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Induction of macrophage-like immunosuppressive cells from mouse ES cells that contribute to prolong allogeneic graft survival.

Kudo H, Wada H, Sasaki H, Tsuji H, Otsuka R, Baghdadi M, Kojo S, Chikaraishi T, Seino K - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; Department of Urology St. Marianna University School of Medicine, Miyamae-ku, Kawasaki City, Kanagawa, Japan.

ABSTRACT
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.

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Characterization of ESCs-derived myeloid cells.(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 Hprt and shown as mean ± SD from three experiments were shown. *P<0.05, **P<0.01.
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pone-0111826-g001: Characterization of ESCs-derived myeloid cells.(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 Hprt and shown as mean ± SD from three experiments were shown. *P<0.05, **P<0.01.

Mentions: To generate myeloid cells from ESCs, we established a differentiation protocol based on sequential stimulation with GM-CSF, M-CSF and IL-4 (Figure 1A). Previous reports suggested that immune-stimulatory dendritic cells (DCs) could be obtained from ESCs or iPSCs with a similar protocol without using lipopolysaccharide (LPS) [14], [15]. On the other hand, other reports have shown that the addition of LPS during the process of inducing bone marrow-derived DCs resulted in the generation of immunosuppressive myeloid cells [16], [17]. However, the role of LPS stimulation in the induction of immunosuppressive cells from PSCs is unknown. Thus, we evaluated the effects of LPS stimulation on the differentiation of E14 ESCs from 129X1/SvJJmsSlc (129) mice. In the culture without LPS, we could obtain immune-stimulatory DCs (ES-DCs) in the floating cell fraction by day 20 as previously reported [14]. On the other hand, the prolonged culture with IL-4 and addition of LPS induced large, irregularly shaped cells which firmly adhered to bacteriologic petri dish surface by day 24 (Fig. 1A). To our knowledge, the adherent clusters in this culture derived from ESCs (and also from iPSCs) have not been formally investigated or reported. As shown later in this paper, these cells show substantial immunosuppressive properties, and thus we refer to them as ESCs-derived suppressor cells (ES-SCs).


Induction of macrophage-like immunosuppressive cells from mouse ES cells that contribute to prolong allogeneic graft survival.

Kudo H, Wada H, Sasaki H, Tsuji H, Otsuka R, Baghdadi M, Kojo S, Chikaraishi T, Seino K - PLoS ONE (2014)

Characterization of ESCs-derived myeloid cells.(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 Hprt and shown as mean ± SD from three experiments were shown. *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214817&req=5

pone-0111826-g001: Characterization of ESCs-derived myeloid cells.(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 Hprt and shown as mean ± SD from three experiments were shown. *P<0.05, **P<0.01.
Mentions: To generate myeloid cells from ESCs, we established a differentiation protocol based on sequential stimulation with GM-CSF, M-CSF and IL-4 (Figure 1A). Previous reports suggested that immune-stimulatory dendritic cells (DCs) could be obtained from ESCs or iPSCs with a similar protocol without using lipopolysaccharide (LPS) [14], [15]. On the other hand, other reports have shown that the addition of LPS during the process of inducing bone marrow-derived DCs resulted in the generation of immunosuppressive myeloid cells [16], [17]. However, the role of LPS stimulation in the induction of immunosuppressive cells from PSCs is unknown. Thus, we evaluated the effects of LPS stimulation on the differentiation of E14 ESCs from 129X1/SvJJmsSlc (129) mice. In the culture without LPS, we could obtain immune-stimulatory DCs (ES-DCs) in the floating cell fraction by day 20 as previously reported [14]. On the other hand, the prolonged culture with IL-4 and addition of LPS induced large, irregularly shaped cells which firmly adhered to bacteriologic petri dish surface by day 24 (Fig. 1A). To our knowledge, the adherent clusters in this culture derived from ESCs (and also from iPSCs) have not been formally investigated or reported. As shown later in this paper, these cells show substantial immunosuppressive properties, and thus we refer to them as ESCs-derived suppressor cells (ES-SCs).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; Department of Urology St. Marianna University School of Medicine, Miyamae-ku, Kawasaki City, Kanagawa, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus