Identification of Long-term Repopulating Potential of Human Cord Blood-derived CD34^-Flt3^- SCID-repopulating Cells by Intra-Bone Marrow Injection

¹ Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka, Japan
² Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland Baltimore, Baltimore, Maryland
³ Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka, Japan; First Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, Japan
⁴ Department of Gynecology and Obstetrics, Fukuda Hospital, Kumamoto, Japan
⁵ Department of Obstetrics and Gynecology, Aizenbashi Hospital, Osaka, Japan
⁶ Department of Microbiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, Japan
⁷ Department of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba, Japan
⁸ First Department of Pathology, Transplantation Center, Kansai Medical University, Moriguchi, Osaka, Japan
⁹ Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University

^* To whom correspondence should be addressed. E-mail: sonoda{at}takii.kmu.ac.jp.

	Abstract

Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34^-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In contrast to murine CD34^- KSL (Kit⁺Sca-1⁺Lineage^-) cells, human CB-derived Lin^-CD34^- cells did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34^- SRCs. Both CD34⁺flt3^+/- cells showed SRC activity. In the CD34^- cell fraction, only CD34^-flt3^- cells showed distinct SRC activity by IBMI. While CD34⁺flt3⁺ cells showed a rather weak secondary repopulating activity, CD34⁺flt3^- cells repopulated many more secondary recipient mice. However, CD34^-flt3^- cells repopulated all the secondary recipients and the repopulating rate was much higher. Next, we cocultured CD34^-flt3^- cells with the murine stromal cell line, HESS-5. After one week, significant numbers of CD34⁺flt3^+/- cells were generated and they showed distinct SRC activity. These results indicated that CB-derived CD34^-flt3^- cells produced CD34⁺flt3^- as well as CD34⁺flt3⁺ SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34^- SRCs, like murine CD34^- KSL cells, do not express flt3. Based on these data, we propose that the immunophenotype of very primitive long-term repopulating human hematopoietic stem cells is Lin^-CD34^-c-kit^-flt3^-.

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