>a<p>Several recipient mice were sacrificed for histological examination.</p><p>n.d., not determined.</p><p>Graft (ESCs) survival.</p

ES-SCs suppress T cell proliferation.

<p>(A) MLR assay: C3H T cells vs. bone marrow-derived DCs. Where indicated, 129 ES-DCs or ES-SCs were added to MLR culture and inhibitory effects were measured by proliferation rate (CFSE cell division). (B) ES-SCs and MLR assay: C3H T cells were stimulated with irradiated bone marrow-derived DCs, and then graded numbers of ES-SCs were added to MLR culture. T cell proliferation was estimated by [³H] thymidine uptake. Results are expressed as mean cpm ± SD. (C) ES-SCs and MLR assay. Several immunosuppressive molecules were blocked by specific inhibitors: Anti-TGFβ mAb (10 µg/mL), anti-PD-L2 mAb (10 µg/mL), _L-NMMA (50 µM), or corresponding isotype-matched controls were added. T cell proliferation was estimated by [³H] thymidine uptake. Results are expressed as mean cpm ± SD. (D) Measurement of NO concentration in the supernatants of ES-DCs or ES-SCs. (E) Specificity of immune suppression by ES-SCs. Splenic T cells from ES-SCs- or PBS-administered C3H mice were stimulated with bone marrow-derived DCs from the indicated mice including C3H (Syn; Syngeneic), 129 (Allo; allogeneic but same background as ES-SCs), and BALB/c (3rd; allogeneic, third party). T cell proliferation was estimated by [³H] thymidine uptake. Data represent mean ± SD of three wells. Similar results were obtained from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p

Characterization of ESCs-derived myeloid cells.

<p>(A) A scheme describes the culture protocol used to obtain floating ES-DCs and adherent ESCs-derived suppressor cells (ES-SCs). EB; embryoid body, M-CSF; macrophage colony-stimulating factor, GM-CSF; granulocyte macrophage colony-stimulating factor. (B) Flow cytometric analysis of cell surface molecular expression on ES-DCs and ES-SCs. Histogram: gray – isotype control, black line – specific antibody. Data are shown as representative of three independent experiments. (C) Quantitative RT-PCR analysis for expression of macrophage- and immunosuppression-related genes in ES-DCs and ES-SCs. Values were normalized to <i>Hprt</i> and shown as mean ± SD from three experiments were shown. *P<0.05, **<i>P</i><0.01.</p