C57bl 6 tg cag egfp

All methods were carried out in accordance with the approved guidelines. Eight-week-old male C57BL/6J mice and C57BL/6-Tg (CAG-EGFP) (GFP-transgenic; GFP-Tg) mice were purchased from Japan SLC (Shizuoka, Japan). GFP-bone marrow chimeric mice were produced by lethal irradiation (9 Gy) and systemic injection with 4.0–6.0 × 10⁶ bone marrow cells isolated from GFP-Tg mice. At 4 weeks after bone marrow transplantation, diabetes was induced via an HFD containing 60% lard (High-Fat Diet 32; Clea Japan, Inc., Tokyo, Japan) or by a single intraperitoneal injection of STZ (150 mg/kg; Wako, Osaka, Japan) dissolved in citrate buffer (pH 4.5). Control mice were given a normal diet or treated with buffer intraperitoneally. After 28 weeks of receiving the HFD, the mice were administered 1.0 × 10⁴ MSCs/g body weight 4 times (HFD-MSC) every 2 weeks, with the controls receiving buffer (HFD-vehicle) (Fig. 1a). At 4 weeks after STZ injection, the mice were administered 1.0 × 10⁴ MSCs/g body weight 2 times (STZ-MSC) every 4 weeks, with the controls receiving buffer (STZ-vehicle) (Fig. 2a). All experimental protocols and studies were approved by the animal experiment committee of Sapporo Medical University (Sapporo, Japan).

Experiment used C57BL/6N (Japan SLC Inc.), C57BL/6-Tg(CAG-EGFP) (Japan SLC Inc.), BALB/cSlc-nu/nu (Japan SLC Inc.), and Lgr5-EGFP-IRES-creERT2 mice (Jackson Laboratory). The animals were housed in environmentally controlled rooms, and all the experimental procedures using animals were approved by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch and performed in accordance with the relevant guidelines and regulations.

All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services, Public Health Services, National Institute of Health, NIH publication no. 86–23, 1985)34 , and approved by the Ethical Committee for Animal Experiments at the University of Tokyo. EGFP-overexpressing C57BL/6 mice (C57BL/6-Tg (CAG-EGFP)) were purchased from Japan SLC Inc. C3H/HeN and C3H/HeJ mice were purchased from CLEA Japan. All mice were allowed access to feed and water ad libitum.

Mouse SSC cultures were established as described previously^{10 (link),31 (link),57 (link)} with minor modifications. In brief, murine testicular cells were prepared from 5-day-old GFP mice (C57BL/6-Tg (CAG-EGFP), Japan SLC, Shizuoka, Japan) by enzymatic digestion using trypsin-EDTA, and cultured on SNL 76/7 STO cell (a kind gift from Dr. A. Bradley, Baylor College of Medicine, USA) feeders in SFM with 10 ng/mL hGDNF (R&D Systems, Minneapolis, MN, USA) and 0.5 ng/mL human FGF2 (Corning). In some experiments, 30 μL/mL of pGDNF were used instead of hGDNF. One day after seeding, weakly attached spermatogonia were collected and cultured on fresh feeders. Medium was changed every other day, and the cells were subcultured every 5–7 days as described previously^{31 (link)}.

C57BL/6J and C57BL/6-Tg (CAG-EGFP) female mice were purchased from Japan SLC (Shizuoka, Japan) and CLEA Japan (Tokyo, Japan). Young mice were 2–3 months-old, whereas those older than 24 months were regarded as aged. Mice with grossly visible tumors, hepatomegaly or splenomegaly were excluded from the study. Some but not all mice were assessed for body weight, liver weight, lung weight and serum ALT levels before the experiments began. For the quantification of serum ALT levels, blood was sampled from the heart and centrifuged to separate serum for ALT measurements using an ALT assay kit (#700,260, Cayman Chemical, Ann Arbor, Michigan) and a microplate reader (PowerScanHT; DS Pharma Biomedical, Osaka, Japan). All experiments were carried out following the guidelines of Osaka University Committee for animal and recombinant DNA experiments. Mice were handled and maintained according to the Osaka University guidelines for animal experimentation.

C57BL/6 (male and female), C57BL/6-Tg(CAG-EGFP) (male and female, Japan SLC) and B6.129P-Cx3cr1^tm1Litt/J mice (male, Jackson Laboratory) were used. All mice were aged 4–10 weeks at the start of each experiment, and were housed in groups of two or three per cage at a temperature of 22 ± 1 °C with a 12-hour light-dark cycle, and were fed food and water ad libitum. All animal experiments were conducted according to the national and international guidelines contained in the ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan) and ‘Regulation of Laboratory Animals’ (Kyushu University) and under the protocols approved by the Institutional Animal Care and Use committee review panels at Kyushu University.