Human Hemopoietic Cells Research Articles

301. CID-Mediated In Vivo Selection: Heterogeneity among Large Animal Models

Animal models are indispensable tools for probing physiological processes, pathological perturbations, and for developing new therapies. Ultimately, animal models must provide useful guideposts for developing gene therapy approaches that are eventually proven effective in humans. Significant differences in results using various gene therapy animal models have been described. However, little or no information exists regarding differences that exist among the available large animal models. Here we evaluated, in the hemopoietic cells of baboons, a system for selection that has previously demonstrated activity in mice, dogs and humans. This system employs a derivative of the thrombopoietin receptor (F36Vmpl), that is conditionally activated in the presence of a small molecule drug called a chemical inducer of dimerization (CID). Whereas cultured mouse, dog and human hemopoietic cells all respond to the mpl signal, we observed only a minor and variable response to the mpl signal in the cultured cells of baboons. Similarly, we have noted significant CID-dependent rises in the frequency of transduced hemopoietic cells in mice and in dogs, however here we show that responses to CID administration in three baboons were modest and variable. A number of factors may account for the disparity between baboons and other species. First, we have noted that GFP expression is attenuated in the red cells of baboons transplanted with the F36VmplGFP vector compared to red cells derived from mice, dogs and humans transduced with the same vector. Since red cells expressing F36Vmpl respond especially well to CID administration in the mouse and dog, an inadequate level of F36Vmpl expression may account for the absence of a red cell response in the baboons. Of note, placing F36Vmpl directly downstream of the LTR (rather than downstream of the IRES) failed to generate more prominent hematological responses to CID administration. A second issue pertains to the susceptibility of the baboon cells to proliferate in response to the murine mpl signal. As noted above, significant CID dependent growth occurs in transduced hemopoietic cells from mice, dogs and humans in response to the murine mpl signal. Whether baboon cells would have responded better to signals emanating from baboon mpl is unknown, however it is noteworthy that mouse and human mpl are indistinguishable with respect to their ability to induce growth of transduced human cord blood cells. A third set of considerations pertains to the optimal concentration of CID necessary to trigger cell growth. Head-to-head comparisons of transduced dog, baboon and human CD34+ cells point to differences in the relative thresholds for triggering mpl-induced growth, and differences between the species with respect to the concentrations at which excessive amounts of CID inhibit cell growth due to excessive occupancy of the drug binding domains. Finally, we have noted major differences in the pharmacokinetic profiles of AP20187 in baboons compared to dogs. These findings have general implications for the evaluation and development of new strategies for gene therapy.

Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells

A new in vitro way of scoring the potential (proliferative- differentiative) of human haemopoietic stem cells (HHSC) is presented. We have applied it in the investigation of HHSC behaviour in normal bone marrow specimens under culture conditions. This system describes a modification of some existing culture assays for primitive HHSC function, and proliferation of the cobblestone area forming cell (CAFC) was determined by counting the cobblestone areas (CA) produced every week. The main steps of the assay are: (a) culture of MNC fraction on pre-established fibroblastic confluent layers from the M2.10B4 mouse cell line; (b) weekly counting of CA over a period of 5 weeks; (c) weekly in situ observation of CA morphology; (d) enumeration of secondary progenitors per CA, in the 5th week. The CAM index, expressing the value of the CA kinetic line slope and the number of secondary progenitors per CA were evaluated as factors indicating HHSC proliferative and differentiative potential, respectively. This culture system has the potential to serve as an in vitro assay of human stem cell transplantation. It could be applied to evaluating the potential of HHSC contained in haemopoietic collections of diverse origin, only in combination with measurements of the starting number of CAFC.

The human granulocyte/macrophage colony-stimulating factor receptor alpha2 isoform influences haemopoietic lineage commitment and divergence.

A number of alternatively spliced isoforms of haemopoietic growth factor receptors (HGFRs) have been described, but their role in human haemopoiesis remains undetermined. We have investigated the relative expression of the alpha1 and alpha2 isoforms of human granulocyte/macrophage colony-stimulating factor receptor (hGM-CSFR) during haemopoietic cell differentiation, and have shown that both subunits are independently regulated during differentiation of CD34+ human haemopoietic progenitor cells. To further investigate these ex-vivo observations, we established a series of murine FDCP mix cell lines, which, as a consequence of the ectopic expression of alpha1 or alpha2 hGM-CSFR, demonstrated differential differentiation responses to hGM-CSF. In this model system, hGM-CSFR-alpha2-expressing cells showed increased hGM-CSF-mediated erythroid/megakaryocytic differentiation compared with hGM-CSFR-alpha1-expressing cells.

Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution

Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described. Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity. Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC(s), which do not possess secondary repopulation capacity. We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation.

Hyaluronan is synthesized by primitive hemopoietic cells, participates in their lodgment at the endosteum following transplantation, and is involved in the regulation of their proliferation and differentiation in vitro

AbstractThe localization of adult hemopoiesis to the marrow involves developmentally regulated interactions between hemopoietic stem cells and the stromal cell–mediated hemopoietic microenvironment. Although primitive hemopoietic cells exhibit a broad repertoire of adhesion molecules, little is known about the molecules influencing the site of cell lodgment within the marrow following transplantation. However, our recent studies indicate that hierarchically dependent patterns of migration of transplanted hemopoietic cells result in the retention of primitive cells within the endosteal and lineage-committed cells in the central marrow regions. Herein, we now demonstrate that these 2 subpopulations exhibit a striking difference in the expression of a cell surface adhesion molecule, with populations enriched for murine and human hemopoietic stem cells expressing the carbohydrate hyaluronic acid (HA). Furthermore, the presence of this glycosaminoglycan appears critical for the spatial distribution of transplanted stem cells in vivo. In addition, we also demonstrate that the binding of HA by a surrogate ligand results in marked inhibition of primitive hemopoietic cell proliferation and granulocyte differentiation. Collectively, these data describe an important yet previously unrecognized role for HA in the biology of primitive hemopoietic progenitor cells.

Isolation and therapeutic potential of human haemopoietic stem cells.

The haemopoietic stem cell (HSC) has long been regarded as an archetypal, tissue specific, stem cell, capable of completely regenerating haemopoiesis after myeloablation. It has proved relatively easy to harvest HSC, from bone marrow or peripheral blood. In turn, isolation of these cells has allowed therapeutic stem cell transplantation protocols to be developed, that capitalise on their prodigious self renewal and proliferative capabilities. Ex vivo approaches have been described to isolate, genetically manipulateand expand pluripotent stem cell subsets. These techniques have been crucial to the development of gene therapy, and may allow adults to enjoy the potential advantages of cord blood transplantation. Recently, huge conceptual changes have occurred in stem cell biology. In particular, the dogma that, in adults, stem cells are exclusively tissue restricted has been questioned and there is great excitement surrounding the potential plasticity of these cells, with the profound implications that this has, for developing novel cellular therapies. Mesenchymal stem cells, multipotent adult progenitor cells and embryonic stem cells are potential sources of cells for transplantation purposes. These cells may be directed toproduce HSC, in vitro and in the future may be used for therapeutic, or drug development, purposes.

Small-molecule-directed mpl signaling can complement growth factors to selectively expand genetically modified cord blood cells.

Efforts toward achieving gene therapy for blood disorders are plagued by low rates of gene transfer into hemopoietic stem cells. Recent studies suggest that this obstacle can be circumvented using selection. One way to achieve selection employs genes that encode receptor-bearing fusion proteins capable of inducing cell growth in response to drugs called chemical inducers of dimerization (CIDs). We have previously shown that genetically modified marrow cells from mice can proliferate for up to a year in culture in response to CID-initiated signals arising from the thrombopoietin receptor (mpl). The sustained growth observed in mouse hemopoietic cells results from an mpl-induced self-renewal of multipotential hemopoietic progenitor cells. In contrast, human hemopoietic cells proliferate only transiently in response to the mpl signal (from differentiation of transduced erythroid and megakaryocytic progenitors), while human myeloid progenitors fail to respond. Here, we show that myeloid progenitors from human cord blood can be induced to proliferate and/or differentiate in response to the mpl signal by providing additional signals via a combination of growth factors. These findings are relevant for the eventual clinical application of CID-regulated cell therapy.

Cyclic ADP-ribose generation by CD38 improves human hemopoietic stem cell engraftment into NOD/SCID mice.

Cyclic ADP-ribose (cADPR) is a potent and universal intracellular calcium mobilizer, recently shown to behave as a new hemopoietic cytokine stimulating the in vitro proliferation of both committed and uncommitted human hemopoietic progenitors (HP). Here, we investigated the effects of cADPR on engraftment of hemopoietic stem cells (HSC) into irradiated NOD/SCID mice. Two different protocols were used: i) a 24 h in vitro priming of cord blood-derived mononuclear cells (MNC) with micromolar cADPR, followed by their infusion into irradiated mice (both primary and secondary transplants); and ii) co-infusion of MNC with CD38-transfected, cADPR-generating, irradiated murine 3T3 fibroblasts. We demonstrated a dual effect of cADPR on human HP in vivo: i) enhanced proliferation of committed progenitors, responsible for improvement of short-term engraftment; ii) expansion of HSC, with increased long-term human engraftment into secondary recipients and a significantly higher expansion factor of CD34+ progenitors in mice co-infused with MNC and CD38+ 3T3 fibroblasts. These results hold promise for the possible therapeutic use of cADPR, and of cADPR-producing stroma, to achieve long-term expansion of human HSC, that is, those HP capable of self-renewal and responsible for repopulation of the bone marrow.

BCR-ABL insufficiency for the transformation of human stem cells into CML

On the basis of numerical and kinetic data, the paper argues against the tenet that BCR-Abl is sufficient for transforming into Chronic Myelogenous leukemia a human hemopoietic stem cell, regardless of degree of pluripotency.

A novel monoclonal antibody recognizing a cation-dependent epitope within the regulatory loop of human beta(1) integrin (CD29).

Cell adhesion receptors of the integrin superfamily can be expressed in different affinity states towards their ligands. It has been previously demonstrated that beta(1) integrins alpha4beta(1) and alpha5beta(1) are expressed in a nonligand binding form by human hemopoietic progenitor cells but can be activated into a ligand binding form by a variety of stimuli including intracellular stimuli generated by cytokine receptors and extracellular stimuli generated by function-activating anti-beta(1) integrin monoclonal antibodies (MAbs). In both instances, the activation of beta(1) integrins is believed to be the result of conformational changes propagating along the beta(1) integrin chain which in turn increase accessibility to the ligand. A cluster of either function-activating or function-inhibiting anti-beta(1) integrin MAbs have been shown to bind within a 12 amino acid long regulatory loop between residues 207 and 218 of the human beta(1) integrin chain. We describe in this report the first MAb (96.9H9) specific for this regulatory loop whose binding is cation-dependent and requires either Ca(2+) or Mn(2+) but not Mg(2+). In addition, the activation of alpha4beta(1) and alpha5beta(1) integrins by 96.9H9 is a two-step process with distinct cation requirements. Whereas Ca(2+) is sufficient to promote binding of the antibody to the beta(1) integrin chain, Mg(2+) is necessary for activating function following 96.9H9 binding. Our data therefore suggest that the regulatory epitope of the human beta(1) integrin chain is flexible with multiple conformations according to the cationic environment.

Purging of Contaminating Breast Cancer Cells From Hematopoietic Progenitor Cell Preparations Using Activation Enhanced Cell Death

Activation enhanced cell death (AECD) involves stimulating cells with growth or activation signals while concurrently blocking calcium influx. In this study, we have evaluated the effect of AECD on human breast cancer cells. MCF-7 or MDA-MB-231 cells treated with Ca2+ influx blockers econazole or ketotifen for 24 h underwent a dose-dependent, irreversible loss of viability, and clonogenicity. Two-hour treatment of these cells with higher concentrations of the drugs also resulted in loss of clonogenicity, but morphological indicators of cell death were apparent only after longer incubation. Loss of clonogenicity could be enhanced almost 10-fold by co-stimulation of the cells with the agonists EGF or bombesin. Econazole was also effective in inducing cell death in multi-drug resistant MCF-7adr cells. Human hemopoietic progenitor cell sensitivity to econazole or ketotifen was evaluated by colony assay. Under conditions resulting in 2.5-3 logs of breast cancer cell loss, 60-70% of hemopoietic progenitors could be recovered. We further evaluated the effect of econazole on breast cancer cells present in mobilized hemopoietic cells obtained from patients undergoing high dose chemotherapy with autologous stem cell support. In six of eight samples evaluated, cytokeratin-positive breast cancer cells could be detected by immunofluorescence microscopy and colony formation. Breast cancer colonies were reduced 60-500-fold or more after exposure to econazole while hemopoietic colonies were typically reduced only 2-fold. In all cases, addition of EGF as an activator either had no evaluable effect or enhanced breast cancer cell loss. We conclude that Ca2+ influx blockade with concurrent EGF stimulation is a promising approach for purging breast cancer cells from hemopoietic progenitor cell preparations.

Transient Disruption of Autocrine TGF-β Signaling Leads to Enhanced Survival and Proliferation Potential in Single Primitive Human Hemopoietic Progenitor Cells

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-beta negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-beta type II receptor (TbetaRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-beta signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TbetaRIIDN and green fluorescence protein in the same CD34(+)CD38(-)Lin(-) cells were constructed. Overexpression of TbetaRIIDN specifically disrupted TGF-beta-mediated signaling. Autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34(+)CD38(-)Lin(-) cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-beta-negative regulatory loop of primitive hemopoietic progenitor cells.

Agar cultures of human clonogenic hemopoietic precursor cells for early diagnosis of some myeloproliferative diseases.

Growth characteristics of human hemopoietic cells in erythremia and chronic myeloid leukemia were studied using agar cultures with and without hemopoietic growth factors. Agar cultures, similarly to cultures on other semisolid media (plasma clot, methylcellulose) can be used for early differential diagnosis of polycythemia vera (erythremia) and secondary erythrocytosis: erythremia, but not erythrocytosis, is characterized by spontaneous (erythropoietin-independent) formation of colonies from erythrocyte precursor cells. Spontaneous colony formation from granulocyte-macrophage precursor cells can serve as an important test for early diagnosis of chronic myeloid leukemia. The study of colony formation from granulocyte-macrophage precursors and of the capacity of bone marrow cells to form colonies from hemopoietic stromal precursor cells revealed new characteristics of the studied myeloproliferative diseases. Presumably, spontaneous colony formation from erythrocytic and myeloid precursors should be regarded as a sign of tumor transformation of the studied hemopoietic cells.

Hierarchy of Stroma-derived Factors in Supporting Growth of Stroma-dependent Hemopoietic Cells: Membrane-bound SCF is Sufficient to Confer Stroma Competence to Epithelial Cells

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34 + cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34 + hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

Enumeration of bone marrow 'homing' haemopoietic stem cells from G-CSF-mobilised normal donors and influence on engraftment following allogeneic transplantation.

Expression of the chemokine receptor CXCR4 on human haemopoietic stem cells (HSC) may play a crucial role in localising these cells to the bone marrow. To evaluate whether CXCR4 expression is clinically relevant we have enumerated CXCR4-positive HSC used for allogeneic transplantation and sought any relationship with the rate of subsequent haemopoietic reconstitution. CD34-positive progenitor cells were isolated from peripheral blood stem cell (PBSC) collections from 16 normal donors. The proportion of cells co-expressing CXCR4 was enumerated and the times to recipient haemopoietic reconstitution measured. The median frequency of CD34-positive cells co-expressing CXCR4 was 41% (range 16% to 76%) and the median number of CXCR4 CD34 double-positive cells infused at transplantation was 2.5 x 10(6) cells/kg (range 0.8-10.3). Patients receiving >2.5 x 10(6) CXCR4 CD34 double-positive cells/kg demonstrated a significant shortening of time to platelet engraftment compared to the recipients of the lower cell doses (10 days vs 14.5 days, respectively, P = 0.02) with all but one of the recipients of the higher cell doses achieving platelet engraftment by day 11. Co-expression of CXCR4 on CD34-positive progenitor cells may be an important determinant of post-transplant engraftment and in our hands transplantation of a minimum of 2.5 x 10(6) CXCR4 CD34 double-positive cells/kg ensured rapid post-transplant platelet recovery.

CD antigens 2001.

CD antigens 2001.

NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor.

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.

Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+ Cells: Comparisons with Tumor Mast-Cell Lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

Naturally-occurring anti-G-CSF antibodies produced by human cord blood B-cell lines infected with Epstein-Barr virus

Naturally occurring antibodies (auto-Abs) recognizing human granulocyte-colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. Mononuclear cells from cord blood samples of different newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. Six different, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD5-, CD10-, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing lambda clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. The secreted Abs did not affect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing.

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells.

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.