ORIGINAL ARTICLE CD34 þ CD38 þ CD19 þ as well as CD34 þ CD38CD19 þ cells are leukemia- initiating cells with self-renewal capacity in human B-precursor ALL Y Kong 1,2,3 , S Yoshida 1,3 , Y Saito 3 , T Doi 3 , Y Nagatoshi 4 , M Fukata 1 , N Saito 1 , SM Yang 2 , C Iwamoto 1 , J Okamura 4 , KY Liu 2 , XJ Huang 2 , DP Lu 2 , LD Shultz 5 , M Harada 1 and F Ishikawa 3 1 Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; 2 Peking University Institute of Hematology, Peking University People’s Hospital, Beijing, China; 3 Research Unit for Human Disease Models, RIKEN Research Center for Allergy and Immunology (RCAI), Yokohama, Japan; 4 Department of Pediatrics, Kyushu National Cancer Center, Fukuoka, Japan and 5 The Jackson Laboratory, Bar Harbor, Maine, USA The presence of rare malignant stem cells supplying a hierarchy of malignant cells has recently been reported. In human acute myelogenous leukemia (AML), the leukemia stem cells (LSCs) have been phenotypically restricted within the CD34 þ CD38 fraction. To understand the origin of malignant cells in primary human B-precursor acute lymphocytic leukemia (B-ALL), we established a novel in vivo xenotransplantation model. Purified CD34 þ CD38 þ CD19 þ , CD34 þ CD38CD19 þ and CD34 þ CD38CD19 bone marrow (BM) or peripheral blood (PB) cells from three pediatric B-ALL patients were intravenously injected into sublethally irradiated newborn NOD/ SCID/IL2rc null mice. We found that both CD34 þ CD38 þ CD19 þ and CD34 þ CD38CD19 þ cells initiate B-ALL in primary recipients, whereas the recipients of CD34 þ CD38CD10 CD19 cells showed normal human hematopoietic repopula- tion. The extent of leukemic infiltration into the spleen, liver and kidney was similar between the recipients transplanted with CD34 þ CD38 þ CD19 þ cells and those transplanted with CD34 þ CD38CD19 þ cells. In each of the three cases studied, transplantation of CD34 þ CD38 þ CD19 þ cells resulted in the development of B-ALL in secondary recipients, demonstrating self-renewal capacity. The identification of CD34 þ CD38 þ CD19 þ self-renewing B-ALL cells proposes a hierarchy of leukemia-initiating cells (LICs) distinct from that of AML. Recapitulation of patient B-ALL in NOD/SCID/IL2rc null recipients provides a powerful tool for directly studying leukemogenesis and for developing therapeutic strategies. Leukemia (2008) 22, 1207–1213; doi:10.1038/leu.2008.83; published online 17 April 2008 Keywords: acute lymphocytic leukemia; stem cells; transplantation Introduction Acute lymphocytic leukemia (ALL) is the most common hematological malignancy in childhood. On the basis of ontogenic classification, pediatric ALL is divided into T-ALL, B-precursor ALL and mature B-ALL. B-precursor ALL accounts for 80–85% of total pediatric ALL cases. 1,2 Recent reports suggest that at least some cases of human leukemia and cancer, including acute myelogenous leukemia (AML), selectively develop from a rare fraction of malignant stem cells. 3–5 Unlike AML, however, whether the malignant clone arises from such a leukemic stem cell fraction has not been clarified in B-precursor ALL. To identify human leukemia stem cells (LSCs), the in vivo leukemia-initiating capacity of purified cells has been evaluated in various xenotransplantation systems using immunocompro- mized mice. These leukemia-initiating cells (LICs) have been considered equivalent to LSCs, although not all studies have demonstrated other properties of stem cells, that is, differentia- tion and self-renewal capacities. In AML, the cell surface phenotype defined by the markers CD34 and CD38, that is, CD34 þ CD38 analogous to normal hematopoietic stem cells (HSCs), have been used to identify LIC-enriched cell popula- tion. 6,7 Although the engraftment of B-ALL CD34 þ cells in NOD/SCID mice has been reported, 8–10 markers for further enrichment of B-precursor ALL-initiating cells have not been identified. CD38 is expressed by a variety of normal and malignant leukocytes and functions in cell adhesion and signaling. In normal hematopoiesis and in AML, its absence on CD34 þ cells highly enriches a primitive self-renewing stem cell population. 6,7 Similarly, Cobaleda et al. 10 have reported that in Ph þ ALL, CD34 þ CD38 cells exclusively initiate leukemia in NOD/SCID recipients. In this study, we aimed to clarify the significance of CD38 expression in B-precursor ALL-initiating cells. For this purpose, we used the newborn NOD/SCID/IL2rg null xenotransplantation model. This model takes advantage of the absence of acquired immunity accompanied by multiple defects in innate immunity in a novel NOD/SCID strain carrying a complete null mutation in the cytokine receptor common g chain. 11 The use of this severely immunocompromised strain overcomes the limitations of existing SCID-repopulating assays using CB17-scid, NOD/SCID and NOD/SCID/b2m null mice in engraftment levels of human normal and primary leukemic cells, and differentiation from normal or leukemic stem cells into progeny. 12,13 Especially, when human cells are intravenously injected into newborn recipients, differentiation and self- renewal capacities are efficiently detected both in normal and malignant hematopoiesis, making this model an ideal system for creating mouse models of primary human hematological malignancies. 12,13 Using the newborn NOD/SCID/IL2rg null xenotransplantation model, we demonstrate that CD34 þ CD38 þ CD19 þ cells as well as CD34 þ CD38CD19 þ cells have the capacities to initiate B-ALL, to infiltrate into non-hematopoietic organs in vivo and to self-renew. Leukemia initiation by self-renewing CD34 þ CD38 þ CD19 þ primary human ALL cells demon- strates a distinct pathogenesis of B-precursor ALL from that of AML, which may provide new insight into the development of Received 1 October 2007; revised 12 February 2008; accepted 10 March 2008; published online 17 April 2008 Correspondence: Dr F Ishikawa, Research Unit for Human Disease Models, RIKEN Research Center for Allergy and Immunology (RCAI), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. E-mail: f_ishika@rcai.riken.jp Leukemia (2008) 22, 1207–1213 & 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00 www.nature.com/leu