Recent Australian experience with hemopoietic stem and progenitor cell expansion.

This review provides insight into two clinical trials conducted with ex vivo manipulated CD34+ cells. The first was an attempt to deliver a gene therapy for treatment of HIV and the second an attempt to improve rates of hemopoietic recovery with ex vivo generated myeloid cells.

Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor.

Mobilized progenitor cells currently represent the most commonly used source of hematopoietic progenitor cells (HPCs) to effect hematopoietic reconstitution following myeloablative chemotherapies. Despite their widespread use, the molecular mechanisms responsible for the enforced egress of HPCs from the bone marrow (BM) into the circulation in response to mobilizing agents such as cytokines remain to be determined. Results of this study indicate that expression of vascular cell adhesion molecule-1 (VCAM-1) is strongly reduced in vivo in the BM during HPC mobilization by granulocyte colony-stimulating factor (G-CSF) and stem cell factor. Two serine proteases, namely, neutrophil elastase and cathepsin G, were identified, which cleave VCAM-1 and are released by neutrophils accumulating in the BM during the course of immobilization induced by G-CSF. The proposal is made that an essential step contributing to the mobilization of HPCs is the proteolytic cleavage of VCAM-1 expressed by BM stromal cells, an event triggered by the degranulation of neutrophils accumulating in the BM in response to the administration of G-CSF.

Mucin-like molecules as modulators of the survival and proliferation of primitive hematopoietic cells.

Current data suggest that interplay between two classes of molecules contributes to the regulation of hematopoiesis: hematopoietic growth factors, which regulate the survival, proliferation, and development of primitive hematopoietic cells and cell adhesion molecules (CAMs), which are responsible for the localization of hematopoiesis to the bone marrow (BM) and for mediating physical association between developing hematopoietic cells and marrow stromal tissue. A range of cell surface molecules representing several CAM superfamilies including integrins, selectins, the immunoglobulin gene superfamily and an emerging family of mucin-like molecules (the sialomucins) are involved in supporting cell-cell and cell-extracellular matrix (ECM) interactions between primitive hematopoietic cells and the stromal cell-mediated hematopoietic microenvironment (HM) of the bone marrow. There is abundant evidence in non-hematopoietic tissues that CAMs are signalling molecules which participate in a range of signal transduction events important not only for regulating cell adhesion and motility, but also for cell growth and survival. Although the signalling functions of CAMs have not been studied extensively in primitive hematopoietic progenitors (HPCs), extrapolation from burgeoning data in other systems is consistent with the hypothesis that hematopoiesis within the BM is regulated by interaction between signals generated locally by CAMs and those elicited by cytokines. Evidence in support of this notion was initially provided by studies on normal HPCs demonstrating cross-talk between members of the integrin superfamily and cytokine receptors. In this article we review recent reports that mucin-like molecules are also signalling molecules on primitive hematopoietic cells and that the signals they deliver potently inhibit hematopoiesis.

PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis.

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.

Stem cell factor as a single agent induces selective proliferation of the Philadelphia chromosome positive fraction of chronic myeloid leukemia CD34(+) cells.

The interaction between p145(c-KIT) and p210(bcr-abl) in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34(+) cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34(+) cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34(+) cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) from CML CD34(+)CD38(+) cells and the more primitive CML CD34(+)CD38(-) cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34(+) cells showed exclusive growth of Ph+ cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of beta1-integrin-mediated adhesion of CML CD34(+) cells to fibronectin was not increased when compared with the effect on normal CD34(+) cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors.

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, G-CSF, and IL-3 were found to be the most important HGF for production of neutrophilic cells. The 4-HGF combination of IL-3, IL-6, G-CSF, and SCF was optimized by performing dose-response experiments and shown to be as potent as 6 HGF for production of nascent CFU-GM and neutrophilic precursors.

Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand.

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.

Detection of minimal residual disease in an AML patient with trisomy 8 using interphase fish.

Patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) often exhibit clonal chromosomal abnormalities. Using a probe for the centromeric region of chromosome 8, fluorescence in situ hybridization (FISH) on interphase cells was used to detect trisomy 8 in an AML patient whose leukemia was characterised by the karyotype 47, XY, +8, del(9) (q21.1q32). We have demonstrated using FISH the presence of the trisomy at all stages of the patient's disease course (including remission, peripheral blood cell harvest and relapse), whereas conventional karyoptypic analysis was only able to detect the trisomy at diagnosis and clinical relapse. We have also shown using immunophenotyping, cell sorting and FISH, that the trisomic cells in this patient were restricted to the CD34+ subset of blood and bone marrow and could not be found in the CD 34-, T or B cell compartment. Overall we have shown FISH to be a rapid, quantitative method for the detection of cells with numerical chromosome abnormalities. FISH analysis of interphase cells provides valuable information on the status of the whole population, rather than just cycling cells, and can be applied successfully to monitor the level of leukemic cells.

Peripheral blood is a source of BCR-ABL-negative pre-progenitors in early chronic phase chronic myeloid leukemia.

Manipulation of autologous bone marrow cells (BM) for transplantation in chronic myeloid leukemia (CML) to enrich for normal cells is a novel approach that may improve survival for patients not suitable for allogeneic transplantation. Limitations of this technique include the reported low frequency of normal stem cells in CML and the difficulties in obtaining sufficient BM for manipulation. To address these problems we compared the apheresis product with the diagnostic bone marrow at diagnosis as a source of primitive BCR/ABL-negative progenitors. We analyzed the CD34+ HLA-DR- and CD34+CD38(-) populations in five CML patients to evaluate the frequency of BCR-ABL-negative progenitors and pre-progenitors in these populations. Progenitor analysis was performed by RT-PCR of individual hemopoietic colonies from a standard CFU-GM assay. Analysis of pre-progenitors involved RT-PCR of secondary colonies derived from a stroma-free pre-CFU assay. Our results show variable levels of BCR-ABL-negative progenitors in the 34+DR- population but very low levels of BCR-ABL-negative progenitors in the 34+38- population in blood. Analysis of pre-progenitors from the 34+DR- fraction of peripheral blood (PB) and BM showed 80-100% and 85-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. Analysis of pre-progenitors from the 34+38- fraction of PB and BM showed 23-100% and 42-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. In summary, pre-progenitors from the 34+DR- and 34+38- populations are predominantly BCR-ABL negative in both marrow and blood at diagnosis. Apheresis product collected at diagnosis is a more abundant sources of BCR-ABL-negative pre-progenitors than BM. Thus, apheresis product could potentially be utilized as a source of BCR-ABL-negative stem cells in CML.

Standardization of the CFU-GM assay using hematopoietic growth factors.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay is used commonly to assess adequacy of progenitor number in bone marrow transplantation. The assay is poorly standardized, resulting in variability of results between and within laboratories. We assessed three variables that contribute to the lack of standardization. The colony-stimulating activity of human placental-conditioned medium (HPCM) was compared with combinations of recombinant hematopoietic growth factors (HGF) in 5 normal bone marrow donors. A protocol for batch testing of fetal calf serum (FCS) is described. In addition, a rigid training program has been introduced to minimize interstaff and intrastaff variability in the counting of colonies. We show that a five-factor combination of interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and stem cell factor (SCF) produces a mean increase of 85% in colony number. Some combinations of three HGF produce similar growth to HPCM, and all four HGF combinations are equivalent or superior to HPCM. Batch testing of FCS shows variability between batches. We show significant interstaff and intrastaff variability between a new and experienced staff member that improves following a period of training. In summary, the use of recombinant HGF in association with a rigorous program of batch testing of FCS and staff training results in a CFU-GM assay that can be standardized between laboratories.

Hollow-fibre affinity cell separation system for CD34+ cell enrichment.

A hollow-fibre immunoadsorption system has been developed for the purification of CD34+ cells from mononuclear cells. This cell separation technique is based on the use of uniform surface fluid shear stress to fractionate cells that attach to the inside surface of hollow fibres. Monoclonal antibody to the CD34 antigen was covalently coupled to the lumenal surface of cuprophan minidialysers (surface area 220 cm2). After the selective adsorption of CD34+ cells (28 min), a depleted fraction was collected at 5 dynes/cm2 followed by washes at 10 and 25 dynes/cm2. Antigen-positive cells were recovered after incubation with chymopapain. The device was tested by using peripheral blood mononuclear cells from seven patients who had received granulocyte colony-stimulating factor and chemotherapy. The average number of cells processed was 1.3 +/- 0.2 x 10(8) (+/- S.E.M.), and the preselection incidence of CD34+ cells ws 1.6 +/- 0.6% (range 0.21-4.13%; n = 7). The enrichment purity was 94.4 +/- 3.1%, and 61 +/- 9% of input CD34+ cells were recovered in the enriched fraction (n = 4). Enrichment resulted in a 3.3 +/- 0.1% log10 depletion of CD34- cells (n = 4). Hollow-fibre affinity cell separation has potential as a medium to large-scale cell enrichment technology.

Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors.

Adhesive interactions with the extracellular matrix of the bone marrow (BM) stroma are of critical importance in the regulation of hematopoiesis. In part, these interactions are presumed to play an important role in retaining CD34+ hematopoietic progenitor cells (HPCs) within the BM environment, in close proximity with BM stromal cells and the cytokines they produce. Evidence of a more direct role for cell adhesion in the regulation of hematopoiesis is provided by recent data showing that adhesive interactions can also provide important costimulatory signals. We have previously shown that normal CD34+ HPCs express high levels of fibronectin (Fn) receptors very late antigen-4 (VLA-4) and VLA-5 in a low-affinity state, which do not allow HPCs to strongly adhere on immobilized Fn, and that cytokines such as interleukin-3, granulocyte-monocyte colony-stimulating factor, and stem cell factor transiently activate these receptors, providing HPCs with an adhesive phenotype on Fn. Thus, knowledge of the functional states of adhesion receptors is critical to our understanding of the physiological mechanisms responsible for the regulation of normal hematopoiesis. Herein, we show that combinations of cytokines that synergize to stimulate the proliferation of CD34+ HPCs result in additive stimulation of the adhesion of these cells to Fn. Thus, the activation level of Fn receptors expressed by normal CD34+ HPCs is highly correlated with their proliferative state, suggesting a functional link between these two events. Therefore, we propose a 2-step model with an initial activation of VLA-4 and VLA-5 generated by cytokine receptors that is followed by a secondary signal resulting from Fn binding to VLA-4 and VLA-5, which may cooperate with those generated by cytokine receptors.

Use of hematopoietic growth factors for in vitro expansion of precursor cell populations.

The increasing availability of recombinant human hematopoietic growth factors for clinical use has encouraged the development of novel approaches to the manipulation of hematopoiesis. Of particular note are the various strategies that have been proposed for ex vivo expansion of primitive hematopoietic cells. The majority of these involve growth of hematopoietic cells enriched in primitive progenitors in stromal cell-free suspension culture systems supported by the addition of various combinations of hematopoietic growth factors. In this article, we review recent progress in this area together with potential clinical applications for this technology.

Ex vivo hematopoietic progenitor cell expansion.

The ability to culture and expand hematopoietic progenitor cells ex vivo has major implications for both bone marrow and stem cell support following marrow ablative or subablative high-dose therapy and for improving the efficiency of retroviral transfection in gene marking and gene therapy. This review focuses on methods for the generation of myeloid progenitor and post-progenitor cells from peripheral blood stem cell collections, with particular emphasis on the characterization of these cells and practical issues associated with their expansion.

Signals from platelet/endothelial cell adhesion molecule enhance the adhesive activity of the very late antigen-4 integrin of human CD34+ hemopoietic progenitor cells.

Adhesive interactions between human CD34+ hemopoietic progenitor cells and bone marrow stromal cells control the localization, proliferation, and differentiation of CD34+ cells. Changes in adhesive interactions may contribute to the mobilization of CD34+ cells to the blood induced by chemotherapy and cytokines. Thus, the identities and functional states of adhesion receptors are critical properties of CD34+ cells. Here, we confirm that the adhesion receptors very late antigen-4 (VLA-4), LFA-1, and platelet/endothelial cell adhesion molecule-1 (PECAM-1) are expressed on the CD34+ cell line KG1a and on CD34+ normal, steady state bone marrow cells. Therapeutically mobilized CD34+ cells express similar levels of PECAM-1 but reduced levels of VLA-4 and LFA-1 in comparison with steady state bone marrow cells. Integrin adhesive activity was measured from the binding of PKH 26- or phycoerythrin-labeled CD34+ cells to FITC-labeled Chinese hamster ovary (CHO) cells expressing vascular CAM-1 (VCAM-1) or intercellular CAM-1, which are ligands for VLA-4 and LFA-1, respectively. Incubation mixtures were analyzed by flow cytometry for the loss of free CD34+ cells and gain of CD34(+)-CHO cell aggregates. VLA-4 mediates the strong and specific adhesion of KG1a cells and bone marrow CD34+ cells to VCAM-1-transfected CHO cells. CD34+ cells mobilized with granulocyte colony stimulating factor (G-CSF) or cyclophosphamide also bind VCAM-1 via VLA-4. The VLA-4-mediated adhesion of all CD34+ cells to VCAM-1 is enhanced by Abs to the coexpressed adhesion receptor PECAM-1, implicating signals transmitted from PECAM-1 as determinants of VLA-4 integrin activity. VLA-4 function in CD34+ cells mobilized with G-CSF or cyclophosphamide is equivalent to steady state CD34+ cells. LFA-1 mediates minimal adhesion between CD34+ cells and intercellular CAM-1 transfected CHO cells and is refractory to PECAM-1 modulation. We infer that VLA-4, but not LFA-1, contributes to the constitutive adhesive phenotype of CD34+ cells. PECAM-1 is probably one of several receptors that control adhesive interactions between hemopoietic progenitors and target cells by regulating the activation states of specific integrins.

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells.

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte-macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.

The use of the APAAP technique as a rapid indicator of peripheral blood progenitor cell levels.

Rapid and sustained engraftment following autotransplantation with peripheral blood stem cells (PBSC) depends on adequate numbers of stem cells and progenitor cells. In this study we have compared the number of myeloid progenitor cells quantitated using the colony forming units-granulocyte macrophage (CFU-GM) clonogenic assay with the number of CD34+ cells estimated both by flow cytometry and by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. We have analysed 15 peripheral blood mononuclear cells (PBMNC) samples from 13 normal subjects and 179 PBMNC from 32 patients undergoing PBSC harvests during the recovery phase of high dose cyclophosphamide chemotheraphy. The number of CD34+ cells measured by the APAAP technique correlated well with the number of CD34+ cells measured by flow cytometry (r = 0.727, p = 0.0001), and also with the number of CFU-GM measured in the clonogenic assay (r = 0.721, p = 0.0001). The APAAP method provides a rapid, reliable measure of progenitor cell levels that can be used to monitor the optimal time to harvest peripheral blood stem cells (PBSC), and to estimate the marrow repopulating ability (MRA) of stem cell preparations used for transplantation.

The mobilization of primitive hemopoietic progenitors into the peripheral blood.

There is considerable interest in the use of peripheral blood progenitor cells (PBPC) for hemopoietic rescue following high dose chemotherapy. Current regimens mobilize CD34+ with variable efficacy and there remains considerable empiricism in the design of these regimens. Some involve myelosuppression, some the administration of various cytokines alone or in combination, while a combination of chemotherapy and cytokines is employed in others. Certain protocols result in mobilization within one week while in others, maximal PBPC levels occur only after several weeks. Thus, procedures required for optimal mobilization of PBPC remain to be defined. An understanding of the mechanisms responsible for mobilization may lead to the development of improved mobilization strategies. Herein we review data that explore the mechanisms involved in the mobilization of PBPC in man. These data demonstrate that mobilization is associated with marked changes in the expression and function of cell adhesion molecules (CAMs) on hemopoietic progenitor cells (HPC), suggesting that the release of HPC into the blood involves a perturbation of the adhesive interactions between these cells and the marrow stroma that, in steady-state conditions, serve to restrict HPC to the bone marrow. Downregulation of c-kit is invariably associated with successful mobilization which, when combined with data from in vitro studies, implies a key role for stem cell factor (SCF) as an orchestrator of mobilization.