Physiologic corticosterone oscillations regulate murine hematopoietic stem/progenitor cell proliferation and CXCL12 expression by bone marrow stromal progenitors.

The role of corticosterone (Cort), the immune system's major stress hormone, in the regulation of hematopoietic stem and progenitor cells (HSPCs) and their dynamic bone marrow (BM) microenvironment is currently unknown. We report that corticotropin-releasing factor receptor 1 (CRFR1) mutant mice with chronically low Cort levels showed aberrant HSPC regulation, having higher HSPC numbers and upregulation of the chemokine CXCL12, phenotypes that were restored by Cort supplementation. Expanded stromal progenitors known to support HSPCs were also observed in these low-Cort-containing mice. A similar phenotype was induced in wild-type (WT) mice by Metyrapone, a Cort synthesis inhibitor. Conversely, high Cort exposure induced HSPC apoptosis, reduced long-term BM repopulation and decreased stromal progenitor cell numbers. We documented circadian oscillations of Cort in WT BM but not in CRFR1 mutant mice, leading to diminished circadian BM CXCL12 fluctuations and increased number of circulating HSPCs in these mice. Finally, low Cort induced expansion of stromal progenitors, CXCL12 expression, HSPC proliferation and BM repopulation capacity, involving Notch1 signaling. This was associated with upregulation of the Notch ligand, Jagged1, in BM myeloid cells. Our results suggest that daily physiologic Cort oscillations are critical for balanced HSPC proliferation and function involving Notch1 signaling and their supportive BM microenvironment.

The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice.

Hematopoietic stem cells are identified based on their functional ability to migrate via the blood circulation of transplanted recipients, to home to the host bone marrow and to durably repopulate this organ with high levels of maturing myeloid and lymphoid cells. While a small pool of undifferentiated stem cells with the potential to repeat the entire process in serially transplanted recipients is maintained within the bone marrow, maturing cells are continuously released into the circulation. In recent years pre-clinical, functional in vivo models for human stem cells have been developed, using immune-deficient mice or pre-immune, fetal sheep as recipients. The mechanism of human stem cell migration, homing and repopulation in transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice as well as the accessory mediators that facilitate these processes, will be reviewed. In particular, the essential roles of the chemokine SDF-1 and its receptor CXCR4 which mediate and regulate stem cell homing and repopulation will be discussed.

Rapid and efficient homing of human CD34(+)CD38(-/low)CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice.

Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34(+)CD38(-/low)CXCR4(+) cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34(-)CD38(-/low)Lin(-) cells were not detected. Moreover, whereas freshly isolated CD34(+)CD38(+/high) cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34(+)CD38(+) cells. Homing of enriched human CD34(+) cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34(+)CD38(-/low)CXCR4(+) cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34(+) cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Galpha(i) proteins, inhibited SDF-1-mediated in vitro transwell migration but not adhesion or in vivo homing of CD34(+) cells. Homing of human CD34(+) cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34(+)CD38(-/low)CXCR4(+) cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.

Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood.

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

The plant lectin FRIL supports prolonged in vitro maintenance of quiescent human cord blood CD34(+)CD38(-/low)/SCID repopulating stem cells.

Ex vivo maintenance of human stem cells is crucial for many clinical applications. Current culture methods rely on optimized combinations of cytokines. Although these conditions provide some level of stem cell support, they primarily induce proliferation and differentiation, resulting in reduced repopulation capacity. The recently identified legume lectin FRIL has been shown to preserve human cord blood progenitors up to a month in suspension culture without medium changes. To test whether FRIL also preserves human SCID repopulating stem cells (SRC), we cultured human CD34(+) cord blood cells in medium containing FRIL, with or without subsequent exposure to cytokines, and tested their repopulating potential. We report that FRIL maintains SRC between 6 and 13 days in culture. Incubation of CD34(+) cells with FRIL results in significantly lower numbers of cycling cells compared with cytokine-stimulated cells. CD34(+) cells first cultured with FRIL for 6 days and subsequently exposed to cytokines for an additional 4 days generated significantly more mononuclear and progenitor cells and higher levels of engraftment in NOD/SCID mice compared with CD34(+) cells cultured with FRIL alone. Similar results were obtained with CD34(+)CD38(-/low) cells, including expansion of SRC that were cultured in FRIL followed by cytokine stimulation. Moreover, CD34(+) cells precultured with FRIL successfully engrafted primary and more importantly secondary recipients with lymphoid and myeloid cells, providing further support that FRIL maintains SRC for prolonged periods.FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications.

beta2 microglobulin-deficient (B2m(null)) NOD/SCID mice are excellent recipients for studying human stem cell function.

Human SCID repopulating cells (SRC) are defined based on their functional ability to repopulate the bone marrow of NOD/SCID mice with both myeloid and lymphoid cell populations. The frequency of SRC in umbilical cord blood cells is 1 in 9.3 x 10(5) mononuclear cells. We report that as few as 8 x 10(4) human cord blood mononuclear cells transplanted into NOD/SCID/B2m(null )mice resulted in multilineage differentiation in the murine bone marrow, revealing a more than 11-fold higher SRC frequency than in NOD/SCID mice. Moreover, as few as 2 to 5 x 10(3) CD34(+) cells recovered from the bone marrow of primary transplanted NOD/SCID mice were sufficient for engrafting secondary NOD/SCID/B2m(null )mice with SRC, suggesting SRC self-renewal. Thus, by using NOD/SCID/B2m(null )mice as recipients, we established a functional assay for human stem cells capable of engrafting the bone marrow of primary and secondary transplanted immune-deficient mice with SRC, providing a model that better resembles autologous stem cell transplantation.

The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice.

Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen-4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen-1 (LFA-1). Treatment of human CD34(+) cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti-LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34(+) cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule-1) and VLA-4/VCAM-1 (vascular adhesion molecule-1). Furthermore, SDF-1-induced polarization and extravasation of CD34(+)/CXCR4(+) cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.

NOD/LtSz-Rag1null mice: an immunodeficient and radioresistant model for engraftment of human hematolymphoid cells, HIV infection, and adoptive transfer of NOD mouse diabetogenic T cells.

Development of a small animal model for the in vivo study of human immunity and infectious disease remains an important goal, particularly for investigations of HIV vaccine development. NOD/Lt mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation readily support engraftment with high levels of human hematolymphoid cells. However, NOD/LtSz-scid mice are highly radiosensitive, have short life spans, and a small number develop functional lymphocytes with age. To overcome these limitations, we have backcrossed the null allele of the recombination-activating gene (Rag1) for 10 generations onto the NOD/LtSz strain background. Mice deficient in RAG1 activity are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. NOD/LtSz-Rag1null mice have an increased mean life span compared with NOD/LtSz-scid mice due to a later onset of lymphoma development, are radioresistant, and lack serum Ig throughout life. NOD/LtSz-Rag1null mice were devoid of mature T or B cells. Cytotoxic assays demonstrated low NK cell activity. NOD/LtSz-Rag1null mice supported high levels of engraftment with human lymphoid cells and human hemopoietic stem cells. The engrafted human T cells were readily infected with HIV. Finally, NOD/LtSz-Rag1null recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly developed diabetes. These data demonstrate the advantages of NOD/LtSz-Rag1null mice as a radiation and lymphoma-resistant model for long-term analyses of engrafted human hematolymphoid cells or diabetogenic NOD lymphoid cells.

The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34(+)CD38(-/low) cells capable of repopulating severe combined immunodeficiency mice.

In vitro maintenance and proliferation of human hematopoietic stem cells is crucial for many clinical applications. Early hematopoietic cells express low levels of FLT-3 and c-kit receptors, as well as the interleukin-6 (IL-6) receptor signal transducing element, gp130, but do not express IL-6 receptor itself. Therefore, we have attempted to maintain human cord blood or bone marrow CD34(+) cells ex vivo in serum-free cultures containing stem cell factor (SCF) and FLT-3 ligand (FL) alone or together with a new recombinant molecule of soluble IL-6 receptor fused to IL-6 (IL6RIL6 chimera). The effect of IL6RIL6 chimera on the proliferation and differentiation of CD34(+) cells was compared with that of each chimera component added separately. The engraftment potential of in vitro-cultured cells was determined using our recently established functional in vivo assay for primitive human severe combined immunodeficiency (SCID)-repopulating cells (SRC). We report here that IL6RIL6 chimera induced significantly higher levels of progenitors and SRC compared with SCF + FL alone or together with IL-6 and soluble IL-6 receptor. IL6RIL6 chimera prolonged in vitro maintenance of SRC for up to 14 days. Stimulation of CD34(+)CD38(-/low) enriched cells with IL6RIL6 chimera maintained the early CD34(+)CD38(-/low) cell subpopulation, which could be detected in vitro for up to 14 days. Moreover, IL6RIL6 chimera preferentially stimulated the growth of early CD34(+)38(-/low) cells, resulting in significantly higher levels of progenitors compared with more mature CD34(+)38(+) cells. Taken together, these findings demonstrate the importance of IL6RIL6 chimera in stimulating the proliferation of early CD34(+). CD38(-)gp130(+)IL-6R(-) cells in vitro and extended maintenance of progenitors and SRC.

Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34(+) cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34(+) and CD34(+)38(-) subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34(+) CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34(+) CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34(+) cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] +/- stem cell factor [SCF] +/- interleukin-6 [IL-6]). Mice that had been injected with >/=20,000 fresh or cryopreserved uncultured CD34(+) cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34(+) CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34(-) cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34(+) CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Differential effects of CD4+ and CD8+ cells on lymphocyte development from human cord blood cells in murine fetal thymus explants.

The possibility that mature lymphocytes play a role in the regulation of human T cell development was studied in the experimental model of fetal thymus organ cultures (FTOC), by reconstituting lymphocyte-depleted murine fetal thymus (FT) lobe with cells isolated from human umbilical cord blood (CB). Cultures were incubated with human cytokines (IL-7, FLT-3 ligand and Steel Factor), or remained untreated. When CD4+, or CD8+ CB cells, were co-cultured with FT explants, they expanded and maintained their original phenotypic markers, with no significant effect of the cytokines. Cultures of human hematopoietic stem cells (CD34+) gave rise to CD4+CD8- cells, which were mainly CD3-, with no indication of further intermediate developmental stages. However, a limited number of CD4+CD8+ (double positive [DP]) cells were detected when the CD34- cells were co-cultured with CD4+ cells from the same CB samples. In contrast, FT with unseparated CB cells resulted in the different CD4/CD8 subsets, and their numbers increased in the presence of cytokines. The appearance of DP cells depended on the presence of either CD4+ or CD8+ cells in the cultured CB samples. Hence, DP cells were not detected when the CB was depleted of CD4+ and CD8- cells ("depCB") before culture, and they appeared when depCB were co-cultured with either CD4+ or CD8+ cells. In contrast, CD4+ cells inhibited the development of CD8+CD3+ cells, and this was most pronounced in the absence of the cytokines. There was no symmetrical down-regulatory effect of CD8+ cells on the development of CD4+CD3+ cells. Addition of IL-15 to the cytokine mixture led to an increased proportion of CD56+ cells in cultures of CD34+ cells. The presence of CD4+, and not CD8+ cells, interfered with this process. Our results thus imply differential effects of CD4+ and CD8+ cells on thymocytopoiesis.

Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.

Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Idiotype-specific inhibition of LFA-1-mediated cell adhesion by anti-idiotype x anti-LFA-1 bispecific antibodies.

Adhesion molecules are involved in lymphoma dissemination. Antibodies to adhesion molecules may block tumor metastasis. However, such antibody treatment may block as well normal functions of the immune system. We tested the hypothesis that a bispecific antibody with specificity for an adhesion molecule and for a tumor specific antigen binds preferentially to tumor cells which coexpress both antigens and hence selectively blocks adhesion. A bispecific antibody was developed by somatic cell hybridization of two hybridomas, one producing a monoclonal antibody against the immunoglobulin idiotypic determinant of the murine B cell lymphoma 38C-13 and the other producing an antibody against the alpha subunit (CD11a) of the adhesion molecule LFA-1. The bispecific antibody, anti-idiotype x anti-LFA-1, was purified by affinity chromatography. The dual specificity of the hybrid hybridoma product was demonstrated by a radioimmunoassay devised for detection of bifunctional activity. The bispecific antibody was shown by flow cytometry to bind efficiently to 38C-13 cells that coexpress idiotype and LFA-1. It bound only weakly to idiotype-negative variants of 38C-13 that express only LFA-1. In binding assays to immobilized ICAM-1, the anti-idiotype x anti-LFA-1 was highly active in blocking 38C-13 cell adhesion. However, it did not effect adhesion of idiotype-negative tumor cells or of normal T lymphocytes. In summary, the bispecific antibody preferentially blocks adhesion of cells that coexpress the tumor specific antigen and the adhesion receptor. The present approach may provide a general way for the selective adhesion blockade of a specific cell population.

Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis.

Many events and requirements of the developmental program of human hematopoietic stem cells have not yet been discovered. A major impediment has been the lack of an appropriate experimental system. At present the conditions for maintaining human stem cells in vitro are not fully known. As a result within a short period the small stem cell pool is lost due to differentiation, making it difficult to examine the correlation between these cells and their function in vivo. Most of our knowledge of hematopoietic stem cells is from animal models in which purified stem cell canididates are assayed based on their functional ability to rescue lethally conditioned recipients. The permanent correction of many genetic disorders of the hematopoietic system requires efficient methods for introducing genes into stem cells in vitro. However, progress has been hindered by the absence of preclinical models that assay the repopulating capacity of primitive human cells. In addition, the development of therapy for malignant diseases also requires assays to identify the target leukemic stem cells based on their ability to initiate the disease. The recent development of methods to transplant or implant both normal and leukemic cells into immune-deficient mice provides the foundation for human stem cell assays. These models assay the repopulating capacity of primitive human cells and provide an important approach to identify and characterize human stem cells, both normal and leukemic. This review focuses on the development of functional assays for normal and leukemic human stem cells and on the new insights that these models are beginning to provide on the organization of the human stem cell hierarchy.

Identification of human juvenile chronic myelogenous leukemia stem cells capable of initiating the disease in primary and secondary SCID mice.

Most juvenile chronic myelogenous leukemia (JCML) cells have limited long-term proliferative capacity, and only a minority of immature cells give rise to colonies in semisolid cultures. Clonogenic JCML progenitors cannot be maintained in culture because they differentiate, and within a few weeks the leukemic clone is lost. This makes it difficult to identify the cell that initiates and maintains the disease in patients. To determine the proliferative capacity of JCML cells in vivo, bone marrow (BM), peripheral blood, or spleen cells from eight patients with JCML either at diagnosis or during treatment were transplanted into sublethally irradiated severe combined immune deficient (SCID) mice. JCML cells from all patients homed to the murine BM and proliferated extensively in response to exogenous stimulation with granulocyte-macrophage colony-stimulating factor. Within a few weeks, highly engrafted mice became ill and cachectic due to infiltration of leukemic cells and secretion of tumor necrosis factor-alpha. Murine BM, spleen, and liver were infiltrated with leukemic blasts, and typical JCML colony-forming progenitors could be recovered. Kinetic experiments demonstrated that only a small minority of transplanted cells homed to the murine BM, and that these cells initiated and maintained the disease in vivo by extensive proliferation and differentiation. To characterize the cell-surface phenotype of the JCML initiating cell (JCML-IC), JCML blood or spleen cells were fractionated on the basis of CD34/CD38 marker expression and transplanted into SCID mice. Only immature CD34+ cells could initiate the disease, while mature CD34- cells did not engraft. Within the CD34+ compartment, there was enrichment for JCML-ICs by immature cells with a CD34+/CD38- stem-cell-like phenotype. Mice transplanted with more mature CD34+/CD38+ populations that also contained clonogenic JCML progenitors were poorly engrafted. These results indicate that the JCML-IC is an earlier stage of development than clonogenic JCML progenitors. Additional evidence that the JCML-IC has stem-cell properties comes from secondary transplant experiments that test the self-renewal capacity. The JCML-IC from all three patients tested could successfully reinitiate the disease in secondary murine recipients. Thus, we have developed a functional in vivo model that replicates many aspects of human JCML, and have used this model to identify and characterize JCML-ICs and their stem-cell properties.