A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver,☆☆

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Abstract

We previously reported a new in vivo model named as “GFP/CCl4 model” for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific “niche” made by CCl4 induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.

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Materials and methods

Mice. C57BL6/Tg14 (act-EGFP) OsbY01 mice (GFP-Tg mice) showed GFP expression in multiple tissue and cells and were kindly provided by Masaru Okabe (Genome Research Center, Osaka University, Osaka, Japan) [13]. C57BL/6 female mice were purchased from Japan SLC (Shizuoka, Japan). AML1 knockout mice were generated as described previously [24]. The genetic background of these mice used in this study was C57BL/6 mice. Male and female mice were mated overnight and female mice were scored based on

Anti-Liv8 antibody detected hematopoietic progenitor cell in fetal liver at E 11.5

Previously we had raised a rat monoclonal antibody, anti-Liv2, which recognized hepatoblasts at E 9.5 [21]. As shown in Fig. 1A, Liv2-positive cells were also detected in fetal liver at E 11.5. Using the antibody developed in this study, Liv8-positive cells were seen in the fetal liver on E 11.5 (Fig. 1B). Fetal liver at E 11.5 functions as a secondary hematopoietic organ [17]. We analyzed whether anti-Liv8 positive cell is associated with hepatoblast or hematopoietic cell. We found

Discussion

The anti-Liv8 antibody is a useful antibody to separate hematopoietic cells and non-hematopoietic cells in adult bone marrow. We found Liv8-positive cells in fetal liver at E11.5, but could not detect no-positive cells in fetal liver of AML1 knockout mice (Fig. 1C) at E 11.5. This result suggested that anti-Liv8-positive cell might be associated with the generation of HSC. We used FACS analysis to understand more about the characterization of Liv8-positive cells in the bone marrow. Around 32%

Acknowledgements

We thank Dr. Masaru Okabe (Genome Research Center, Osaka University) for the gift of GFP transgenic mice and Mr. Jun Oba for his excellent support for immunohistochemistry.

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    Abbreviations: BMC, bone marrow cell; CCl4, carbon tetrachloride; FAH, fumarylacetoacetate hydrolase; GFP, green fluorescent protein; EGFP, enhanced GFP; GFP-Tg mice, C57BL6/Tg14 (act-EGFP) OsbY01 mice; HSC, hematopoietic stem cell; E, embryonic day; MSC, mesenchymal stem cells; MAPC, multipotent adult progenitor cell.

    ☆☆

    This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 13470121 to Shuji Terai, Isao Sakaida, and Kiwamu Okita, and No. 13770262 to Shuji Terai) for translational research from the Ministry of Health, Labor and Welfare (H-trans-5 to Shuji Terai, Isao Sakaida, Hiroshi Nishina, and Kiwamu Okita).

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    Request for Anti-Liv8 contact to Dr. Hiroshi Nishina, Department of Physiological Chemistry, Graduate School of Pharmaceutical Science, University of Tokyo, Hongo 7-3-1, Bunkyoku, Tokyo 113 0033, Japan.

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