Additional file 1: Figure S1. of Genetic diversity of Mycobacterium tuberculosis isolates from Tochigi prefecture, a local region of Japan

Geographical location of Tochigi Prefecture in Japan. Tochigi is one of the inland prefectures of the Northern portion of the Kanto region. Its population on March 1, 2007, was 2,014,931 persons. The area of Tochigi prefecture is approximately 6,400 km2, making it the 20th largest in Japan, but the largest in the Kanto region. (PPTX 127 kb

Additional file 4: Figure S2. of Genetic diversity of Mycobacterium tuberculosis isolates from Tochigi prefecture, a local region of Japan

Associations between patient gender and Mycobacterium tuberculosis lineages. The stacked bar charts show the percentages of M. tuberculosis lineages by (A) countries of birth and (B) sampling period. The numbers of isolates are indicated on the graphs. (PPTX 133 kb

Additional file 2: Table S1. of Genetic diversity of Mycobacterium tuberculosis isolates from Tochigi prefecture, a local region of Japan

Geographic analysis of lineages of M. tuberculosis isolates obtained from foreign- and Japanese-born patients living in the North, Central, and South regions of Tochigi Prefecture. (DOCX 31 kb

Additional file 3: Table S2. of Genetic diversity of Mycobacterium tuberculosis isolates from Tochigi prefecture, a local region of Japan

Associations between patient gender and lineages of M. tuberculosis isolates obtained from foreign- and Japanese-born patients. (DOCX 31 kb

Controlling Immune Rejection Is a Fail-Safe System against Potential Tumorigenicity after Human iPSC-Derived Neural Stem Cell Transplantation

<div><p>Our previous work reported functional recovery after transplantation of mouse and human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) into rodent models of spinal cord injury (SCI). Although hiPSC-NS/PCs proved useful for the treatment of SCI, the tumorigenicity of the transplanted cells must be resolved before they can be used in clinical applications. The current study sought to determine the feasibility of ablation of the tumors formed after hiPSC-NS/PC transplantation through immunoregulation. Tumorigenic hiPSC-NS/PCs were transplanted into the intact spinal cords of immunocompetent BALB/cA mice with or without immunosuppressant treatment. <i>In vivo</i> bioluminescence imaging was used to evaluate the chronological survival and growth of the transplanted cells. The graft survival rate was 0% in the group without immunosuppressants versus 100% in the group with immunosuppressants. Most of the mice that received immunosuppressants exhibited hind-limb paralysis owing to tumor growth at 3 months after iPSC-NS/PC transplantation. Histological analysis showed that the tumors shared certain characteristics with low-grade gliomas rather than with teratomas. After confirming the progression of the tumors in immunosuppressed mice, the immunosuppressant agents were discontinued, resulting in the complete rejection of iPSC-NS/PC-derived masses within 42 days after drug cessation. In accordance with the tumor rejection, hind-limb motor function was recovered in all of the mice. Moreover, infiltration of microglia and lymphocytes was observed during the course of tumor rejection, along with apoptosis of iPSC-NS/PC-generated cells. Thus, immune rejection can be used as a fail-safe system against potential tumorigenicity after transplantation of iPSC-NS/PCs to treat SCI.</p></div

Representative <i>in vivo</i> images and quantitative analysis of photon counts derived from grafted hiPSC-NS/PCs.

<p>(A) Bioluminescence images of representative mice at 0, 21, 97, and 142 days after human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation. Upper panel: BALB/cA mouse with immunosuppressant treatment (FK506 plus anti-cluster of differentiation (CD) 4 monoclonal antibody (mAb); With-IS group); middle panel: BALB/cA mouse with immunosuppressant treatment, followed by discontinuation of immunosuppressants 100 days later (IS off group); lower panel: BALB/cA mouse without immunosuppressant treatment (Without-IS group). (B) Quantitative analysis of photon counts derived from grafted hiPSC-NS/PCs. Graft survival rate was 100% (n = 17/21, three animals died and one animal was sacrificed by day 21) in BALB/cA mice with immunosuppressant treatment (FK506 plus anti-CD4 mAb), versus 0% (n = 0/6) in BALB/cA mice without immunosuppressant treatment. After discontinuing the administration of FK506 and anti-CD4 mAb, all the grafted cells were rejected by day 164 and drastic reductions in signal intensity were observed. Data represent the mean value ± the standard error of the mean. (*p<0.05; Friedman’s test followed by Dunn’s post-hoc test.)</p

Lymphoid population in peripheral blood.

<p>Peripheral blood cells were analyzed using fluorescence-activated cell sorting-based flow cytometry. Data were gated on cluster of differentiation (CD) 4-positive or CD8-positive T-cell subsets. CD4-positive T-cells were depleted immediately after administration of FK506 plus anti-CD4 monoclonal antibody, but recovered after discontinuing immunosuppressant treatment. IS(+), with immunosuppressants; IS(−), without immunosuppressants.</p

Tumor formation by grafted hiPSC-NS/PCs in the mouse spinal cord.

<p>Representative hematoxylin-eosin (HE)-stained (A) and human nuclear antigen (HNA)-stained (B) images of sagittal sections of spinal cord at 79 days after cell transplantation. HE staining revealed a biphasic tumor pattern with high and low cell density areas. The high cell density area contained compact bipolar cells with rosenthal fibers, whereas the low cell density area contained loose-textured multipolar cells with microcysts. The low cell density area surrounded by the square box is shown at higher magnification to the left of the image. Immunostaining for glial fibrillary acidic protein (GFAP), HNA, GFP, nestin, β-III tubulin (C, D, E), and Oligo-1 (F). The human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NSC)-derived tumors mainly consisted of undifferentiated cells that stained positively for nestin, with low numbers of differentiated cells (e.g., β-III tubulin-positive neurons, GFAP-positive astrocytes, and Oligo-1-positive oligodendrocyte precursor cells). Nestin-positive cells were located in the center of the tumor, whereas differentiated cells were localized to the tumor margin (D, E, F). The boxed area in (C) corresponds to the higher magnification images in (D). Tumors contained a paucity of octamer-binding transcription factor (Oct) 4-positive (G) and Ki-67-positive (H) cells. The Ki-67 index was 7.0%. Scale bars in A–C, 500 μm; D–H, 100 μm.</p

Fate of hiPSC-NS/PCs transplanted into the mouse spinal cord.

<p>Fate of hiPSC-NS/PCs transplanted into the mouse spinal cord.</p

Detection of bioluminescence and fluorescence signals in lentivirally transfected 253G1-NS/PCs <i>in vitro</i>.

<p>Phase-contrast (A) and fluorescence (B) images of a neurosphere derived from tumorigenic 253G1 induced pluripotent stem cells. Neural stem/progenitor cells (NS/PCs) differentiated into β-III tubulin-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes <i>in vitro</i> (C). Bioluminescence imaging was used to detect bioluminescence signals in various numbers of 253G1-NS/PCs (0, 1.5 × 10⁵, 3 × 10⁵, 6 × 10⁵, and 1.2 × 10⁶ cells per well) (D). A direct linear correlation was found between cell numbers and photon counts <i>in vitro</i> (E). Scale bars in A–C, 1,000 μm.</p