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Postnatal hematopoietic stem (and progenitor) cells (HS(P)Cs) are especially vulnerable to oxidative stress, leading to early hematopoietic senescence and/or malignant transformation. Elevated intracellular reactive oxygen species (ROS) can, among others, oxidize nucleotides, and thus can result in genotoxicity and mutagenesis if left unrepaired. Oxidized bases, as well as other spontaneous single base modifications, are recognized and repaired by the base excision repair (BER) pathway. Hence, the BER pathway is crucial to maintain genome integrity. In contrast to other DNA repair pathways however, the role of BER in maintaining HSPC functionality remains enigmatic, chiefly because knockout (KO) of BER genes is in many cases embryonic lethal. BER is a complex multi-step repair process. After initial removal and excision of the damaged base, the apurinic/apyrimidinic (AP) site is processed by the AP endonuclease (APEX1) enzyme. At this point, the BER pathway branches into 2 sub-pathways, namely the short-patch (SP-BER; wherein DNA polymerase beta (Polβ), Ligase III (Lig3) together with X-ray repair cross-complementing protein 1 (Xrcc1) are active) and the long-patch BER (LP-BER; wherein Lig1, Flap Structure-Specific Endonuclease 1 (Fen1), and sometimes Polβ are active) for the repair synthesis and the gap filling steps. In this study we wished to address the role of BER in adult hematopoiesis. Therefore, we used CRISPR-Cas9 to KO different BER genes in adult bone marrow (BM) HS(P)Cs, including two genes common to the BER (sub-)pathway(s) (Apex1 and Polβ) as well as one gene in the SP-BER (Xrcc1) and one gene in the LP-BER (Lig1) pathway. The effect thereof was evaluated on HS(P)C repopulation in vivo as well as on HS(P)C expansion during long-term in vitro culture (using the culture medium described by Wilkinson et al., Nature 2019). All CRISPR-Cas9 experiments were validated using a second sgRNA targeting the selected BER genes. Lig1-KO caused in vivo HSPC dysfunction: at 20 weeks post-transplantation, significantly less Lig1 KO cells were observed in the committed progenitor (HPC) and lineage committed (Lin +) BM compartments. By contrast, KO of Xrcc1 had only minor effects on HS(P)C repopulation, but we observed increased HSC expansion and myeloid biased differentiation in some recipient mice, which might correspond to clonal hematopoiesis and is consistent with the finding of XRCC1 loss-of-function mutation in myelodysplastic patients (Joshi et al, Ann Hematol 2016). Knockout of Polβ did not affect hematopoiesis in vivo or in vitro. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HS(P)Cs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less Apex1 deficient cells were detected in the different blood lineages and nearly no CRISPR-Cas9 KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of Apex1 KO BM cells was observed. APEX1 has two major functional activities, namely its nuclease activity, involved in BER, and its redox activity (also called Ref-1 function) important in reducing oxidized transcription factors and therefore implicated in transcriptional regulation. However, little is known regarding the nuclease and Ref-1 function(s) in primary adult hematopoietic cells. We therefore cultured BM HS(P)Cs for 1 week in the continuous presence of 2 distinct chemicals blocking the APEX1 nucleases, or 2 different chemicals inhibiting specifically the Ref-1 function. We demonstrated that both APEX1 functions are essential for hematopoiesis, even if the 2 functions appear to support the survival, expansion and maintenance of HS(P)Cs through different mechanisms. While the Ref-1 function was essential for proliferation (as both Ref-1 inhibitors cause cell cycle arrest) of all the lineages (including the Lin + cells), both inhibitors of the nuclease function affected more the expansion/survival of the less committed HS(P)Cs without leading to any cell cycle arrest. In conclusion, this study demonstrates for the first time the important role of BER genes in adult hematopoiesis, often deregulated in cancer, including hematopoietic malignancies. We observed a particularly severe phenotype upon loss of Apex1 in adult HSPCs, and ongoing studies (such as RNA sequencing analysis) should provide novel insights in underlying mechanisms of APEX1 deficiencies in HS(P)Cs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation.

Directional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3(-/-) HSPCs, while it affected GPC1(-/-) HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3(-/-) but not GPC1(-/-) BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.

Multipotent adult progenitor cells (MAPCs) are marrow-derived pluripotent stem cells with a broad differentiation potential. We sought to identify factors that affect adoptively transferred MAPCs. In vitro, MAPCs expressed low levels of major histocompatibility complex (MHC) antigens, failed to stimulate CD4(+) and CD8(+) T-cell alloresponses, and were targets of NK cytolysis. To study in vivo biodistribution, we labeled MAPCs with luciferase for sequential quantification of bioluminescence and DsRed2 for immunohistochemical analysis. C57BL /6 MAPCs were infused intravenously into C57BL /6, Rag-2(-/-) (T- and B-cell-deficient), and Rag-2(-/-)/IL-2Rgamma(c)(-/-) (T-, B-, and NK-cell-deficient) mice. In C57BL /6 mice, MAPCs were transiently detected only in the chest compared with long-term persistence in T- and B-cell-deficient mice. NK depletion reduced MAPC elimination. Because the lungs were the major uptake site after intravenous injection, intra-arterial injections were tested and found to result in more widespread biodistribution. Widespread MAPC biodistribution and long-term persistence were seen in irradiated recipients given allogeneic marrow and MAPCs; such MAPCs expressed MHC class I antigens in tissues. Our data indicate that the biodistribution and persistence of reporter gene-labeled MAPCs are maximized after intra-arterial delivery or host irradiation and that T cells, B cells, and NK cells contribute to in vivo MAPC rejection. ispartof: Blood vol:107 issue:10 pages:4182-4188 ispartof: location:United States status: published

Hematopoietic stem cells (HSC) are cells with self-renewing multilineage differentiation potential. Although engraftment in xenogeneic recipients can be used to measure human HSC, these assays do not allow assessment of individual progenitors. We developed an in vitro assay that allows the identification of a single human bone marrow progenitor closely related to HSC, which we termed "Myeloid-Lymphoid Initiating Cell," or ML-IC, because it is capable of generating multiple secondary progenitors that can reinitiate long-term myeloid and lymphoid hematopoiesis in vitro. The assay is done in contact with murine AFT024 fetal liver stromal cells and with Flt3-Ligand, stem cell factor, and interleukin-7. In this assay, 0.2% to 1.7% of Lin -/34(+)/DRdim cells could generate 1 to 3 long-term culture initiating cells (LTC-IC) as well as 1 to 4 NK-IC after 4 to 6 weeks. In addition, this assay measures contribution of net-progenitor conservation and net-progenitor proliferation over time, providing insight in the fate of individual LTC-IC and NK-IC. This assay will prove useful to enumerate the number of very primitive human progenitors with multilineage differentiation potential, as well as to evaluate future ex vivo culture conditions. ispartof: Blood vol:93 issue:11 pages:3750-6 ispartof: location:United States status: published

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph-) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph- progenitors are highly enriched in the CD34(+)HLA-DR- fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph- progenitor or stem cells, leaving the unanswered question whether Ph- CD34(+)HLA-DR- progenitors are normal. To show the clonal nature of Ph- CD34(+)HLA-DR- CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34(+)HLA-DR+ and CD34(+)HLA-DR- progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA-, CD34(+)HLA-DR- progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34(+)HLA-DR- derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph-, BCR/ABL mRNA- and polyclonal state in the CD34(+)HLA-DR- and CD34(+)HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34(+) cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph-, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34(+)HLA-DR- cells that are Ph- and BCR/ABL mRNA- are polyclonal and therefore benign. This population is suitable for autografting in CML. ispartof: Blood vol:93 issue:1 pages:284-92 ispartof: location:United States status: published

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P

The aims of this study were (1) to evaluate the effect of intermediate (cyclophosphamide alone) or intensive (mitoxantrone, cytosine arabinoside, cyclophosphamide) priming on the cytogenetic response in mobilized bone marrow (BM) or peripheral blood (PB) progenitors in patients with chronic myelogenous leukemia (CML), (2) to determine the incidence of cytogenetic remissions after mobilized progenitor transplantation in CML, and (3) to determine the effect of in vivo priming on the ability to select Philadelphia chromosome–negative (Ph-negative) CD34+HLA-DR− cells from mobilized BM or PB in quantities sufficient for transplantation. Between February 1994 and March 1997, 44 patients were enrolled in three sequential protocols. Although the duration of neutropenia after only cyclophosphamide mobilization was shorter, clinical morbidity for the intermediate and intensive priming protocols was similar. Cytogenetic responses in mobilized PB progenitors were similar after mobilization with either intermediate or intensive chemotherapy. The degree of Ph negativity in the mobilized product correlated with disease stage at the time of mobilization (early chronic phase [ECP] > late CP > accelerated phase). Cytogenetic responses after transplantation with mobilized progenitors obtained after the different regimens were similar. The cytogenetic status of the graft predicted the cytogenetic status of marrow obtained 3 weeks after transplantation whereas cytogenetic responses 3, 6, and 12 months after transplantation correlated with the number of BCR/ABL–negative CD34+HLA-DR−cells, but not the number of Ph-negative metaphases in the graft. In patients with ECP CML, mobilized PB collections yielded significantly more CD34+HLA-DR− cells than from steady state or mobilized BM. CD34+HLA-DR− cells were Ph negative and polyclonal (X-chromosome inactivation) in the majority of ECP CML patients, before and after mobilization and irrespective of the mobilization regimen. Because infusion of large numbers of Ph-negative CD34+HLA-DR− cells predicted superior outcome after transplantation, approaches in which CD34+HLA-DR− cells are selected from mobilized PB may result in longer lasting and clinically significant cytogenetic responses after transplantation. © 1998 by The American Society of Hematology.

Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34(+) cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34(+) cells for 3 to 5 weeks. Coculture of more mature CD15(+)/CD14(-) myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34(+) nor CD15(+)/CD14(-) cells produced IL-6, G-CSF, IL-1beta, or tumor necrosis factor alpha. When CD34(+) cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34(+) cells or when CD34(+) cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit-granulocyte-macrophage-stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34(+) progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant. ispartof: Blood vol:91 issue:10 pages:3724-33 ispartof: location:United States status: published

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors. ispartof: Blood vol:91 issue:9 pages:3414-22 ispartof: location:United States status: published

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to β1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210BCR-ABL, interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210BCR-ABL in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34+HLA-DR+(DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated β1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210BCR-ABL levels in CML CD34+ cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ABL gene rearrangement. The only curative therapy is allogeneic transplantation. Although autologous transplants may prolong survival, most patients relapse because of disease persisting in the host and in the graft. Continued administration of chemotherapy after transplant could reduce the incidence of relapse provided that the autograft can be protected by transfer of a drug-resistance gene. However, CML autografts will almost certainly contain malignant stem cells that will also be rendered drug-resistant. The presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CML. We thus hypothesized that transfer of a vector that combines a drug-resistance gene with anti-BCR/ABL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX)-resistant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS sequences directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and MO7e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210BCR/ABL protein levels by 6- to 10-fold in most cells. This subsequently led to the restoration of normal function of BCR/ABL cDNA+ cells: they grew significantly slower in the presence of interleukin-3 (IL-3); they underwent apoptotic cell death when cultured without IL-3; and they had restored expression and function of adhesion receptors. These effects were specific, because LasBD-containing AS sequences directed at the b3a2 BCR/ABL breakpoint did not affect p190BCR/ABL-containing cells. LasBD also rendered 20% to 30% of primary Ph− and Ph+ CD34+ cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resistant Ph+ CD34+ cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitro and for more than 70 days in vivo. Finally, LasBD decreased tumorigenicity of 32DBCR/ABL cells in vivo by 3 to 4 logs. In conclusion, the tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD can suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prove to be an extremely useful adjunct in autologous transplantation for CML.

We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+ HLA-DR- and CD34+ CD38- and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+ HLA-DR+ cells (rich in mature LTC-IC) and CD34+ HLA-DR- cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1alpha) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+ HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+ HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+ HLA-DR- mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+ HLA-DR- PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1alpha were added to noncontact cultures (96 +/- 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of "mature" CD34+ HLA-DR+ LTC-IC with a limited proliferative capacity, primitive CD34+ HLA-DR- LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1alpha for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM. ispartof: Blood vol:89 issue:11 pages:3991-7 ispartof: location:United States status: published

Chronic myelogenous leukemia (CML) progenitors show decreased adhesion to stroma and fibronectin (FN) through beta 1 integrin receptors. We have previously shown that interferon-alpha (IFN-alpha) restores beta 1 integrin-mediated adhesion of CML progenitors to stroma. Because beta1 integrins transmit proliferation inhibitory signals from the microenvironment to normal hematopoietic progenitors, we hypothesized that decreased integrin-mediated adhesion of CML progenitors contributes to their continuous proliferation when in contact with stroma and that IFN-alpha treatment, by restoring integrin-mediated adhesion, also restores integrin-mediated microenvironmental inhibition of CML progenitor proliferation. We show here that, in contrast to normal colony-forming cells (CFC), the percentage of malignant CML CFC in S-phase was not significantly reduced following coculture with stromal layers. However, IFN-alpha treatment resulted in a significant reduction in the proliferation of CML CFC on coculture with stroma. This effect was not because of a direct antiproliferative effect of IFN-alpha on CML CFC because the proliferation of IFN-alpha treated CML CFC kept in suspension culture was not reduced. We examined the role of restored signaling through beta 1 integrin receptors in IFN-alpha induced inhibition of CML progenitors in two sets of experiment. In the first set of experiments, we demonstrated that proliferation of IFN-alpha-treated CML CFC, but not untreated CML CFC, was significantly reduced following coculture with 33/66-kD and 75-kD FN fragments, recognized by alpha 4 beta 1 and alpha 5 beta 1 integrins respectively. In a second set of experiments, we demonstrate that direct stimulation of integrin receptors by crosslinking with blocking antibodies to alpha 4, alpha 5, and beta 1 integrins and secondary goat antimouse antibodies resulted in significant reduction in proliferation of normal and IFN-alpha treated CML progenitors but not untreated CML CFC. These studies indicate that CML hematopoietic progenitors are unresponsive to beta 1-integrin mediated proliferation inhibition and that IFN-alpha not only restores beta 1 integrin-mediated adhesion but also beta1-mediated microenvironmental inhibition of CML progenitor proliferation. These observations may explain, at least in part, the therapeutic efficacy of IFN-alpha in CML. ispartof: Blood vol:87 issue:9 pages:3883-91 ispartof: location:United States status: published

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2-10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2-10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2-10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2-10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors. ispartof: Blood vol:87 issue:8 pages:3229-36 ispartof: location:United States status: published

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML. ispartof: Blood vol:87 issue:6 pages:2450-8 ispartof: location:United States status: published

Although it is well established that BMP4 plays an important role in the development of hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis and how. Here, we describe a novel mechanism by which BMP4 regulates homing of murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment of murine BM derived c-kitLin-Sca-1 (KLS) and CD150CD48-KLS cells for up to 5 days in vitro prevented the culture-induced loss of Integrin-α4 (ITGA4) expression as well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmia-associated transcription factor (MITF), known to induce ITGA4 expression. Elevated ITGA4 expression significantly enhanced HSPC attachment to bone marrow stromal cells, homing and long-term engraftment of the BMP4 treated cells compared with the cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM derived Lin-CD34 cells in culture, which was associated with improved homing potential. Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution.Cytokine-induced loss of murine as well as human HSPC homing during ex vivo culture can be prevented by addition of BMP4. In HSPCs, BMP4 directly regulates Integrin-α4 expression through SMAD-independent p38 MAPK-mediated signaling.

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC-IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth-inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14-cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14-mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte-macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM-CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G-CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM-CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-1 alpha (IL-1 alpha) can act as both a hematopoietic growth factor and a stimulant of cellular and humoral immune responses. To promote acceleration of hematologic recovery and induce immune antitumor activity, we initiated a phase I/II dose escalation trial of 6-hour daily infusions of recombinant human IL-1 alpha after autologous transplantation. Forty patients with Hodgkin's disease (n = 9) and non- Hodgkin's lymphoma (n = 31) transplanted with unmobilized autologous peripheral blood stem cells or bone marrow stem cells received daily 6- hour infusions of IL-1 alpha (day 0 to day +13) at daily doses between 0.1 to 10 micrograms/m2/d; 7 patients received only 7 planned days of IL-1 alpha (day 0 through 6). Most patients received all 14 days of therapy, although 5 patients discontinued treatment early (after 1 to 6 doses) because of fever and severe chills. Toxicity included IL-1 alpha- related fever (occurring on a median of 9 of 14 treatment days), fatigue, and severe chills. Hypotension was dose-limiting and led to discontinuation of IL-1 alpha in both patients receiving 10 micrograms/m2/d. IL-1 alpha-treated patients receiving 3.0 micrograms/m2/d (the maximum tolerated dose) achieved neutrophil recovery (absolute neutrophil count greater than 500/microL) significantly earlier (median, 12 days; range, 11 to 27) than untreated control patients or those receiving IL-1 alpha at 0.1 to 1.0 micrograms/m2/d (median, 27; range, 9 to 63; P < .0001). In addition, the IL-1 alpha patients' bone marrows at day +14 were significantly enriched with committed myeloid progenitor cells. Strong trends to earlier freedom from red blood cell (P = .06) and platelet (P = .09) transfusions were also noted after IL-1 alpha treatment. This earlier hematopoietic engraftment after 3.0 micrograms/m2/d IL-1 alpha allowed earlier hospital discharge (median, 25 v 37 days for control or low- dose IL-1 alpha patients [P < .0001]) and a concomitant reduction (by $38,000) in median hospital charges (P = .01). The clinical toxicities of IL-1 alpha infusion are substantial, though not life-threatening. The accelerated hematopoiesis and immune response activation observed in this trial suggest the value of its further investigation in controlled trials and perhaps in combination with other hemopoietins after transplantation.

Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer (“stroma-free”) or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane (“stroma-noncontact”). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a “stroma-free” culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in “stroma-noncontact” cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in “stroma-noncontact” cultures supplemented with or without IL-3 than in “stroma-free” cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.

We have adapted the stroma-dependent long-term bone marrow culture (LTBMC) system to study the development of human natural killer cells (NK) from the CD34+/HLA-DR- (CD34+/DR-) BM mononuclear cell (BMMNC) population. The CD34+/DR- population does not express any known antigens associated with myeloid or lymphoid lineage and has been shown by us and others to contain primitive hematopoietic progenitors capable of both self-renewal and differentiation to myeloid lineage. CD34+/DR- cells obtained from normal human BM by fluorescence-activated cell sorting were plated on allogeneic, irradiated BM stromal layers. After 5 weeks of culture in the presence of media containing recombinant interleukin-2 and human serum, 147- +/- 21-fold expansion of cells with the morphologic appearance of large granular lymphocytes was observed. Cultured cells (84.8% +/- 1.5%) expressed the characteristic CD56+/CD3- phenotype of NK. A proportion of CD56+/CD3- cells expressed other markers of lymphoid lineage that have been associated with mature NK, including CD2 (7.8% +/- 1.2%), CD7 (19.5% +/- 2.8), CD8 (3.1% +/- 1.0%), and CD16 (4.5% +/- 1.3%). The cultured cells did not express other antigens associated with T-lymphocyte (CD3, CD5, T-cell receptor [TCR] alpha/beta and TCR gamma/delta), B-lymphocyte (CD19), myeloid (MY8, CD33, and CD71), or monocytoid (CD14 and CD15) lineage and did not express the CD34 antigen associated with hematopoietic progenitors present on the starting population. This NK population was cytotoxic against both K562 (E:T 20:1; 79% +/- 1.9%) and Raji (E:T 20:1; 38% +/- 5.7%) target cell lines. The NK progenitor frequency in the CD34+/DR- cell population determined by limiting dilution of CD34+DR- on stromal layers followed by a functional chromium release assay against K562 targets was 1:169 +/- 50 CD34+/DR- cells. The data suggest that human LTBMC developed to study myeloid differentiation can be modified to study the origin and development of the NK and possibly other lymphoid lineages. Modified cultures show that cells with morphologic, phenotypic, and functional characteristics of NK can be derived from a population of BMMNC with the phenotype of primitive hematopoietic progenitors and without phenotypic evidence of lymphoid- or myeloid- lineage commitment. Further studies will address the cell of origin and the ontogeny of human NK and other lymphoid lineages.

We have studied the mechanisms underlying expansion of recombinant interleukin-2 (rIL-2)-stimulated natural killer (NK) cells in vitro. A population of NK cells expressing the CD56+/CD3-phenotype (98.9% +/- 0.42%) was obtained from normal human peripheral blood mononuclear cells (PBMNC) by fluorescence-activated cell sorting (FACS). Culture of NK cells in media containing rIL-2 (1,000 U/mL) for 18 days resulted in a population of activated NK cells (ANK) with significantly enhanced cytotoxicity, but only 2.6 +/- 0.56-fold expansion of cell number compared with the starting NK population. Culture of starting NK populations and autologous PBMNC in a Transwell system (Costar, Cambridge, MA), providing separation of the two cell populations by a 0.4-microns pore membrane, resulted in a dose-dependent increase in fold expansion of ANK (expansion = 19.9 +/- 4.0-fold; P < .001; n = 22) significantly greater than that observed when NK were cultured alone. Further experiments using the Transwell system showed that the stimulatory effect of autologous PBMNC on ANK progenitor proliferation resides in the CD14+ monocyte fraction (maximal expansion = 14.5 +/- 1.5-fold; n = 17) and not in the CD5+ T-lymphocyte or CD19+ B- lymphocyte fractions. Direct coculture of purified NK and autologous monocytes in the same compartment, thus permitting cell-cell contact, resulted in significantly greater expansion of the ANK population (30.6 +/- 4.7-fold expansion, P < .001; n = 10) than that observed when NK and monocytes were separated by the Transwell membrane. Finally, depletion of PBMNC of cells bearing CD5 and CD8 by panning on antibody- coated plastic flasks resulted in a starting cell population enriched for NK progenitors and for monocytes. Cultures of this resultant population for 18 days in the presence of rIL-2 yielded an ANK population similar to that obtained when CD56+/CD3- cells obtained by FACS were cocultured with autologous monocytes. These results suggest that proliferation of ANK requires autologous monocytes and is in part mediated by humoral factors, but is enhanced when NK and monocytes are in direct cell-cell contact. Depletion of cells bearing CD5 and CD8 from PBMNC is a single efficient method for obtaining a starting population capable of producing large numbers of ANK in culture that may lead to new therapeutic uses for the ANK population.

Long-term bone marrow cultures support both differentiation and conservation of primitive human hematopoietic progenitors in the absence of exogenous cytokines. It is believed that hematopoiesis in such cultures requires direct contact between hematopoietic progenitors and stroma. In the present study, we demonstrate that primitive progenitors physically separated from the stromal layer by a 0.45- microns microporous membrane continue to generate differentiated progenitors for at least 8 weeks. Moreover, primitive progenitors are conserved to a greater extent under these conditions, as when cultured in direct contact with the stroma. However, excessive production of granulocyte-macrophage progenitors occurs when primitive progenitors are not allowed to interact directly with the stroma. Thus, direct contact between hematopoietic and stromal cells is not required for either differentiation or conservation of primitive hematopoietic progenitors but is essential for the regulated production of mature blood elements. These findings can now be used to define the role of diffusible factors and cell-cell or cell-extracellular matrix adhesion events in the regulation of conservation, proliferation, and differentiation of primitive human hematopoietic progenitors in vitro.

Chronic myelogenous leukemia (CML) is a lethal malignancy of the human hematopoietic stem cell. Here we report that coexistent benign, primitive hematopoietic progenitors can be distinguished from their malignant counterparts in CML bone marrow by differences in cell surface antigen expression. Selection of bone marrow cells expressing the CD34 antigen but lacking the HLA-DR antigen results in recovery of small lymphocyte-like blasts, which initiate and sustain production of myeloid clonogenic progeny in vitro. Secondary clonogenic cells derived at week 1, 5, and 8 from long-term bone marrow cultures (LTBMCs) initiated with primitive progenitors, which lack HLA-DR antigens, exhibit neither the Philadelphia chromosome (Ph1) nor the corresponding bcr/abl mRNA characteristic of CML. In contrast, clonogenic cells recovered at week 1, 5, and 8 from LTBMCs initiated with the CML HLA- DR+ population contain Ph1 and express bcr/abl mRNA. This observation indicates that it may be possible to select a population of viable, exclusively benign hematopoietic stem cells from CML bone marrow capable of repopulating the hematopoietic compartment following autologous bone marrow transplantation.

It is here reported that mesenchymal stem cells known to give rise to limb-bud mesoderm can, at the single-cell level, also differentiate into cells of visceral mesoderm and can be expanded extensively by means of clinically applicable methods. These cells were named mesodermal progenitor cells (MPCs). MPCs were selected by depleting bone marrow mononuclear cells from more than 30 healthy human donors of CD45+/glycophorin-A (GlyA)+ cells. Cells were cultured on fibronectin with epidermal growth factor and platelet-derived growth factor BB and 2% or less fetal calf serum. It was found that 1/5 × 103CD45−GlyA− cells, or 1/106 bone marrow mononuclear cells, gave rise to clusters of small adherent cells. Cell-doubling time was 48 to 72 hours, and cells have been expanded in culture for more than 60 cell doublings. MPCs are CD34−, CD44low, CD45−, CD117 (cKit)−, class I–HLA−, and HLA-DR−. MPCs differentiated into cells of limb-bud mesoderm (osteoblasts, chondrocytes, adipocytes, stroma cells, and skeletal myoblasts) as well as visceral mesoderm (endothelial cells). Retroviral marking was used to definitively prove that single MPCs can differentiate into cells of limb bud and visceral mesoderm. Thus, MPCs that proliferate without obvious senescence under clinically applicable conditions and differentiate at the single-cell level not only into mesenchymal cells but also cells of visceral mesoderm may be an ideal source of stem cells for treatment of genetic or degenerative disorders affecting cells of mesodermal origin.

Most insights into the molecular mechanisms underlying transformation by the p210(BCR/ABL) oncoprotein are derived from studies in which BCR/ABL cDNA was introduced into hematopoietic or fibroblast cell lines. However, such cell line models may not represent all the features of chronic myelogenous leukemia (CML) caused by additional genetic abnormalities and differences in the biology of cell lines compared with primary hematopoietic progenitor and stem cells. A primary human hematopoietic progenitor cell model for CML was developed by the transduction of b3a2 BCR/ABL cDNA in normal CD34(+) cells. Adhesion of BCR/ABL-transduced CD34(+) cells to fibronectin was decreased, but migration over fibronectin was enhanced compared with that of mock-transduced CD34(+) cells. Adhesion to fibronectin did not decrease the proliferation of BCR/ABL-transduced CD34(+) cells but decreased the proliferation of mock-transduced CD34(+) cells. This was associated with elevated levels of p27(Kip) in p210(BCR/ABL)-expressing CD34(+) cells. In addition, the presence of p210(BCR/ABL) delayed apoptosis after the withdrawal of cytokines and serum. Finally, significantly more and larger myeloid colony-forming units grew from BCR/ABL than from mock-transduced CD34(+) cells. Thus, the transduction of CD34(+) cells with the b3a2-BCR/ABL cDNA recreates most, if not all, phenotypic abnormalities seen in primary CML CD34(+) cells. This model should prove useful for the study of molecular mechanisms associated with the presence of p210(BCR/ABL) in CML. ispartof: Blood vol:97 issue:8 pages:2406-12 ispartof: location:United States status: published

[Comment on Yoder et al, page 1801][1]In this issue of Blood , Yoder and colleagues take a step forward in resolving the existing controversy on the origin and functional definition of endothelial progenitor cells (EPCs). The authors demonstrate that the mononuclear cell fraction of peripheral blood

Osteoblasts are one of the important cellular components of the niche for hematopoietic stem cells (HSCs) in mammalian bone marrow (BM). Integrin receptors not only play a key role in HSC adhesion within the BM niche but also transfer regulatory signals from the microenvironment to HSCs. Periostin (Postn or osteoblast specific factor-1; OSF-1) is expressed in osteoblasts in addition to many other tissues, and acts as a ligand for Integrin-αvβ3 (ITGAV-B3). We identified POSTN as an important regulator of the cell cycle in adult murine HSCs. POSTN inhibited culture induced proliferation of HSCs thereby decreasing the total number of cells following 2-5 day culture of primitive HSCs, identified as CD150+CD48-Lin-Sca-1+c-kit+ (CD150 KLS) cells with SCF and TPO, while increasing the proportion of long-term (LT-) HSCs. Culture for 5 days with POSTN decreased the short-term (ST-) engraftment of progeny of 200 CD150 KLS cells, while significantly increasing LT- engraftment of the donor derived cells. A significant fraction of CD150 KLS cells expressed ITGAV as well as ITGB3. POSTN did not affect proliferation of HSCs in vitro following blocking of ITGAV with neutralizing antibodies. Among the important cell cycle regulators, we found an increase in p27kip1 expression in HSCs. Preliminary studies on possible signaling mechanisms involved, showed that POSTN inhibits Akt phosphorylation, known to mediate inhibition of both expression and activation of p27Kip1. Intravenous infusion of recombinant POSTN protein significantly decreased proliferation of hematopoietic progenitors as shown by Brdu incorporation and Hoechst/Pyronin staining. Interestingly, POSTN infusion also led to an increase in the number of KLS as well as CD150 KLS cells in the BM. Studies on characterization of the hematopoietic system of Postn-/- mice are underway. To further determine the role of ITGAV in HSCs, we used blocking antibodies against ITGAV and performed homing and engraftment studies. No effect on either homing potential or engraftment of ST- and LT- engraftment was seen. However, the competitive repopulation of ITGAV- CD150 KLS cells was significantly lower that that of ITGAV+ CD150 KLS cells (isolated using non-blocking antibodies). Therefore, our studies confirm the importance of ITGAV expression on primitive HSCs as well as presents POSTN as an important cell cycle regulator in the hematopoietic system. Disclosures: No relevant conflicts of interest to declare.

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) have successfully been used as stem cells for both autologous and allogeneic transplants. However, little is known concerning the absolute number and phenotype of primitive progenitors, such as long-term culture-initiating cells (LTC-IC) in mobilized PBPC. The aim of our study was to evaluate the capacity of G-CSF to mobilize LTC-IC in the PB of normal individuals and to evaluate the phenotypic and functional characteristics of G-CSF mobilized LTC-IC. G-CSF was administered to 29 healthy volunteers at 7.5 micrograms or 10 micrograms/kg/d subcutaneously (SC) for 5 consecutive days and PBPC were harvested on day 6. Mobilization with G-CSF increased the absolute number of week 5 LTC-IC in PB 60-fold, while the number of CD34+ cells and committed colony forming cells (CFC) was increased sevenfold to 12-fold. The frequency of CFC and week 5 LTC-IC in CD34+ cells selected by fluorescence-activated cell sorter (FACS) from mobilized PBPC was 2 +/- 0.3-fold and 9 +/- 2.2-fold higher respectively than in CD34+ cells selected from unmobilized PBMNC. CFC were enriched in the CD34+ CD38+ and CD34+ HLA-DR+ populations. The absolute number of LTC-IC present in CD34+ CD38- and CD34+ HLA-DR- cells selected by FACS from either mobilized PBPC, unmobilized PBMNC or steady state bone marrow (BM) was similar (0.5% to 2%). In contrast to unmobilized PBMNC or steady state BM CD34+ CD38+ and CD34+ HLA-DR+ cells, which contain less than 0.1% LTC-IC, CD34+ CD38+ and CD34+ HLA-DR+ cells sorted from mobilized PBPC contained 0.5% to 5% of cells capable of sustaining hematopoiesis in long-term cultures for 5 weeks. However, 90% to 95% of LTC-IC present in mobilized CD34+ CD38+ and CD34+ HLA-DR+ cells were not able to sustain hematopoiesis for 8 weeks, while 30% of CD34+ CD38- and CD34+ HLA-DR- LTC-IC present in mobilized PBPC could sustain hematopoiesis for at least 8 weeks. This suggests that the majority of CD34+ CD38+ and CD34+ HLA-DR+ week 5 LTC-IC represent progenitors at an intermediate state of differentiation. We conclude that G-CSF effectively mobilizes LTC-IC in the blood of normal individuals. Although a fraction of these cells has functional characteristics similar to those of steady state PBMNC or BM LTC-IC, more than 85% of mobilized PBPC LTC-IC are CD34+ CD38+ and CD34+ HLA-DR+, capable of sustaining hematopoiesis for 5 weeks, but not for 8 weeks. The functional and phenotypic characterization of primitive and more mature populations of LTC-IC in mobilized PBPC should prove extremely useful in future studies examining the role of these progenitors in engraftment following transplantation. ispartof: Blood vol:88 issue:6 pages:2033-42 ispartof: location:United States status: published

Abstract 725 Directional migration is an important factor that determines homing of transplanted hematopoietic stem cells (HSC). The SDF-1α-CXCR4 axis is one of the most important determinants of this process. CD26, expressed on various cell types, leads to proteolytic cleavage of SDF-1α, which leads to the inactivation of its chemokine activity. We identified tissue factor pathway inhibitor (TFPI) as an inhibitor of CD26. Culture of murine bone marrow derived c-kit+Lin−Sca-1+ (KLS) cells or human umbilical cord blood derived Lin−CD34+ cells with TFPI significantly inhibited CD26 activity, which resulted in significantly increased migration towards SDF-1α. Moreover, TFPI treatment of murine KLS cells led to significant improvement in homing following intravenous injection and long-term reconstitution upon transplantation in competitive repopulation assays. We found that the effects of TFPI were mediated by the heparan sulphate proteoglycan, Glypican-3 (Gpc3). TFPI directly bound Gpc3; TFPI did not affect CD26 activity, migration or homing of Gpc3−/− KLS cells, but affected Gpc1−/− KLS cells similar to the wild type. Finally, KLS cells from Gpc3−/− but not Gpc1−/− mice homed significantly less following IV injection. Hence, we present a novel molecule that can be used in an HSC specific manner to enhance HSC migration, homing and engraftment. Disclosures: No relevant conflicts of interest to declare.

A recent transcriptome analysis of human umbilical cord blood and bone marrow (BM) CD34+CD33−CD38−ckit+Rholo (Rholo) vs. CD34+CD33−CD38−ckit+Rhohi (Rhohi) cells identified the gene RASSF8, to be more highly expressed in the hematopoietic stem cell (HSC) rich compartment (Rholo) versus committed progenitor cells (Rhohi). In a subsequent screen for the function of differentially expressed genes in hematopoiesis, the knockdown of RASSF8 using morpholinos in zebrafish embryos resulted in decreased blood formation and reduced expression of early (scl and gata1) and late (hbae1 and lcp1) hematopoietic markers. To test the function of RASSF8 in mammalian hematopoiesis, we overexpressed RASSF8 cDNA in lineage depleted murine bone marrow cells (Lin−) by retroviral transduction using the MSCV-RASSF8-IRES-GFP vector (rMIG-RASSF8). Sorted transduced GFP+ cells were cultured in serum-free medium (supplemented with SCF, TPO, Flt3L and Il-3) for three to five days. No significant differences were seen in overall cell expansion, cell death (7-AAD staining) or cell proliferation (thymidine incorporation assay and propidium iodide staining) between rMIG-RASSF8 transduced cells and cells transduced with the control vector (rMIG). However, we noted a significant accumulation of the stem cell enriched cKit+Lin−Sca1+ (KLS) population in rMIG-RASSF8 transduced cells (43+/−9%) compared to control cells transduced with rMIG (13+/−8%, p=0.003; n=4) after five days. The total number of colony forming cells (CFCs) produced by 750 rMIG-RASSF8 transduced Lin− cells (24+/−7) was significantly lower than in rMIG transduced cells (54+/−8, p=0.008; n=3). CFCs from rMIG-RASSF8 transduced cells were morphologically smaller and more compact, consistent with the morphology of primitive blast-forming colonies. Competitive repopulation assays using 25 000 rMIG-RASSF8 or rMIG transduced C57/Bl6 CD45.1 Lin- cells versus 100 000 C57/Bl6 CD45.2 mononuclear BM cells were performed to evaluate HSC engraftment potential. Four weeks after transplantation only 1+/−0.96% CD45.1 cells were found in the peripheral blood (PB) of the animals receiving rMIG-RASSF8 transduced cells vs. 23+/−12% CD45.1 cells in animals that had received rMIG transduced cells (p< 0.05; n=3). Likewise, PB analysis at 3 months demonstrated nearly no reconstitution of rMIG-RASSF8 transduced CD45.1 cells (1+/−1.7%) vs 30+/−23% rMIG transduced CD45.1 cells (p=0.02). In conclusion, constitutive overexpression of RASSF8 increases the retention/expansion of KLS cells in vitro, blocks differentiation of progenitors giving rise to CFC and inhibits engraftment of HSC. These studies thus suggest that short term overexpression of RASSF8 might ultimately lead to new methods of HSC expansion.

There is increasing evidence that the immune system can promote cytopenia in myelodysplastic syndromes (MDS) to a variable extent. The interaction between CD40 and its natural ligand CD40L (CD154) plays a critical role in the regulation of humoral and cellular immune responses. The aim of this study was to investigate the role of the CD40/CD40L interaction in the pathogenesis of MDS. For this purpose, we looked at the presence of the CD40 receptor and its ligand in MDS peripheral blood (PB) and bone marrow (BM), and we studied the in vitro effects of inhibition and stimulation of the CD40 receptor on MDS monocytes. 1) Expression of CD40/CD40L. We analysed PB samples of 40 patients with MDS (19 RCMD, 15 RCMD-RS, 3 RAEB-1, 3 5q- syndrome) and 29 age-matched control subjects by flow cytometry. We observed that MDS patients have significantly higher percentages of circulating CD40+/CD14+ cells (8.34 % +/− 1.28 vs. 2.78 % +/− 0.31, p=0.0004) and CD40L+ T helper cells (4.44 % +/− 0.52 vs. 2.16 % +/− 0.22, p=0.001). In addition, we found higher percentages of CD40+/CD14+ cells (9.47 % +/− 1.96 vs. 3.53 % +/− 0.63, p=0.03) and CD40L+ T helper cells (5.13 % +/− 0.86 vs. 2.60 % +/− 0.53, p= 0.008) in BM of 22 patients (1 RA, 11 RCMD, 4 RCMD-RS, 5 RAEB-1 and 1 5q- syndrome) compared to 11 donor samples. 2) Stimulation of CD40. Selected CD14+ cells from PB of 21 patients (1 RA, 12 RCMD, 6 RCMD-RS, 2 RAEB-1) and 25 control subjects were stimulated for 24h with LPS or an agonistic anti-human CD40 antibody (clone 64, PanGenetics NV, The Netherlands). We observed that anti-CD40 induced significantly higher TNF-alpha levels in patients than in controls (436 vs. 64 pg/mL, p=0.008), whereas LPS induced comparable TNF-alpha levels. 3) Blocking of CD40. We cultured 5x105 BM mononuclear cells of 22 patients (1 RA, 12 RCMD, 3 RCMD-RS, 4 RAEB-1 and 2 5q- syndrome) in methylcellulose supplemented with growth-factors in the presence or absence of 5D12 (antagonist chimeric monoclonal anti-human CD40 antibody, PanGenetics NV, The Netherlands). The presence of 5D12 significantly increased colony-formation at day 14 (140 vs. 112, p=0.002). 5D12 did not affect colony formation in CD14-depleted cultures, nor in controls. In conclusion, we have demonstrated elevated levels of CD40 receptor on PB and BM monocytes of patients with MDS compared to age-matched control subjects. In addition, we have demonstrated increased levels of its ligand CD40L on T helper cells in PB and BM. Stimulation of CD40 on monocytes from patients induces significantly higher TNF-alpha levels, a cytokine known to inhibit colony formation. Moreover, we demonstrated that inhibition of this CD40 receptor with 5D12 can increase colony formation and that its effect is specific monocyte-dependent. Our observation together with the proven safety of 5D12 in phase 1 trials for patients with inflammatory bowel disease, might justify a phase I trial of 5D12 to improve cytopenia in patients with MDS.

We investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5′ untranslated region (UTR) (SurUTR) and sequences flanking the initiation codon (SurATG) of zebrafish survivin-1 gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:GFP) embryos. In embryos co-injected with SurUTR and SurATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5′UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Hematopoietic cell development at 18 hpf, before the onset of circulation, was unaffected in the survivin MO injected embryos. In conclusion, survivin is involved in angiogenesis during zebrafish development and is under VEGF regulation.

The Wnt/β-catenin pathway is important for maintenance of the undifferentiated state of human embryonic stem cells (ESCs), hematopoietic stem cells (HSCs) and other stem cells. We here tested if β-catenin activation, by inhibiting GSK3 β with BIO (6-Bromoindirubin-3′-oxime), affects the differentiation status of murine multipotent adult progenitor cells (mMAPCs). mMAPCs were cultured with or without 0.1–2 μ M BIO, LIF, EGF and PDGF-BB, at low density (plating density of 100/cm2 and cells passed every 2 days) or at high confluency (like ESCs). Quantitative RT-PCR (Q-RT-PCR) was done weekly for the ESC-specific pluripotency transcripts, Oct4, Rex1, Nanog, UTF1 and E-Ras, and lineage commitment genes, vWF, VE-cadherin, Sox1, Nestin, CK19, Albumin, HNF3β and AFP. After 2–4 weeks, cells were analyzed by FACS for ESC/MAPC specific antigens, and by immunohistochemsitry for Oct4, β-catenin and E-cadherin protein. Furthermore, cells were differentiated into endothelial cells and hepatocytes by using VEGF-A and HGF/FGF4, respectively for 2 weeks. Q-RT-PCR was used to demonstrate lineage differentiation. BIO caused a dose-dependent clustering of mMAPCs from 4 days after treatment at both cell densities. FACS phenotype of low- and high-density mMAPCs was not affected by BIO. Nanog mRNA was not detected in any of the cell populations. However, Oct4, Rex1, vWF, and CK19 mRNA levels decreased in a BIO dose-dependent manner in mMAPCs maintained at low density, while they acquired higher levels of Sox1 mRNA (P=NS). At high-density, Oct4 and Rex1 mRNA level decreased in a BIO dose-dependent manner for the first week. On the other hand, 0.1 μ M BIO-treatment induced higher levels of Oct4 and Rex1 in cells maintained at high density. β-catenin and E-cadherin protein was highly expressed in clusters of low-density mMAPCs induced by BIO as well as BIO-treated mMAPCs maintained at ESC densities. In both cell densities, only mMAPCs treated with 1 or 2 μM BIO contained large dense mMAPCs clusters that expressed very high levels of β-catenin and E-cadherin. There was no statistical difference of differentiation potential between BIO conditions and cell densities. BIO may not allow culture of mMAPCs without loss of Oct4 as it did for ESCs, but induces clusters like ESCs in a BIO-dose dependent manner. High concentrations of BIO enhances β-catenin and E-cadherin protein expression. BIO may induce lineage commitment of mMAPCs. Despite loss of Oct4 mRNA and protein, MAPCs treated with BIO continue to have the ability to differentiate. Ongoing studies are testing whether BIO treated cells maintained at high density also maintain in vivo engraftment and differentiation potential.

Hematopoietic stem cells (HSCs) are functionally defined by their capacity to home to the bone marrow microenvironment, proliferate and differentiate to restore normal hematopoiesis in a myeloablated recipient; however, the molecular determinants of these processes are not well understood. By comparing the gene expression profiles of highly purified human HSC-enriched and HSC-depleted cell populations, and subsequently validating the hematopoietic function of a subset of these differentially expressed genes using zebrafish (Danio rerio), we previously identified human Sprouty 1 (SPRY1), an evolutionarily conserved antagonist of RTK signaling, as a potential regulator of mammalian HSC development and/or cell-fate decisions. To directly assess the role of SPRY1 in mammalian HSC and hematopoietic progenitor cell (HPC) function, we constructed a dual-promoter lentiviral vector to co-express SPRY1 and green fluorescent protein (GFP) (SPRY1-LV) and a control lentiviral vector to express GFP alone (GFP-LV) in the CD34+ fraction of human umbilical cord blood (UCB). While the enforced expression of SPRY1 in CD34+ UCB cells had no effect on the frequency or morphology of colonies generated in short-term in vitro colony-forming cell (CFC) assays (SPRY1-LV = 27.6 ± 12.3% and GFP-LV = 28.4 ± 20.0%; n = 3), it profoundly inhibited the capacity of UCB CD34+ cells to engraft in the bone marrow NOD-SCID mice in vivo. The estimated frequency of week 11 SCID-repopulating cells (SRC) (± 1 standard error) for SPRY1-LV (n = 13 mice) and GFP-LV (n = 15 mice) cells was 1 in 12,678 cells (6,167 – 26,064) and 1 in 3,412 cells (2,272 – 5,124), respectively, as determined using limiting dilution conditions and Poisson statistics. Furthermore, in 14 day cultures designed for the ex vivo expansion and/or maintenance of primitive hematopoietic cells, ectopic expression of SPRY1 in CD34+ UCB cells dramatically inhibited the expansion of total nucleated cells (SPRY1-LV = 37.9 ± 10.1 fold; GFP-LV = 71.1 ± 5.8 fold; n = 3; p

Sorting nexin 5 (SNX 5) is a member of the sorting nexin family, a diverse group of cellular trafficking proteins unified by the presence of a phospholipid binding domain (PHOX) which is involved in protein-protein interactions. While the function of SNX5 is unknown, there is evidence from yeast two hybrid studies that SNX5 binds to FANCA, and SNX5 is one of the 20 genes located on a region on mouse chromosome 2 associated with accelerated stem cell aging. We have recently shown that SNX5 is significantly higher expressed in CD34+CD33-CD38-Rho+ (Rhohi) cells from umbilical cord blood (UCB) and bone marrow (BM), depleted of SCID repopulating cells (SRCs) than in CD34+CD33-CD38-cKIT+ Rho- cells (Rholo), containing all SRCs. A large scale high throughput functional analysis was carried out in zebrafish and genes differentially expressed between Rholo and Rhohi cells were knocked down using morpholino (MO) antisense oligonucleotides. Morpholinos are a useful tool which can specifically inhibit the translation of target mRNA. 16 out of 70 genes knocked down, including snx5, demonstrated decreased circulating blood in the zebrafish injected. Further analysis has shown that MO knock down of snx5 in zebrafish (n=152) results in hematopoietic failure with normal vasculature, with normal expression of scl and gata-1 by in situ hybridization, indicating a defect at or beyond the hematopoietic stem cell stage. Quantitative RT-PCR was used to further confirm the phenotype. Levels of hemoglobin, l-plastin and myeloperoxidase mRNA in snx5 morphants compared to uninjected controls, were significantly lower, consistent with a multi-lineage hematopoietic differentiation defect. The decrease in circulating blood could be partially rescued by overexpressing human SNX5 cDNA, confirming specificity of the morphant phenotype. To determine whether SNX5 might be linked to Fanconi anemia (FA), we measured levels of SNX5 mRNA and protein in EBV transformed cell lines from patients with Fanconi-complementation group A (FANCA), -complementation group C (FANCC) or unknown complementation group (FANC-NX). We have found no significant difference between SNX5 mRNA expression in 11 cell lines compared to Jurkat controls. However, levels of SNX5 protein appeared lower in FANCA cells suggesting a possible role of SNX5 in FANCA, which is being evaluated further. Ongoing studies evaluating the role of SNX5 in normal human hematopoiesis will also be presented.

Here, we show that Multi-potent Adult Progenitor Cells (MAPCs) can be derived from adult and fetal swine bone marrow. Swine MAPC (swMAPC) contain initially multiple different progenitors and mature cells; however, cultures are homogenous by 50 population doublings and can grow past 150 population doublings. SwMAPCs are CD44, CD45, and MHC-I, II negative, express Oct3a/4 at levels close to those seen in embryonic stem cells, and have telomerase activity leading to no telomere shortening with expansion. We also have very solid evidence that swMAPCs differentiate into most mesoderm, neuroectoderm, and endoderm lineages as demonstrated by a significant up-regulation of transcription factors and other lineage specific proteins in a time dependent fashion similar to development, measured by Q-RT-PCR as well as immunohistology. In addition, swMAPCs induced to the endothelial lineage form vascular tubes, to the hepatic lineage produce albumin and urea, and to the smooth muscle lineage display significant calcium flux in response to smooth muscle agonists. In addition, we are investigating the swMAPC differentiation into cardiomyocytes. Preliminary data indicates swMAPCs express TBX5, Nkx2.5, and GATA6 by day 7 at significant levels. As we have seen for rodent MAPCs, when swMAPCs are allowed to grow at high density, Oct3a/4 levels drop to undetectable ranges, the cells take on a mesenchymal stem cell (MSC) phenotype, and the differentiation potential is lost. When replated under low density conditions, oct3a/4 expression or differentiation capacity can not be re-induced, and cells remain MSC like. Therefore, we suggest that Oct3a/4 is not an in vitro culture phenomena but is already present in swMAPCs and cannot be recovered once lost with standard culturing techniques.

Summary The clinical utility of an ex-vivo expanded stem cell population is dependent on both safety and efficacy. As an initial step toward ensuring that expanded adult stem cell populations are safe, we harvested bone marrows from four different strains of rat and cultured mononuclear cells under low serum media shown previously to allow for propagation of Multipotent Adult Progenitor Cells (MAPCs). We then determined the karyotype and tri-lineage potential for these expanding populations at numerous population doublings. We observe that an average of 6.7% of cells within these cultures are tetraploid and 2.7% of cells show other karyotypic alterations. In spite of this low level of karyotypic instability, no persistent chromosomal abnormalities are present in these cultures after more than 100 population doublings. As measured by qPCR and indirect immunofluoroescence for lineage specific marker genes, these cells have tri-lineage potential and are therefore considered to be MAPCs. These data support and extend previous conclusions that MAPC cultures with a nearly normal karyotype can be routinely established and expanded from rat bone marrow providing important standards for performance and safety in pre-clinical disease models.

In an effort to identify novel regulators of hematopoiesis, we identified a Sprouty family member, SPRY1, significantly enriched (>1.5 fold, p-value < 0.03) in human hematopoietic stem cells (HSC) compared to more committed hematopoietic progenitor cells (HPC) using Affymetrix-based global gene expression profiling (see Eckfeldt et al. abstract). Predominantly FGF, but also VEGF, and EGF signaling induces expression of Sprouty family members (SPRY1-4). SPRY proteins form heterodimers with other Sprouty members along with homodimers. They and are intracellular antagonists and agonists of receptor tyrosine kinase signaling via the RAS/MAPK pathway. SPRY proteins are best characterized as antagonists of the FGF, EGF, and VEGF pathways. As part of a high-throughput functional analysis of the genes identified by human gene array in zebrafish, we evaluated the role of the only identified zebrafish homologue for Sprouty, SPRY4, in zebrafish hematopoiesis. Using morpholino (MO) antisense technology we knocked-down expression of zebrafish SPRY4 during early zebrafish development in a double transgenic GATA1:dsRed/Fli1:eGFP zebrafish line. The SPRY4 morpholino injected fish (SPRY4[superscript]MO[/superscript]) showed an almost complete loss of blood with normal vasculature, which was confirmed by injection of a second unique morpholino designed against SPRY4. [italics] In situ [/italics]hybridization of the hematopoietic markers SCL, GATA1 and Ikaros were all significantly reduced or absent at multiple developmental stages in the SPRY4[superscript]MO[/superscript] embryo. Together with the normal development of the vasculature, this data indicates SPRY4 acts early in hematopoiesis and after the specification of the hemangioblast. [italics] In situ[/italics] hybridization for SPRY4 demonstrated expression in the otic vesicle, tail bud and lateral plate mesoderm, the first site of hematopoietic stem cells. Coinjection of SPRY morpholino and low doses of VEGF morpholino did not rescue the SPRY blood phenotype, indicating that the blood phenotype is not likely through the VEGF pathway. Previous gain-of-function experiments injecting SPRY4 mRNA showed an expansion of hematopoietic progenitors (Furthauer et al. Development 2001), a phenotype we confirmed with overexpression of SPRY4 cDNA. Experiments are ongoing to overexpress human SPRY1 or zebrafish SPRY4 cDNAs in mammalian cells to determine if the hematopoietic phenotype correlates with the zebrafish data. Additional genes differentially expressed in human HSC and HPC evaluated via the high throughput zebrafish screen will also be presented. This is the first description of functional validation of a gene from a human microarray in zebrafish, an essential step in obtaining meaningful in vivo data from global gene profiling studies.

We have previously reported that multipotent adult progenitor cells (MAPCs) can be isolated from the bone marrow of rodents and humans. Here we describe isolation of similar cells, fetal swine MAPCs (fsMAPCs) from fetal swine bone marrow (BM). BM was obtained from 5 fetal swine donors. BM mononuclear cells (BMMNCs) were plated into fibronectin-coated T150 flasks at 2x104 cells/cm2 in 58% LG-DMEM and 40% MCDB-201, supplemented with ITS, LA-BSA, 10−8 M dexamethasone, 10−4 M ascorbic acid 2-phosphate, 2% fetal bovine serum (FBS), 10ng/ml EGF and 10ng/ml PDGF-BB. Small clusters of cells appeared by day 5, were detached with 0.25% trypsin-EDTA on day 7 and sub-cloned at 1 cell/well in 96 well plates. Once populations of small cells appeared (40% of wells), they were culture expanded by plating at 500 cells/cm2. Cells were replated at 500cells/cm2 once they divided 2–3 times. Population doubling (PD) time was 24h for the initial 30–40 PDs, and 48 h at higher PDs. 3/5 fetal swine populations have been maintained for more than 100 PDs without cytogenetic abnormalities. For two of the populations, telomere length was evaluated on day-0, 40PDs and 80PDS, and no telomere shortening was seen. The phenotype of cultured cells before 30 PDs is CD45 and MHC class I negative, CD44 and MHC class II positive, while the phenotype after 60 PDs was CD44, CD45, MHC class I and II negative. FsMAPCs, cultured on Matrigel with fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), differentiated into epitheloid cells that expressed cytokeratin 18 (CK18), hepatocyte nuclear factor-1 (HNF-1), albumin protein on day 14–28. Cells differentiated for 21 days secreted 10pg/cell/hour urea (Urea Production Test), as is seen for primary hepatocytes. More functional tests for hepatocytes are ongoing, including LDL uptake, glycogen storage, cytochrome-p450 activity. When treated with vascular endothelial growth factor (VEGF), fsMAPCs differentiated into endothelium-like cells that expressed CD31, CD34, VEGF-Receptor, vascular endothelial cadherin (VE-Cadherin) and von willebrand factor (VWF) protein and mRNA at day 14–28, formed vascular tube on extracellular matrix and took up LDL. Neuroectoderm differentiation studies are ongoing. Finally, we have preliminary evidence that fsMAPCs may differentiate into cells with cardiomyocyte markers. FsMAPCs, plated at 1,500 cells/cm2, were cultured with dickkopf-1 (Dkk-1) and 0.75% dimethyl sulfoxide (DMSO) for 1 day, followed by bone morphogenetic protein-2 (BMP-2), FGF-8, FGF-4, Dkk-1 for 21 days. Cells acquired muscle morphology after d6. By day 21, cells expressed cardiac Troponin T, cardiac myosin heavy chain (cMHC), phospholamban protein, and Nkx2.5, Cardiac Troponin T, cMHC, GATA-4, atrial natriuretic peptide (ANP) mRNA. Although suggestive that the cells may differentiate into cardiomyocytes, they did not beat regularly. Electrophysiology studies are ongoing to determine whether the cells have functional characteristics of cardiomyocytes.

MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion>> Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.

We have previously described the isolation of MAPCs, from human, mouse and rat bone marrow. MAPCs can differentiate into most mesodermal cell types as well as cells with neuroectodermal and endodermal features. However, culture of MAPCs remains laborious and difficult. To address the difficulties in MAPC culture initiation and maintenance, we performed multi-parameter analysis of different steps along the way. First, we evaluated whether collection of bone marrow influences isolation. We found that cells that initiate MAPC cultures appear to be present in higher frequency near growth plates, and near the endosteum. Cells from the middle part of the bone shaft generally can only be cultured short-term and rarely form MAPC lines. Hence, including the ends of the bones and very vigorous flushing of the marrow from the cavity are important. A second parameter we evaluated is cell density both during the initial phases of isolation, i.e. before depleting cells by MACS column, and afterwards. Initial plating before column depletion should be done at relatively higher density, i.e. between 2 and 3x104 cells/cm2. However, after MACS depletion, cell density needs to be kept between 2-5x102 cells/cm2. As MAPC are inclined to form clusters, and it is imperative that cell-cell contact is kept at minimum, it is not sufficient to assure global cell density, but assure that cells are distributed evenly throughout culture vessels. However, maintenance at too low cell densities significantly inhibits proliferation and eventually cells die. This suggests first that paracrine factors produced by the cells are important for their self-renewal, but that cell-cell contact factors, either alone or by secondary release of cytokines, induce cell differentiation. Initial evaluation of gene array studies comparing MAPC maintained at ideal density vs. allowed to grow at high density indicates differential expression of the main three developmental signal pathways, TGFb/BMP, Notch and wnt signaling. These are now being evaluated to determine whether relaxation of the density requirement can be done without loss of potency and self-renewal ability. We also tested the effect of trypsinization. MAPC are very lightly attached cells, whereas differentiating cells are larger and more adherent. Hence short term trypsinization (less than 30sec) and a few gentle taps loosens up the small MAPCs whereas it does not detach the larger cell population. Long-term exposure to trypsin is furthermore toxic to MAPCs; therefore immediate dilution of trypsin with a large volume of MAPC medium is required. On a practical note, as MAPC are small (9–12μm) and are present as a lose layer on top of the pellet, one needs to take care that they are not inadvertently disposed off when centrifuging. We have also found that different fetal calf serum (FCS)-lots significantly affect MAPC growth. In general, FCS that causes faster growth induce differentiation, loss of multi-lineage differentiation potential, and induce at much higher frequency cytogenetic abnormalities, including tetraploidy and aneuploidy. This is more common in mouse than rat MAPC cultures. Hence, as has been shown for hematopoietic progenitors, MSCs and ESCs, screening of serum lots is needed to optimize MAPC culture. A final vital factor is the CO2 and O2 concentration in which cells are maintained. MAPC die under alkaline conditions, hence optimally CO2 concentrations are kept between 5.5– 6%C02. Likewise, we have started to use 5% O2, as this decreases the frequency of cytogenetic abnormalities that occur. Whether this also supports better self-renewal is currently being studied.

Smooth muscle cells (SMC) play a key role in vascular development and morphogenesis of blood vessels defined by a noncontractile, or synthetic SMC phenotype. Mature SMCs respond to vascular injury/repair by phenotypic modulation to a synthetic state, and hence are also involved in various clinical disorders including atherosclerosis and cancer. We have induced relatively homogenous differentiation of SMC-like cells from multipotent adult progenitor cells (MAPC). MAPCs have been isolated from murine, rat and human bone marrow. MAPCs can differentiate into mesodermal, neuroectodermal, and endodermal cell types. Here we report that serum free medium supplemented with PDGF-BB and TGF-b1 allows differentiation of rat MAPCs to a population of cells expressing smooth muscle specific markers, as determined by Q-RT-PCR (a-SMC actin, calponin, SM-22a, myocardin, and smooth muscle heavy chain myosin),and immunohistochemistry (a-SMC actin staining in >90% cells and calponin staining in ~50% of cells, co-localization localized with defined stress fibers). Such SMCs can subsequently be passed for at least 15 passages (26 doublings) in the presence of FCS and PDGF-BB and TGF-b1, without loss of phenotypic characteristics. RT-PCR analysis for genes of other mesodermal lineages, including cardiac muscle and endothelium expressed genes (Nkx, Mef-2D, PCAM, and vWF) was negative. To characterize the function of the SMC-like cell population, calcium handling and in-vitro remodeling properties were studied. Passage 4 SMC-like cells responded to endothelin-1, norepinepherine, oxytocin, arg-vasopressin, and bradykinin with an increase in intracellular calcium ion concentration, consistent with a smooth muscle phenotype. To assess the remodeling potential, passage 4 cells were entrapped into fibrin matrix hemispheres as previously described for neonatal rat SMC. After 5 weeks in culture, hydroxyproline assays quantified total collagen as (86.8 +/− 9.3 ug) approximately 50% the amount reported using neonatal SMCs. In addition, the remodeled hemispheres demonstrated defined mechanical properties (tensile strength= 0.20 MPa, modulus = 0.65 MPa), similar to what we have previously observed when neonatal rat SMCs are entrapped in fibrin disks. Masson Trichrome staining demonstrates areas of tissue organization characterized by fibrils and collagen production. While further characterization of this population is needed to determine their developmental plasticity, the data is suggestive of a vascular SMC-like cell population derived from MAPCs that demonstrates functional smooth muscle characteristics and could be used to tissue engineer blood vessels.

Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

MAPC are non-hematopoietic stem cells with the capacity to form most, if not all, cell types of the body. To date, the observations of homing of the MAPC have been limited to post mortem analyses. As MAPC may be useful in cellular therapies, our goal was to map their biodistribution in live organisms. To determine the real-time organ-specific homing pattern of donor MAPC, MAPC (from BM of C57BL/6J-rosa26 mice) were co-nucleofected with cDNAs encoding the red fluorescent protein DsRed2 and luciferase, using the Sleeping Beauty (SB) transposon system. Non-viral gene transfer mediated by SB is potentially advantageous to viral gene transfer because transposons may be less immunogenic since no viral proteins are present, and they are relatively easy to produce. DsRed2 and luciferase genes were cloned into plasmid vectors containing the transposase recognition sequences flanking the reporter genes (pT/CAGGS-DsRed2; pT/CAGGS-Luciferase). MAPC (106) were co-nucleofected (Amaxa, setting T-20, buffer T) with 5mcg of each marker plasmid and the SB transposase plasmid (p/CMV-HSB2) at a 1:50 ratio. 19% of MAPC expressed DsRed2 7 days after nucleofection. The MAPC were FACS sorted (1 cell per well) for cells with the highest DsRed2 expression. All MAPC tested expressed both DsRed2 and luciferase, suggesting that co-nucleofection is an efficient means of delivery of two plasmids. Two transgenic MAPC clones selected for further analysis were confirmed to be euploid by cytogenetic analysis, and maintained differentiation potential into the three germ layers. To verify transgene integration by transposition, the genomic sites of transposon integration were determined using splinkerette PCR. In the genome of MAPC clone 1, DsRed2 transposed in two sites on chromosome 5. One integration site (5qA3) was in the 3′ untranslated region of activin receptor interacting protein 1 (Acvrinp1). In clone 2 DsRed2 transposed into a single site on chromosome 10, in an intron of a gene termed SHPRH, which encodes a putative protein with SNF2/helicase and PHD-finger domains. To investigate the real time kinetics of MAPC population after infusion, 5 x 106 DsRed2 and luciferase positive MAPC (clone 2) were infused via tail vein into 8-week-old Rag2/IL-2Rgc−/− mice (T-, B- and NK-immunodeficient mice were used as a recipient to minimize the likelihood that the host would reject donor MAPC). Using whole body imaging (Xenogen) we were able to follow the distribution of the luciferase-marked MAPC over a period of 10 weeks. In addition, using DsRed2 expression the donor MAPC-derived cells in whole lung and in lung cryosections were identified. In summary, we show for the first time stable gene expression in adult stem cells using Sleeping Beauty transposon mediated non-viral gene transfer. These results show that MAPC-based cellular therapies can be monitored in vivo and suggest that transposon-based technology may be an attractive alternative to viral based gene delivery and therapy.

Cyclin dependent kinase inhibitors (CDKIs), p27Kip1 and p21Cip1, function as cell cycle inhibitors when located in the nucleus. When localized to the cytoplasm, these CDKIs can function as anti-apoptotic molecules by sequestering/preventing the activation of pro-apoptotic proteins such as ASK1 and procaspase-3. Our lab has reported elevated cytoplasmic CDKIs and decreased nuclear CDKIs in hematopoietic cells expressing BCR/ABL, the oncogene found in more than 90% of cases of chronic myeloid leukemia. Within the past decade, STI571 has been shown highly promising for CML treatment. However, there is increasing evidence suggesting that the drug might function more as a suppressor of proliferation and less as a promoter of cell death. In the current studies, we differentiate the effect of STI571 on proliferation vs. survival by tracking the subcellular increase/decrease in CDKIs using MO7e cells engineered to express BCR/ABL. To determine if a correlation exists between STI571 resistance and levels of cytoplasmic anti-apoptotic CDKIs, we also investigated changes in levels of nuclear vs. cytoplasmic CDKIs in LAMA84 -S (sensitive to STI571) vs. LAMA84-R (resistant to STI571). And lastly, we tested whether activation of TRAIL would enhance cell death in STI571-resistant cells. STI571 treatment increases nuclear CDKIs, correlating directly with a decrease in proliferation of MO7e/p210 cells. However, the high levels of cytoplasmic CDKIs in MO7e/p210bcr/abl was not modulated following STI571 treatment and cell death was not prominent, unless growth factors were removed. Moreover, cytoplasmic p21Cip co-immunoprecipitated with ASK1 and procaspase 3. When compared with LAMA-S cells, LAMA-R cells expressed even higher levels of cytoplasmic CDKIs. Treatment with STI571 decreased cytoplasmic CDKIs in LAMA84-S cells and resulted in cell death. As hypothesized, LAMA84-R cells did not show reduction in cytoplasmic CDKIs and did not enter apoptosis. However, when treated with STI571 and TRAIL, LAMA84-R cells showed a decrease in cytoplasmic CDKIs, and increase in apoptosis. Based on these observations, we conclude that: 1. BCR/ABL expression reduces nuclear CDKIs but increases cytoplasmic CDKIs. 2. STI571 treatment restores nuclear CDKIs and reduces cell proliferation of BCR/ABL expressing cells under physiological conditions. 3. Treatment of STI571+TRAIL reduces cytoplasmic CDKIs and increases cell death of BCR/ABL expressing cells, most notably, the STI571-resistant cells. In conclusion, we show that the imbalance between nuclear (cell cycle inhibitor) and cytoplasmic (cell survival enhancer) CDKIs exist in BCR/ABL-hematopoietic cells.

We generated a homogeneous population of cells with cytotoxic activity termed “adherent lymphokine-activated killer” (ALAK) cells from the peripheral blood of nine patients in the chronic phase of Ph1 positive chronic myelogenous leukemia (CML). The selective enrichment of CML ALAK cells depended on their propensity to adhere to plastic and proliferate when cultured in the presence of recombinant interleukin-2 (rIL-2) for 14 days. Culture of peripheral blood mononuclear cells under these conditions resulted in growth of a uniform population of cells with morphologic characteristics of large granular lymphocytes. The NKH1+/CD3- phenotype associated with IL-2-stimulated natural killer (NK) cells was present on 79% +/- 9% of cells. Absence of colony formation in conditions promoting the growth of CFU-GEMM indicated that the CML ALAK population was not contaminated with viable hematopoietic progenitors. Cytogenetic analysis of the CML ALAK population revealed 119/120 Ph1 negative metaphases and l/120 Ph1 positive metaphase in six patients. Southern blot analysis of CML ALAK failed to demonstrate a bcr gene rearrangement in seven patients known to have a bcr gene rearrangement in myeloid cells. Comparison of ALAK populations derived from peripheral blood of CML patients and normals revealed similar cytotoxicity against the NK-sensitive K562 cell line (104 +/- 36 LU v 88 +/- 19 LU; P = NS) and the NK-resistant Raji cell line (93 +/- 26 LU v 98 +/- 28 LU; P = NS). The proliferative capacity of CML ALAK cells (101 +/- 33 fold expansion) exceeds the growth potential of the normal ALAK cells (22.3 +/- 3 fold expansion; P = .02). Direct comparison of equal numbers of CML ALAK cells and a CML LAK cell population produced by incubation of peripheral blood mononuclear cells in rIL-2 for 14 days without adherence revealed that the CML LAK population had significantly lower lytic activity against K562 and Raji cell lines. We are able to expand CML peripheral blood mononuclear cells to provide a population of ALAK cells with potent cytotoxic activity. The CML ALAK population is relatively homogeneous, not contaminated with viable stem cells, not derived from a malignant lineage, and more cytotoxic than equal numbers of CML LAK cells. Further studies are underway to determine if this ALAK population may be effective in autologous killing of chronic myelogenous leukemia stem cells.