Stem Cells

CD34+CD38+CD19+ as well as CD34+CD38−CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL


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+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+CD38+CD19+, CD34+CD38−CD19+ and CD34+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/IL2rγnull mice. We found that both CD34+CD38+CD19+ and CD34+CD38−CD19+ cells initiate B-ALL in primary recipients, whereas the recipients of CD34+CD38−CD10−CD19− cells showed normal human hematopoietic repopulation. The extent of leukemic infiltration into the spleen, liver and kidney was similar between the recipients transplanted with CD34+CD38+CD19+ cells and those transplanted with CD34+CD38−CD19+ cells. In each of the three cases studied, transplantation of CD34+CD38+CD19+ cells resulted in the development of B-ALL in secondary recipients, demonstrating self-renewal capacity. The identification of CD34+CD38+CD19+ 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/IL2rγnull recipients provides a powerful tool for directly studying leukemogenesis and for developing therapeutic strategies.


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, 4, 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 immunocompromized 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, differentiation and self-renewal capacities. In AML, the cell surface phenotype defined by the markers CD34 and CD38, that is, CD34+CD38− analogous to normal hematopoietic stem cells (HSCs), have been used to identify LIC-enriched cell population.6, 7 Although the engraftment of B-ALL CD34+ cells in NOD/SCID mice has been reported,8, 9, 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+ cells highly enriches a primitive self-renewing stem cell population.6, 7 Similarly, Cobaleda et al.10 have reported that in Ph+ ALL, CD34+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/IL2rγnull 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 γ 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/β2mnull 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/IL2rγnull xenotransplantation model, we demonstrate that CD34+CD38+CD19+ cells as well as CD34+CD38−CD19+ 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+CD38+CD19+ primary human ALL cells demonstrates a distinct pathogenesis of B-precursor ALL from that of AML, which may provide new insight into the development of novel strategies for the treatment of pediatric B-precursor ALL. Furthermore, the use of anti-human CD19 antibody may discriminate normal HSCs and LSCs within CD34+CD38− stem fraction, enabling autologous BM transplantation without LIC contamination in patients with B-precursor ALL.

Materials and methods


NOD.Cg-PrkdcscidIL2rgtmlWjl/Sz (NOD/SCID/IL2rγnull) mice11 were developed at The Jackson Laboratory (Bar Harbor, ME, USA). The NOD/SCID/IL2rγnull strain was established by backcrossing a complete null mutation of the γ chain locus onto the NOD.Cg-Prkdcscid strain. These mice have been bred and maintained under defined flora with irradiated food at the animal facility at RIKEN Research Center for Allergy and Immunology (RCAI). All experimental procedures were performed according to the guidelines established by the Institutional Animal Committee at RCAI.

Cell purification and xenogeneic transplantation

Bone marrow (BM; Cases 1 and 3) or peripheral blood (PB; Case 2) samples were obtained from three pediatric patients with newly diagnosed B-precursor ALL after written informed consent. The patient ages at the time of diagnosis were 6 years, 3 months and 10 months old, respectively, for Cases 1, 2 and 3. The white blood cell count at the time of diagnosis were 260.0, 134.0 and 196.0 ( × 109 ml−1), respectively, for Cases 1, 2 and 3. MLL rearrangement was identified in Cases 2 and 3. BM and PB mononuclear cells (MNCs) were isolated by density centrifugation using lymphocyte separation medium (ICN Biomedicals, Oh, USA). BMMNCs or PBMNCs were stained with mouse anti-human CD10, CD19, CD3, CD4, CD8, CD34 and CD38 monoclonal antibodies (BD Immunocytometry, San Jose, CA, USA). Samples were analyzed and sorted using FACSAria (Becton Dickinson, San Jose, CA, USA). Nonviable cells were excluded by 7-aminoactinomycin D (BD Immunocytometry) staining. Within the viable CD3−CD4−CD8− BMMNCs or PBMNCs, CD34+CD38−CD19+, CD34+CD38+CD19+ and CD34+CD38−CD10−CD19− populations were sorted and injected into sublethally irradiated (1.5 Gy) NOD/SCID/IL2rγnull mice through the facial vein within 48 h of birth. The purity of each cell population was higher than 97%. As control, normal human HSCs (Lin−CD34+CD38− cells) were purifed from cord blood MNCs and intravenously transplanted into NOD/SCID/IL2rγnull newborns. Cord blood was obtained from Tokyo Cord Blood Bank after written consent was obtained from donors, and experimental plans were evaluated at IRB.

Evaluation of hematopoietic chimerism by flow cytometry

Starting 4 weeks after transplantation, PB was harvested from retro-orbital plexus of the recipients every 2–4 weeks. Recipients were killed when they became moribund and their BM, spleen and PB were evaluated for the repopulation of human normal or leukemic cells with mouse anti-human CD3, CD4, CD8, CD10, CD19, CD20, CD33, CD34, CD38, CD41a, CD45, HLA-DR and surface IgM monoclonal antibodies (BD Immunocytometry). Multicolor flow cytometric analyses were performed using FACSAria or FACSCanto II (Becton Dickinson). Engraftment of human B-ALL was defined by the frequency of the hCD45+hCD19+ cells.

Histological analysis

The liver, kidney and spleen tissues of the recipients were fixed with 4% paraformaldehyde for 1 h, dehydrated with 70% ethanol, embedded in paraffin and 5 μm sections were prepared. Hematoxylin-eosin staining was performed on each tissue section derived from the recipient mice. Immunostaining with mouse anti-human CD19 primary antibody (AbD SeroTec, Oxford, UK) and Cy3-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) was performed after dehydration with graded alcohol and antigen retrieval with heated citrate buffer. Each section was examined using light microscopy (Zeiss Axiovert 200, Carl Zeiss, Germany) and laser scanning confocal microscopy (Leica TCS, Leica, Germany) to identify the infiltrating B-ALL cells.

Serial transplantation

For serial transplantation, either sorted human CD45+CD34+CD38+CD19+ cells or magnetic bead-enriched human CD34+ cells (Miltenyi Biotec, Germany) were obtained from the recipient BM and spleen, and 104–106 purified cells were intravenously transplanted into newborn NOD/SCID/IL2rγnull mice. Human engraftment was evaluated in the serially transplanted recipients at 4–12 weeks post-transplantation.

Statistical analysis

Continuous variables were expressed as mean±s.d. The data were analyzed by SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA).


Phenotypic characterization of primary human B-ALL cells

It is essential to understand leukemogenesis of B-precursor ALL, the most common hematological malignancy in children. While AML-initiating cells and Ph+ ALL-initiating cells are highly enriched within the CD34+CD38− population, the significance of CD38 expression in B-precursor ALL-initiating cells has not been clarified. To address this question, we first examined the phenotypic characteristics of B-precursor ALL cells in three patient samples. We analyzed the frequency of each fraction expressing CD34, CD38, CD10 and CD19 antigens by multicolor flow cytometry (Figure 1). As expected, considerable heterogeneity in the expression of cell surface antigens was observed in the three patient samples examined. Within the CD3−CD4−CD8− BM or PB MNC populations, consistent with the diagnosis of B-precursor ALL, CD34+ cells accounted for 90.5±3.6%. CD34+CD38− and CD34+CD38+ cells accounted for 5.2±6.0 and 85.3±7.7%, respectively. In contrast with normal BM and cord blood samples, CD19+ cells accounted for 66.9±44.4 and 93.1±11.1% of CD34+CD38− and CD34+CD38+ populations, respectively. The CD34+CD38−CD10−CD19− subfraction was present in low frequency (0.18±0.17%) in all three samples tested. The complete phenotype is shown in Figure 1.

Figure 1

Phenotypic analysis of primary B-precursor ALL cells. BM (Cases 1 and 3) and PB (Case 2) from three cases of B-precursor ALL were analyzed for the expressions of CD34 and CD38 within T-cell-depleted MNCs. Within CD34+CD38− and CD34+CD38+ populations, the expression patterns of CD10 and CD19 were analyzed. Three subfractions (CD34+CD38−CD19+, CD34+CD38+CD19+ and CD34+CD38−CD10−CD19− cells) from each sample were transplanted into newborn NOD/SCID/IL2rγnull mice. ALL, acute lymphocytic leukemia; BM, bone marrow; MNCs, mononuclear cells; PB, peripheral blood; TCD, T-cell-depleted.

Both CD34+CD38+CD19+ and CD34+CD38−CD19+ primary human B-ALL cells have long-term engraftment and leukemia-initiating capacity

On the basis of phenotypic characterization above, we simultaneously purified CD34+CD38+CD19+, CD34+CD38−CD19+, CD34+CD38−CD10−CD19− cells from B-precursor ALL patient BM (Cases 1 and 3) and PB (Case 2), and intravenously transplanted these purified populations into sublethally irradiated NOD/SCID/IL2rγnull newborns. The information on each recipient is summarized in Table 1. The engraftment of human B-ALL cells was monitored by flow cytometric analysis of hCD45+CD19+ cells in the recipient PB. In contrast with previous reports on AML7 and Ph+ ALL,10, 14 the injection of either CD34+CD38−CD19+ or CD34+CD38+CD19+ cells resulted in efficient engraftment of human B-ALL (Table 1). The engraftment of B-ALL in the PB of recipient mice following injection of CD34+CD38+CD19+ cells was seen for long term, with human B-ALL cells being observed to increase over time for up to 15 weeks post-transplantation. Purified CD34+CD38−CD10−CD19− cells did not initiate B-ALL in the recipients, but showed engraftment of normal human hematopoietic cells.

Table 1 Serial transplantation of primary human B-ALL CD34+CD38+CD19+ and CD34+CD38-CD19+ cells

When the recipient mice exhibited ruffled fur and lethargy, we killed them to analyze the engraftment levels of human B-ALL in the BM and the spleen. As observed in the PB, BM and spleen showed efficient engraftment of ALL both in the recipients transplanted with CD34+CD38+CD19+ cells and those transplanted with CD34+CD38−CD19+ cells (Table 1 and Figure 2). The significance of CD38 expression on leukemia-initiating capacity is totally different between adult AML and pediatric B-precursor ALL.

Figure 2

Both CD34+CD38−CD19+ and CD34+CD38+CD19+ B-ALL cells have the ability to reconstitute B-ALL in vivo. Flow cytometric analyses of BM from representative recipients of each cell fraction from each case are shown. Left panels: gated on hCD45+ cells; middle panels: gated on hCD34+CD38− cells; right panels: gated on hCD34+CD38+ cells. For Case 1, 6.5 × 104 CD34+CD38+CD19+ and CD34+CD38−CD19+ were transplanted. For Case 2, 5.0 × 104 CD34+CD38+CD19+ and 4.0 × 104 CD34+CD38−CD19+ cells were transplanted. For Case 3, 2.5 × 104 CD34+CD38+CD19+ and 6.5 × 104 CD34+CD38−CD19+ cells were transplanted. ALL, acute lymphocytic leukemia; BM, bone marrow.

Transplanted primary human B-ALL cells infiltrate into recipient organs

As we demonstrated leukemia-initiating capacity both in CD34+CD38−CD19+ and CD34+CD38+CD19+ cells, we next performed histological analyses of the recipient organs to examine the infiltration of B-ALL cells in the liver, kidney and spleen. In the liver and the kidney, infiltration of monomorphic MNCs was detected with hematoxylin-eosin staining in the recipients transplanted either with CD34+CD38+CD19+ or CD34+CD38−CD19+ cells derived from all three cases (Figure 3). When compared with the organs from normal human HSC recipient, there are sheets and clusters of monomorphic MNCs in the organs of the B-ALL recipients. The infiltrating cells exhibited cellular morphology similar to that of cells engrafted in the spleen. These cells also expressed CD19 on their surface, suggesting that they are human B-ALL cells. No infiltrating CD19+ cells were detected in the liver and kidney of the normal human HSC recipient. The degree of infiltration in the recipients transplanted with CD34+CD38−CD19+ cells and those transplanted with CD34+CD38+CD19+ cells was not significantly different.

Figure 3

Both CD34+CD38−CD19+ and CD34+CD38+CD19+ B-ALL cells infiltrate into recipient organs. Hematoxylin-eosin staining and anti-human CD19 antibody labeling of the liver, kidney and spleen of a recipient transplanted with CD34+CD38+CD19+ cells and a recipient transplanted with CD34+CD38−CD19+ cells. Similarly stained and labeled liver, kidney and spleen sections from a recipient of normal human cord blood CD34+CD38− cells are included as controls. Nuclei were stained with DAPI. ALL, acute lymphocytic leukemia; DAPI, 4,6-diamidino-2-phenylindole. Scale bars represent 20 μm in HE staining and 50 μm in CD19 immunostaining.

Primary human B-ALL CD34+CD38+CD19+ cells possess self-renewal capacity

As the ALL-initiating capacity and the ability to infiltrate recipient organs were confirmed in the CD34+CD38+CD19+ as well as the CD34+CD38−CD19+ population, we examined the self-renewal capacity of CD34+CD38+CD19+ and CD34+CD38−CD19+ populations by serial transplantation. From CD34+CD38+CD19+ recipients, we intravenously transplanted 104–106 sorted CD45+CD34+CD38+CD19+ cells or enriched CD34+ cells from the primary (all three cases) and secondary (Cases 2 and 3) recipients into secondary and tertiary newborn NOD/SCID/IL2rγnull recipients. In all the secondary and tertiary recipients, high levels of hCD45+CD19+ engraftment were seen (Table 1a). Similarly, when 2 × 105–2.8 × 106 enriched CD34+ cells from CD34+CD38−CD19+ primary recipients (Cases 1 and 2) were transplanted into secondary recipients, high levels of hCD45+CD19+ engraftment were found (Table 1b). These findings suggest that CD34+CD38+CD19+ and CD34+CD38−CD19+ cells not only initiate leukemia, but also possess self-renewal capacity.

CD34+CD38−CD10−CD19− B-ALL cells exhibit normal multilineage differentiation capacity

In normal hematopoiesis and in AML, the CD34+CD38− population is highly enriched for self-renewing stem cells. Transplantation of either CD34+CD38−CD19+ or CD34+CD38−CD10−CD19− cells derived from B-ALL BM resulted in the development of hCD19+ cells in the recipient PB. However, 7/7 recipients transplanted with CD34+CD38−CD19+ cells died of leukemia, whereas 0/3 transplanted with CD34+CD38−CD10−CD19− cells developed disease. While almost all the engrafted CD19+ cells in the PB of recipients transplanted with CD34+CD38−CD19+ cells express CD34 on their surface and lack CD20 and surface IgM expression, the engrafted CD19+ cells in the PB of recipients transplanted with CD34+CD38−CD10−CD19− cells express CD20 and surface IgM, not CD34, on their surface (Figure 4). In addition, human myeloid and platelet development is also detected only in the recipients transplanted with CD34+CD38−CD10−CD19− cells (Figure 4). CD34+CD38−CD10−CD19− cells derived from B-ALL BM cells are highly enriched with normal HSCs, suggesting that the expression of CD19 distinguish LICs from normal HSCs within the CD34+CD38− population.

Figure 4

CD34+CD38−CD10−CD19− B-ALL cells show multilineage normal hematopoietic differentiation in vivo. Representative flow cytometric analysis of PB mononuclear cells demonstrating that the majority of human CD45+CD19+ cells in a representative recipient of CD34+CD38−CD19+ B-ALL cells express CD34 but lack CD20, CD10 and surface IgM expression. In contrast, mature human B cells, myeloid cells and platelets can be detected in a representative recipient transplanted with 2.0 × 103 CD34+CD38−CD10−CD19− cells at 4 months post-transplantation. ALL, acute lymphocytic leukemia; Mono/DC, monocytes and dendritic cells; Neu, neutrophils; PB, peripheral blood; plt, platelets.


We have recently established the newborn NOD/SCID/IL2rγnull mouse transplantation model that supports significantly higher engraftment levels of human normal HSCs and primary AML stem cells compared with the NOD/SCID/β2mnull mice.12, 13 In this study, we describe the efficient engraftment of primary human pediatric B-precursor ALL using the newborn NOD/SCID/IL2rγnull mouse transplantation model. This is the first report that purified CD34+CD38+CD19+ ALL cells efficiently engraft, initiate leukemia, and self-renew in vivo. Although only three individual patient samples were analyzed, the consistent engraftment in primary and secondary recipients by CD34+CD38+CD19+ cells provides new insights into the leukemogenesis of ALL.

Previous studies have described the engraftment of purified primary B-ALL cells in adult NOD/SCID recipients. Cobaleda et al.10 demonstrated that CD34+CD38− Ph+ ALL cells, but not CD34+CD38+ cells, engrafted and initiated leukemia in adult NOD/SCID recipients. In contrast, in pediatric B-precursor ALL, we found that the CD34+CD38+CD19+ ALL cells, as well as CD34+CD38−CD19+ cells, are able to efficiently engraft, infiltrate into non-hematopoietic organs and self-renew in vivo. B-precursor ALL and Ph+ ALL have distinct biological and clinical characteristics, as the former is the most common leukemia in the pediatric population and the latter is more common in adults. The finding that the hierarchical structure of B-precursor ALL, as defined by the surface phenotype of LSCs, is distinct from that of Ph+ ALL may reflect the biological differences between these two types of lymphoid malignancy. In particular, CD34+CD38+CD19+ cells predominantly reproduced themselves, rather than giving rise to heterogenous cell fraction such as CD34+CD38−CD19+ or CD34− ALL cells. As FACS discrimination gate border between CD38− and CD38+ cells within CD34+ fraction is not clearly delineated, it is not possible to totally exclude the possibility of contamination even with high levels of purity achieved with cell sorting. Additionally, limiting dilution and serial transplantation studies are required to fully define the differences between these CD34+CD38−CD19+ and CD34+CD38+CD19+ populations in B-precursor ALL. In addition, whether there are differences in the LIC phenotype and the function between these two subtypes of B-ALL remains to be determined. LICs in other subtypes of ALL (for example, Ph+ ALL and mature B-ALL) and various subtypes of B-precursor ALL, based on genetic abnormalities, need to be examined in the NOD/SCID/IL2rγnull newborn transplantation model to address this question.

In this study, we have successfully demonstrated that in human primary B-ALL, CD38 expression is irrelevant in defining a leukemogenic population, but rather the presence or absence of CD19 segregates the populations with malignant or normal repopulating capacity within the CD34+CD38− cell population. The role of CD19 as a marker to identify the clonogenic B-ALL precursor cells has been examined in the past. In t(4;11)-positive and t(9;22)-positive high-risk pediatric ALL, the leukemia-specific translocations have been identified in CD34+CD19− as well as CD34+CD19+ ALL cells.13 The reconstitution of the myeloid lineage occurring from the purified CD19−, but not CD19+, cells has been reported in TEL–AML1 fusion-positive and Ph+ ALL.15 Similarly, only CD34+CD19− and CD34+CD10− cells were found to engraft when sorted populations from t(9;22) and t(4;11)-negative B-precursor ALL were transplanted into NOD/SCID recipients.8 Here, we report the reconstitution of mature human B cell and human platelets in addition to myeloid cells from CD34+CD38−CD10−CD19− primary human B-ALL cells. The discrepancy in the engraftment and proliferation of normal myeloid as well as lymphoid lineages in these studies, compared with that in our findings, may be due to the superior sensitivity of engraftment and capacity for normal human hematopoietic development in the newborn NOD/SCID/IL2rγnull transplantation system. Although a previous publication has raised a concern for the possibility of LSC contamination in autologous stem cell graft in ALL,16 our finding suggests that by CD34+CD38−CD10−CD19− purification, the potential contamination of autologous stem cell graft by B-ALL LSCs may be avoidable.

The finding that it is possible for both CD34+CD38+CD19+ and CD34+CD38−CD19+ cells to act as the LICs in B-precursor ALL may be an important pathophysiological difference between AML and ALL. Delineation of the molecular basis of these differences between CD34+CD38− and CD34+CD38+ B-ALL-initiating cells may allow us to develop targeted therapy specific for primitive LICs that elude current antileukemia treatment strategies.


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This work was supported by grants from Ministry of Education, Culture, Sports, Science and Technology of Japan (FI) and National Institutes of Health (LDS).

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Correspondence to F Ishikawa.

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Kong, Y., Yoshida, S., Saito, Y. et al. CD34+CD38+CD19+ as well as CD34+CD38−CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL. Leukemia 22, 1207–1213 (2008).

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  • acute lymphocytic leukemia
  • stem cells
  • transplantation

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