In vivo expansion of the endogenous B-cell compartment stimulated by radiation and serial bone marrow transplantation induces B-cell leukaemia in mice.

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5(+) B cells. This B-cell lineage is established during ontogeny and replenished by the process of self-renewal. Spontaneous and induced leukaemias that frequently affect this lineage are thought to arise as a result of the frequent cell division required to maintain the population throughout adulthood and in response to repeated exposure to environmental antigens. In a series of bone marrow transplant (BMT) experiments performed in B6D2F1 mice, B-cell leukaemia occurred in recipients of serially transplanted syngeneic bone marrow. This study was therefore designed to determine the frequency and phenotype of the observed leukaemia. Male donor cells were initially transplanted into lethally irradiated female hosts and secondary (2 degrees ) BMT was performed at 3 months. At 1, 2, 3 and 16 months following primary (1 degrees ) BMT, and when 2 degrees BMT recipients developed leukaemia, animals were sacrificed and their tissues extensively examined. These analyses confirmed a host-derived CD5(+) transplantable B-cell leukaemia that was initiated in 50% of 1 degrees BMT recipients. With serial passage, the leukaemia became more aggressive and lost CD5 expression, suggesting transformation to a high-grade leukaemia/lymphoma. This previously unreported observation suggests that the combination of radiation and subsequent serial transplantation induces a proliferative stress to the host B-cell compartment that is causative in leukaemic transformation.

Preserved long-term repopulation and differentiation properties of hematopoietic grafts subjected to ex vivo expansion with stem cell factor and interleukin 11.

BACKGROUND: The ex vivo expansion of hematopoietic grafts has been proposed as an efficient procedure for improving the hematological recovery of recipients. The fate of the long-term repopulating cells during the ex vivo manipulation of the graft is, however, a critical issue in ex vivo expansion protocols and ultimately will define the applicability of this new technology in hematopoietic transplants. METHODS: The repopulating ability of mouse hematopoietic samples was determined by means of bone marrow (BM*) competition assays, using congenic strains that express the pan-leukocyte Ly-5.1 and Ly-5.2 antigens. The generation of potential changes in the repopulating properties of human hematopoietic samples subjected to ex vivo expansion was determined by comparing the engraftment of fresh and ex vivo-manipulated CD34+ cord blood cells in irradiated nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. RESULTS: Under our optimized conditions of mouse BM incubation (stem cell factor plus interleukin-11, either with or without macrophage inflammatory protein-1alpha or Flt3 ligand), both the short-term and the mid-term repopulating ability of the ex vivo-expanded samples were significantly improved when compared with fresh samples. In the long-term, no changes in the repopulation and differentiation properties of the graft were observed as a result of the ex vivo expansion process. As deduced from the analysis of NOD/SCID mice transplanted with fresh and ex vivo expanded human CD34+ cord blood cells, the in vitro stimulation mediated by SCF/IL-11/FLT3L was capable of preserving the ability of the grafts to repopulate the lympho-hematopoiesis of recipients for at least 3 months. CONCLUSION: These results indicate that under our optimized conditions of ex vivo expansion, the amplification of the hematopoietic progenitors responsible for the short- and mid-term repopulating properties of the graft can take place without compromising the long-term lympho-hematopoietic repopulating properties.

CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens.

We have previously demonstrated that CD34+ cells, selected from peripheral blood progenitor cells (PBPC), can be expanded in ex vivo culture and can be infused in tandem with unmanipulated PBPC with little or no toxicity. In this study, four patients (two non-Hodgkin's lymphoma (NHL), two multiple myeloma (MM)) received myeloablative conditioning prior to stem cell rescue using ex vivo expanded cells alone. The two patients with NHL received cyclophosphamide and total body irradiation (CY/TBI) and the two patients with MM, busulphan and melphalan (Bu/M). One case received an inadequate CFU-GM dose, despite expansion, and in one case the expanded cells were contaminated. No definitive conclusions may therefore be drawn concerning engraftment in these two cases. However, the other two cases received high doses of committed progenitors. Following infusion of the expanded material, all four patients failed to show sustained neutrophil engraftment and none showed evidence of platelet engraftment. Back-up, unmanipulated PBPC were therefore infused on days 14, 34, 32 and 28 and subsequently all four cases achieved satisfactory engraftment of both neutrophils and platelets. In conclusion, we feel that, CD34+ cells, expanded ex vivo using the conditions described in this report, may not provide durable engraftment following fully myeloablative conditioning.

Cloning and characterization of a novel murine beta chemokine receptor, D6. Comparison to three other related macrophage inflammatory protein-1alpha receptors, CCR-1, CCR-3, and CCR-5.

The beta-chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) is chemotactic for many hemopoietic cell types and can inhibit hemopoietic stem cell (HSC) proliferation, effects mediated through G-protein coupled heptahelical receptors. We have isolated cDNAs for seven chemokine receptors, CCR-1 to -5, MIP-1alphaRL1, and a novel cDNA, D6. Chinese hamster ovary cells expressing CCR-1, -3, -5, and D6 bound 125I-murine MIP-1alpha: the order of affinity was D6 > CCR-5 > CCR-1 > CCR-3. Each bound a distinct subset of other beta-chemokines: the order of competition for 125I-murine MIP-1alpha on D6 was murine MIP-1alpha > human and murine MIP-1beta > human RANTES approximately JE > human MCP-3 > human MCP-1. Human MIP-1alpha and the alpha-chemokines did not compete. Like other chemokine receptors, D6 induced transient increases in [Ca2+] in HEK 293 cells upon ligand binding. D6 mRNA was abundant in lung and detectable in many other tissues. Bone marrow cell fractionation demonstrated T-cell and macrophage/monocyte expression of D6, and CCR-1, -3, and -5. Moreover, we could detect expression of CCR-3, CCR-5, and to a greater extent D6 in a cell population enriched for HSCs. Thus, we have characterized four murine beta chemokine receptors that are likely involved in mediating the pro-inflammatory functions of MIP-1alpha and other chemokines, and we present D6, CCR-3, and CCR-5 as candidate receptors in MIP-1alpha-induced HSC inhibition.

Macrophage Inflammatory Protein-1 Alpha: A Paradox?

Macrophage inflammatory protein - 1 alpha (MIP-1α) is an 8kD protein which tends to form aggregates and is a member of the C-X-C chemokine subfamily, being a pro-inflammatory agent and is chemotactic for lymphocytes. MIP-1α has been shown to inhibit haemopoietic progenitor cell proliferation in vivo and in vitro and to reduce haemopoietic stem cell loss with improved neutrophil recovery after radiotherapy or chemotherapeutic agents in vivo.

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow.

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.

CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.

PURPOSE: The objectives of this phase I study were to assess the feasibility of using cryopreserved peripheral-blood progenitor cells (PBPC) for large-scale CD34 selection and subsequent expansion, and the safety of their use for reinfusion following chemoradiotherapy. PATIENTS AND METHODS: For 10 patients with nonmyeloid malignancy, an aliquot from a PBPC harvest was recovered from liquid nitrogen, and CD34 selected using the Isolex system (Baxter Healthcare, Newbury, United Kingdom) and expanded for 8 days ex vivo in a medium free of animal proteins but supplemented with autologous serum, stemcell factor (SCF), interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin. RESULTS: The mean increase for cell number was 21-fold, for colony-forming units-granulocyte/macrophage (CFU-GM) 139-fold, and for burst-forming units-erythroid (BFU-E) 114-fold. The expanded cells were reinfused in tandem with unmanipulated material (> or = 25 x 10(4) CFU-GM/kg). The patients did not experience any adverse effects immediately on cell infusion or within 48 hours. The 10 index patients were compared with 10 historical controls for parameters of myelosuppressive morbidity. In this small study, there were no differences in either neutrophil or platelet recovery between the patients who received expanded cells and historical controls. CONCLUSION: These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.

Specificity and reciprocity in the interactions between TGF-beta and macrophage inflammatory protein-1 alpha.

TGF-beta and macrophage inflammatory protein-1 alpha (MIP-1 alpha) appear to share a number of biologic properties. We have been attempting to examine the interactions between these two peptides in the hope of gaining an insight into the basis for the apparent functional redundancy. Our earlier observations have indicated that TGF-beta is a potent down-regulator of MIP-1 alpha and MIP-1 beta expression in bone marrow macrophages and also of MIP-1 alpha receptor numbers on FDCPmix cells. We now demonstrate that the interplay between TGF-beta and MIP-1 alpha beta is relatively specific, in that only MIP-1 alpha and MIP-1 beta appear to be potently suppressed by TGF-beta, and that this suppressive activity is restricted to the direct TGF-beta isoforms. Activin and the bone morphogenetic proteins (BMPs) appear to be inactive in this regard. We also demonstrate the existence of an endogenous TGF-beta-mediated block that acts to minimize MIP-1 alpha expression in TGF-beta-expressing macrophages. This coupled with the observations that MIP-1 alpha can induce expression of TGF-beta suggests to us that the complex interactions between MIP-1 alpha and MIP-1 beta and the direct TGF-beta isoforms (beta 1, beta 2, and beta 3) act to ensure minimized MIP-1 alpha beta expression and maximized TGF-beta expression. However, such interplay may also be dependent on the local cytokine or inflammatory profile to which the cells are exposed.

Inhibitors of haemopoiesis and their potential clinical relevance.

Current cytotoxic treatment regimens are most frequently dose-limited by the problem of myelotoxicity, and this could theoretically be prevented or reduced by the use of stem-cell inhibitors, since protection of this compartment during treatment could result in a more favourable outcome in terms of bone-marrow regeneration. Several negative stem-cell regulators have been identified, including macrophage inflammatory protein-1 alpha, transforming growth factor-beta, tumour necrosis factor-alpha, tetrapeptide and pentapeptide. All of these molecules have been shown to inhibit the proliferation of normal haemopoietic progenitors in bone marrow, and stem-cell protection from cytotoxic agents both in vitro and in vivo has been demonstrated. The potential use of inhibitors for the purging of tumour cells from stem-cell grafts is suggested by the observation that there is a differential response between normal and leukaemic progenitors to some inhibitors.

Requirement of MIP-1 alpha for an inflammatory response to viral infection.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a chemokine that has pro-inflammatory and stem cell inhibitory activities in vitro. Its biologic role in vivo was examined in mice in which the gene encoding MIP-1 alpha had been disrupted. Homozygous MIP-1 alpha mutant (-/-) mice were resistant to Coxsackievirus-induced myocarditis seen in infected wild-type (+/+) mice. Influenza virus-infected -/- mice had reduced pneumonitis and delayed clearance of the virus compared with infected +/+ mice. The -/- mice had no overt hematopoietic abnormalities. These results demonstrate that MIP-1 alpha is an important mediator of virus-induced inflammation in vivo.

Perturbations of multiple hemopoietic lineages in retrovirus-induced murine malignant histiocytosis.

A fatal systemic proliferation of malignant histiocytes resembling human malignant histiocytosis was induced in susceptible mice following infection with the murine retrovirus malignant histiocytosis sarcoma virus (MHSV). It is shown that MHSV additionally caused profound alterations of erythropoiesis, granulocytopoiesis and thrombocytopoiesis, and in the hemopoietic stem cell compartment. In the erythroid lineage, MHSV induced a normocytic peripheral anemia, which was paralleled by an unphysiologic, multifocal clonal expansion of erythroid blasts in the spleen. These cells were not transformed and appeared to have a maturation defect since blood reticulocytes did not increase above control values. Moreover, MHSV exerted cytopathic effects on neutrophilic granulocytes and megakaryocytes, since their numbers transiently decreased in the spleen, and agranulocytosis and thrombocytopenia was observed in the blood. Nonetheless, regeneration was found in both lineages at later stages of the infection, which was accompanied by a terminal granulocytosis. The number of lineage-committed and multipotential colony-forming cells in the CFU-S assay increased transiently, but decreased to very low levels in the final stages of the disease. Thus, the studies demonstrate that the same etiologic agent, MHSV, had different effects on hemopoietic cells, which included malignant transformation, hyperproliferative and cytopathic effects.

Aggregation of the chemokine MIP-1 alpha is a dynamic and reversible phenomenon. Biochemical and biological analyses.

Macrophage inhibitory protein (MIP)-1 alpha is a potent inhibitor of hemopoietic stem cell proliferation and is a member of a family of pro-inflammatory mediators, the chemokine family. This molecule along with other members of the chemokine family exists as a peptide of 8 kDa but has a strong tendency for noncovalent extensive self-aggregation. As this aggregation may interfere with biological activity, we have produced nonaggregating variants of MIP-1 alpha which display a range of molecular sizes. The mutants, produced by sequential neutralization of carboxyl-terminal acidic residues, display native molecular masses representative of tetramers, dimers, and monomers. Intriguingly when these mutants are assessed in comparison with native MIP-1 alpha for bioactivity in vitro, they are seen to be equipotent in both stem cell assays and in monocyte shape-change assays, suggesting that there is no requirement for aggregation in either of these biological contexts. This indicates that the aggregated MIP-1 alpha and the aggregated mutants spontaneously disaggregate under assay conditions and ultimately function as monomers. We have further demonstrated the ability of MIP-1 alpha to disaggregate spontaneously in dilute solution by enzyme-linked immunosorbent assay analysis of fractions obtained from gel filtration of varying concentrations of MIP-1 alpha. The aggregation of MIP-1 alpha is therefore a dynamic and reversible phenomenon which has little, if any, impact on bioactivity in vitro.

Efficient isolation of human CD34 positive hemopoietic progenitor cells by immune panninga.

In this study we have assessed the use of soybean agglutinin (SBA) and CD34 microcellector devices for the selection of CD34 positive hemopoietic progenitor cells. Burst forming unit-erythroid (BFU-E), colony forming unit-granulocyte/macrophage (CFU-GM) and the recently developed multipotential human colony forming unit-type A (CFU-A) clonogenic assays were used to measure progenitor numbers in the starting mononuclear cell (MNC), the SBA negative, the nonadherent CD34 negative and the adherent CD34 positive fractions during panning. CFU-A progenitors were present at a relatively high incidence in the MNC fraction (220 per 10(5) MNC) and were enriched 15-fold in the adherent CD34 positive fraction. This progenitor incidence and enrichment were similar to those of CFU-GM and BFU-E. The mean recovery for CD34 positive cells was 2.3 x 10(6) cells per marrow aspirate. Analyses by flow cytometry demonstrated that 1-5% of input MNC were CD34 positive, that the purity of the CD34 fraction was approximately 80% and that the calculated recovery for CD34 positive cells was 61%. Recoveries for CFU-GM, BFU-E and CFU-A were between 18 and 40%. CFU-A progenitors were found exclusively in the adherent CD34 positive fraction, whereas a significant proportion of both CFU-GM and BFU-E were present in the nonadherent CD34 negative fraction. We propose that the Applied Immune Sciences (AIS) flasks preferentially bind the cells which express CD34 most strongly and that this is reflected in the finding of primitive CFU-A only in the CD34 positive fraction, with lineage-restricted progenitors found in both CD34 positive and negative fractions. This hypothesis is strengthened by data on long-term bone marrow cultures in which the CD34 positive fraction is better able to maintain output of CFU-GM compared with the CD34 negative fraction. In conclusion, relatively pure populations of CD34 positive cells may be rapidly and efficiently isolated from bone marrow samples with good recovery. The isolated cells show enhanced colony forming capacity in standard clonogenic assays and in the multipotential CFU-A assay.

Contrasting effects of rh-MIP-1 alpha and TGF-beta 1 on chronic myeloid leukemia progenitors in vitro.

In chronic myeloid leukemia (CML) an abnormality at the stem cell level results in unregulated expansion of myeloid progenitors. The mechanism underlying this uncontrolled proliferation remains unclear. An in vitro clonogenic assay which detects the human counterpart of the murine colony forming unit (CFU) CFU-A/CFU-S day 12 was described in a report of our recent findings. CML bone marrow samples were found to proliferate in the CFU-A assay, producing colonies morphologically indistinguishable from normal controls. The bcr/abl transcripts were sought in the RNA from individual colonies using the polymerase chain reaction (PCR). For the five CML samples tested to date, the majority of CFU-A colonies at diagnosis or in early chronic phase were found to be bcr/abl positive. For normal controls both macrophage inflammatory protein-1 alpha (MIP-1 alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibited the proliferation of CFU-A colonies when directly added to the assay. In contrast, CML progenitors responded normally to TGF-beta 1, but showed no response to MIP-1 alpha. In suicide assays, for five normal bone marrow samples, CFU-A progenitors induced into S-phase returned to a quiescent state after treatment with MIP-1 alpha. CML progenitors demonstrated inherently high cycle status which showed no definite response to MIP-1 alpha. However, TGF-beta 1 resulted in quiescence of CML progenitor cycling. In conclusion, the primitive progenitors from CML samples were inhibited normally by TGF-beta 1 but showed no response to MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor beta: is it a downregulator of stem cell inhibition by macrophage inflammatory protein 1 alpha?

Transforming growth factor beta 1 (TGF-beta 1) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have recently been identified as potent inhibitors of hemopoietic stem cell proliferation. From previous studies, these molecules appear to have similar functions in the control of stem cell proliferation. This study was designed to investigate the relationship, if any, between these two negative regulators in an attempt to elucidate possible distinctive roles for each within the hemopoietic system. We report here that both MIP-1 alpha and TGF-beta are capable of inhibiting the same stem cell population (colony-forming unit [CFU]-A/CFU-S) with similar potencies. We further show that TGF-beta potently inhibits MIP-1 alpha gene expression in bone marrow-derived macrophages, the presumed source of MIP-1 alpha in the bone marrow. This inhibition is not specific to MIP-1 alpha in that expression of MIP-1 beta, a related molecule that does not exhibit potent stem cell inhibitory properties, is inhibited in a similar manner. The inhibition of MIP-1 alpha gene expression is also seen as a reduction in MIP-1 alpha protein production, which markedly decreases 24 h after treating RAW 264.7 cells, a murine macrophage cell line, with TGF-beta. These in vitro results suggest that in the presence of active TGF-beta in vivo, and in the absence of upregulators of MIP-1 alpha transcription, very little MIP-1 alpha will be produced. To address how MIP-1 alpha's target cells, the stem cells, would respond to TGF-beta, and the consequently low levels of MIP-1 alpha produced, we analyzed the effect of TGF-beta on MIP-1 alpha receptor levels on FDCP-MIX cells, a murine stem cell line. We show that TGF-beta (100 pM) reversibly downregulates MIP-1 alpha receptor levels on these cells to a maximum of 50-70% after 24 h. This level of downregulation does not change upon increasing the concentration of TGF-beta or the length of exposure of the cells to TGF-beta. Scatchard analysis shows that TGF-beta downregulates MIP-1 alpha receptor numbers with no change in affinity of the remaining receptors. These results suggest that TGF-beta may be capable of interfering with MIP-1 alpha's role as a stem cell inhibitor. Indeed, they suggest that in the presence of active TGF-beta in vivo, MIP-1 alpha is at best a weak contributor to the overall physiological inhibition of stem cells.

Hemopoietic stem cell inhibitor (SCI/MIP-1 alpha) also inhibits clonogenic epidermal keratinocyte proliferation.

The maintenance and regulation of continuously renewing tissues is ultimately controlled at the level of stem-cell proliferation. We have recently identified a reversible inhibitor of hemopoietic stem-cell proliferation (stem-cell inhibitor [SCI]), which is identical to the macrophage inflammatory protein, MIP-1 alpha, a 69-amino-acid heparin-binding cytokine. To test the cell/tissue specificity of the inhibition of proliferation by SCI/MIP-1 alpha, we have investigated its activity on epidermal keratinocytes, the principal cell type of another continuously renewing tissue. Here we show that SCI/MIP-1 alpha inhibits the proliferation of epidermal keratinocytes in vitro and that the MIP-1 alpha mRNA is present in epidermal Langerhans cells but not in keratinocytes. This suggests an important growth regulatory function for SCI/MIP-1 alpha in keratopoiesis, as well as hemopoiesis, and may also indicate a novel role for the epidermal Langerhans cell. As SCI/MIP-1 alpha can inhibit the proliferation of embryologically distinct precursor cells, this raises the possibility that it may also function in a number of other tissues.

Characterization of a receptor for macrophage inflammatory protein 1 alpha and related proteins on human and murine cells.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a potent stem cell inhibitor and a member of a large and expanding family of related cytokines. In an effort to understand the molecular basis of the activities of MIP-1 alpha, we have sought to characterize the cellular receptors for this molecule. Our results demonstrate the presence of abundant MIP-1 alpha receptors on both human and murine cells. The receptor on K562 cells can bind a range of members of the MIP-1 alpha family and may thus be a general MIP-1 alpha family receptor. Murine FDCPmix cells also bind a range of members of this peptide family, although the receptor(s) that they express appear somewhat more selective for peptides capable of displaying stem cell inhibitory properties. The human and murine receptors do not bind members of the related interleukin 8 family of peptides and are thus distinct from the recently cloned interleukin 8 receptor. We suggest that the receptor on the murine cell is a candidate for the receptor responsible for articulating stem cell inhibitory signals following MIP-1 alpha binding.

Mixed colony formation in vitro by the heterogeneous compartment of multipotential progenitors in human bone marrow.

The physiology of the human haemopoietic primitive progenitor populations can be studied in normal and disease states by clonal in vitro cultures in which the primitive progenitor cells proliferate and differentiate to form mixed colonies. For many applications it is essential that such assays detect a high proportion of primitive progenitor cells. We describe an in vitro assay which detects a high incidence of human CD34+ multipotential progenitor cells. Bone marrow mononuclear cells (MNC) or selected CD34+ cells were plated at low cell concentrations in semisolid agar cultures with synergizing growth factor combinations. The optimum growth factor combination of conditioned medium from Mia PaCa-2 cells (Mia-CM), recombinant granulocyte-macrophage colony-stimulating factor and recombinant stem cell factor (SCF) supported the formation of macroscopic (> or = mm) colonies (97% of which were multilineage), at an average incidence of 250/10(5) MNC. The colony-forming cells (human colony-forming unit, type A) detected, showed a low cycling status (7.3%) and the macroscopic colonies had a high replating efficiency (46%), reflecting their probable primitive nature. This assay should prove invaluable, for studies on the regulation of proliferation of the multipotential compartment and in studies involving the assessment of these cells in transplantation and neoplastic disease.

The haemopoietic stem cell: properties and control mechanisms.

All the cells of the immuno-haemopoietic system derive ultimately from a single pluripotent stem cell through processes of commitment and differentiation. The stem cell is also likely to be capable of extensive self renewal. Many factors which potentially control these processes have been identified and characterised both in vitro and in vivo. We discuss the nature of the haemopoietic stem cell and also the factors which have been identified as potential stem cell regulators. We also draw parallels from embryonal stem cell differentiation to derive a model of intrinsic determination of the haemopoietic stem cell. The possible role of developmental regulators, such as homeobox containing genes, are discussed in the context of differentiation commitment of the haemopoietic stem cell.