Naomi Nishio

Neutrophil depletion delays wound repair in aged mice

One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice.

Paraxial Mesodermal Progenitors Derived from Mouse Embryonic Stem Cells Contribute to Muscle Regeneration via Differentiation into Muscle Satellite Cells

Pluripotent embryonic stem (ES) cells hold great potential for cell‐based therapies. Although several recent studies have reported the potential of ES cell‐derived progenitors for skeletal muscle regeneration, how the cells contribute to reconstitution of the damaged myofibers has remained elusive. Here, we demonstrated the process of injured muscle regeneration by the engraftment of ES cell‐derived mesodermal progenitors. Mesodermal progenitor cells were induced by a conventional differentiation system and isolated by flow cytometer of platelet‐derived growth factor receptor‐α (PDGFR‐α), a marker of paraxial mesoderm, and vascular endothelial growth factor receptor‐2 (VEGFR‐2), a marker of lateral mesoderm. The PDGFR‐α+ population that represented the paraxial mesodermal character demonstrated significant engraftment when transplanted into the injured muscle of immunodeficient mouse. Moreover, the PDGFR‐α+ population could differentiate into the muscle satellite cells that were the stem cells of adult muscle and characterized by the expression of Pax7 and CD34. These ES cell‐derived satellite cells could form functional mature myofibers in vitro and generate myofibers fused with the damaged host myofibers in vivo. On the other hand, the PDGFR‐α−VEGFR‐2+ population that showed lateral mesodermal character exhibited restricted potential to differentiate into the satellite cells in injured muscle. Our results show the potential of ES cell‐derived paraxial mesodermal progenitor cells to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation. This knowledge could be used to form the foundation of the development of stem cell therapies to repair diseased and damaged muscles.

Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells

BackgroundInduced pluripotent stem (iPS) cells are the novel stem cell population induced from somatic cells. It is anticipated that iPS will be used in the expanding field of regenerative medicine. Here, we investigated whether implantation of fetal liver kinase-1 positive (Flk-1+) cells derived from iPS cells could improve angiogenesis in a mouse hind limb model of ischemia.ResultsFlk-1+ cells were induced from iPS cells after four to five days of culture. Hind limb ischemia was surgically induced and sorted Flk-1+ cells were directly injected into ischemic hind limbs of athymic nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk-1+ cells compared with control mice, which were transplanted with vehicle, as evaluated by laser Doppler blood flowmetry. Transplantation of Flk-1+ cells also increased expression of VEGF mRNA in ischemic tissue compared to controls.ConclusionsDirect local implantation of iPS cell-derived Flk-1+ cells would salvage tissues from ischemia. These data indicate that iPS cells could be valuable in the therapeutic induction of angiogenesis.

Establishment of induced pluripotent stem cells from aged mice using bone marrow-derived myeloid cells

If induced pluripotent stem (iPS) cells are to be used to treat damaged tissues or repair organs in elderly patients, it will be necessary to establish iPS cells from their tissues. To determine the feasibility of using this technology with elderly patients, we asked if it was indeed possible to establish iPS cells from the bone marrow (BM) of aged mice. BM cells from aged C57BL/6 mice carrying the green fluorescence protein (GFP) gene were cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for 4 days. Four factors (Oct3/4, Sox2, Klf4 and c-Myc) were introduced into the BM-derived myeloid (BM-M) cells. The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low. However, we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice, which carried GFP. Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining. The iPS cells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice, and can be differentiated to three germ layers in vitro. By co-culturing with OP9, the BM-M-iPS cells can be differentiated to the myeloid lineage. The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF, and lost expression of Nanog and Pou5f1, at least in part, due to methylation of their promoters. On the contrary, Tnf and Il1b gene expression was upregulated and their promoters were hypomethylated.

Dextran sulphate sodium increases splenic Gr1+CD11b+ cells which accelerate recovery from colitis following intravenous transplantation

While Gr1+CD11b+ cells are known to regulate immune responses and accumulate in most cancer tissues, the function of Gr1+CD11b+ cells in inflammation is poorly understood. We investigated the role of Gr1+CD11b+ cells in a dextran sulphate sodium (DSS)‐treated mouse model of ulcerative colitis (UC). C57BL/6 mice were treated with 2% DSS in drinking water for 5 days. Disease progression and recovery were assessed by body weight, disease activity index score (DAI) score and colon length. Splenic Gr1+CD11b+ cell number was greatly increased during the recovery phase of DSS‐induced colitis. DSS‐derived splenic Gr1+CD11b+ cells were administered intravenously to recipient (C57BL/6) mice during the early phase of DSS treatment. The transplanted splenic DSS‐induced Gr1+CD11b+ cells improved DSS‐induced colitis and promoted efficient colonic mucosal healing. We found that the CD11b+ single positive cells increased in the course of DSS‐induced colitis in lamina propria. The transplantation of splenic Gr1+CD11b+ cells induced feedback suppression of myeloid‐lineage cell development. Namely, the transplantation of splenic Gr1+CD11b+ cells greatly suppressed the migration of CD11b+ single positive cells to the lamina propria. Further, transplantation of Gr‐1+CD11b+ cells greatly suppressed the increase of the same population, especially during the late phase of DSS colitis both in spleen and bone marrow.

Differentiation of induced pluripotent stem cells to thymic epithelial cells by phenotype

Thymic epithelial cells (TECs) are present in both cortical and medullary thymic areas, and have crucial roles in functional T‐cell development. In this study, we studied the differentiation of induced pluripotent stem cells (iPSCs) to TEC. When iPSC were cultured for 4 days in collagen IV‐coated dishes in the presence of both activin A and lithium chloride (LiCl), the cells differentiated to definitive endoderm through mesendoderm. Further treatment with Fgf8 followed by Fgf7, Fgf10 and BMP4 differentiated iPSC to thymic epithelial progenitor cells (TEPCs) by phenotype. Gene expression of Hoxa3, Pax1 and Pax9 was observed and cell surface proteins EpCAM1 and MTS24 were detected at day 14 of iPSC differentiation. TEPCs differentiated to medullary TECs (mTECs) by phenotype following the addition of receptor activator nuclear factor B ligand with LiCl. Thus, we successfully induced efficient differentiation from mouse iPSC to TEPCs and mTEC by phenotype using chemically defined conditions.

Antibodies to wounded tissue enhance cutaneous wound healing.

The wound repair process is a highly ordered sequence of events that encompasses haemostasis, inflammatory cell infiltration, tissue regrowth and remodelling. Wound healing follows tissue destruction so we hypothesized that antibodies might bind to wounded tissues, which would facilitate the engulfment of damaged tissues by macrophages. Here, we show that B cells, which produce antibodies to damaged tissues, are engaged in the process of wound healing. Splenectomy delayed wound healing, and transfer of spleen cells into splenectomized mice recovered the delay in wound healing. Furthermore, wound healing in splenectomized nude mice was also delayed. Transfer of enriched B220+ cells by magnetic beads accelerated wound healing in splenectomized mice. We detected immunoglobulin G1 (IgG1) binding to wounded tissues by using fluorescein isothiocyanate‐labelled anti‐IgG1 6–24 hr after wounding. Splenectomy reduced the amount of IgG1 binding to wounded tissues. Immunoblotting studies revealed several bands, which were reduced by splenectomy. Using immunoprecipitation with anti‐IgG bound to protein G we found that the intensity of several bands was lower in the serum from splenectomized mice than in that from sham‐operated mice. These bands were matched to myosin IIA, carbamoyl‐phosphate synthase, argininosuccinate synthase, actin and α‐actinin‐4 by liquid chromatography tandem mass spectrometry analysis.

iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis

Angiogenic cell therapy represents a novel strategy for ischemic diseases, but some patients show poor responses. We investigated the therapeutic potential of an induced pluripotent stem (iPS) cell sheet created by a novel magnetite tissue engineering technology (Mag-TE) for reparative angiogenesis. Mouse iPS cell-derived Flk-1+ cells were incubated with magnetic nanoparticle-containing liposomes (MCLs). MCL-labeled Flk-1+ cells were mixed with diluted extracellular matrix (ECM) precursor and a magnet was placed on the reverse side. Magnetized Flk-1+ cells formed multi-layered cell sheets according to magnetic force. Implantation of the Flk-1+ cell sheet accelerated revascularization of ischemic hindlimbs relative to the contralateral limbs in nude mice as measured by laser Doppler blood flow and capillary density analyses. The Flk-1+ cell sheet also increased the expressions of VEGF and bFGF in ischemic tissue. iPS cell-derived Flk-1+ cell sheets created by this novel Mag-TE method represent a promising new modality for therapeutic angiogenesis.

Up-Regulation of Gr1+CD11b+ Population in Spleen of Dextran Sulfate Sodium Administered Mice Works to Repair Colitis

Dextran sulfate sodium (DSS) is commonly used in rodent IBD models to chemically induce acute intestinal inflammation. The acute course of colitis includes colon tissue damages and recovery from wounded tissues. As skin wound repair was delayed by splenectomy, we asked whether splenectomy would induce the delay of colonic wound healing. In splenectomized mice, body weight recovery, disease score and colon length were delayed. Surprisingly we found a great increase of Gr1+CD11b+ cells in spleen and bone marrow of DSS-administered mice. Anti-Gr-1 antibody treatment worsened the DSS- administered colitis. These results indicate that Gr1+CD11b+ cells induced by DSS worked to repair colon wound healing and repair colitis.

Gadd34 induces autophagy through the suppression of the mTOR pathway during starvation.

Several types of cellular stress induce expression of growth arrest and DNA damage protein 34 (Gadd34). Autophagy occurs under both basal conditions and conditions of stress, such as starvation. Gadd34 and autophagy are both induced under starvation conditions. In this study we found that starvation induced the expression of Gadd34, reduced mTOR activity, and induced autophagy in wild type mice, but not Gadd34 KO mice. Gadd34 bound to and dephosphorylated pTSC2 at Thr1462. Dephosphorylation of TSC2 during the starvation time period leads to the suppression of mTOR, which is a potent inhibitor of autophagy. We concluded that starvation-induced Gadd34 suppresses mTOR and, thereby, induces autophagy.