Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice.

The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3epsilon cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. I. Morphologic characterization.

A new human breast carcinoma cell line (PMC42) was established from a pleural effusion from a woman with metastatic breast cancer. The cells were significantly pleomorphic even after 3.5 years in continuous culture. Eight different cell types could be characterized morphologically in monolayer culture. Cells cloned in agar and replated in monolayer culture were equally heterogenous. Cells also grew in suspension in papillary clusters that structurally resembled glandular organoids. Electron microscopy confirmed the differentiated structure of these cells in which many of the features of lactating breast tissue were evident. It is proposed that this is a culture derived from a malignant breast stem that has retained its ability to differentiate in vitro.

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. II. Characterization of cells growing as organoids.

A new human breast carcinoma cell line (PMC42) has been further characterized. The cells can grow either as monolayers or as floating cords of cells. The cords grow in suspension for long periods but may spontaneously attach and grow out to form a typical PMC42 monolayer. Ultrastructurally, the cells resemble breast ductal cells in many respects. Both epidermal growth factor (EGF) and prolactin induce ultrastructural changes, and lipid production is stimulated markedly by both factors. EGF also promoted the attachment of the floating cords and the growth of cells from these cords as monolayer cultures. The karyotype of the cord cells is different from that previously described for the monolayer cultures. Cord cells are hypodiploid (mode 39), whereas the monolayer cultures are subtriploid (mode 66). Although the ploidy is different, the karyotypes are related with 9 marker chromosomes being common to both populations. In addition, cultures in which cords have attached and in which cells are growing out as monolayers are bimodal with 10-20% of the cells becoming pseudotetraploid with a mode of 77.

Transport and metabolism of 1-beta-D-arabinofuranosylcytosine in human ovarian adenocarcinoma cells.

1-beta-D-Arabinofuranosylcytosine (araC) is an effective drug in the i.p. therapy of ovarian carcinoma but little is known of its transport and metabolism in this tumor. Influx of araC at 1 microM into cultured human ovarian carcinoma cells (CI 80-13S) was largely inhibited by nanomolar concentrations of the nucleoside transport inhibitor, nitrobenzylthioinosine, while the residual influx (approximately 10%) was inhibited only by micromolar concentrations of nitrobenzylthioinosine. There was a two fold greater density of specific [3H]nitrobenzylthioinosine binding to the nucleoside transporters on the ovarian than on cultured human leukemic cells (RC2a). Calculated turnover rates of the nucleoside transporter for 1 microM araC were 5-fold less in ovarian than in leukemic cells. The major metabolic product of araC was 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) which accumulated in the ovarian cells to levels half those achieved in the leukemic cells. AraC was the major product of araCTP degradation in ovarian cells consistent with a pathway (araCTP--------araCMP----araC) which is different from that previously found in leukemic cells (araCTP--------araCMP----araUMP----araU). Despite these differences, ovarian carcinoma cells show substantial accumulation of araCTP from extracellular araC.

Reversal of the multidrug resistance phenotype with cremophor EL, a common vehicle for water-insoluble vitamins and drugs.

A polyethoxylated castor oil, Cremophor EL, which is used as a vehicle for p.o. and i.v. administration of water-insoluble compounds in humans, can reverse the multidrug resistance (MDR) phenotype at doses which are likely to be readily achievable clinically. Using flow cytofluorometric analysis of daunorubicin (DNR) uptake as a measure of the expression of the MDR phenotype, Cremophor EL (1:10(3] in the growth medium increased intracellular DNR in an MDR cell line, R100 cells, to levels similar to that observed in the drug-sensitive parental cells, CCRF-CEM. A similar Cremophor EL-induced increase in DNR uptake was also observed in an unrelated MDR cell line derived from K562 cells. Cremophor EL (less than or equal to 3:10(4] did not inhibit the growth of CCRF-CEM cells or its vinblastine-resistant derivative, R100 cells, but would significantly increase the sensitivity of R100 cells to both vinblastine and DNR. Also Cremophor EL did not increase the sensitivity of normal bone marrow progenitor cells cultured in vitro to high concentrations of vinblastine. Cremophor EL may prove to be a relatively pharmacologically inactive addition to chemotherapeutic protocols which may be able to reverse the MDR phenotype in tumors and also help to prevent the selection of MDR cell variants from within a tumor cell population during chemotherapy.

c-Myb is critical for murine colon development.

The mammalian colon develops from a simple tube of undifferentiated cells into a complex, highly ordered organ, with a continuously self-renewing epithelial layer. We have previously described c-Myb expression in the epithelia of murine and human colon crypts and documented increased expression in colorectal adenocarcinoma cells. To investigate the role of c-Myb in colonic epithelium development, we have used embryos with a disrupted c-myb gene. Prior to the in utero death of these embryos at E15, we excised colon tissue and transplanted it under the kidney capsule of recipient mice to allow further development and cyto-differentiation. Compared to the colons of wildtype and heterozygous littermates, the c-myb homozygous knockout colon is highly irregular with a disordered epithelium and abnormal crypts. In addition, the expression of Bcl-2, a known target of c-Myb, is reduced and apoptosis is increased, indicating a critical requirement for c-Myb in normal colon development.

Age-related changes in extramedullary hematopoiesis in the spleen of normal and perturbed osteopetrotic (op/op) mice.

Occlusion of the marrow cavity by excessive bone formation in young osteopetrotic (op/op) mice results in a significant reduction in the space available for hematopoiesis. At this time, splenomegaly is evident, and the spleen is a site of significant extramedullary hematopoiesis. In vitro clonal assays of spleen cell suspensions in young op/op mice demonstrated a 22-fold elevation in the content of high proliferative potential colony-forming cells (HPP-CFC) (4 weeks of age) and a 14-fold elevation in the content of committed progenitors responsive to colony-stimulating factor-1 (CSF-1) (6 weeks of age). Flow-cytometric analysis also demonstrated a shift in the myeloid:lymphoid cell ratio in the spleens of young op/op mice, with a 35% reduction in the number of B220+ cells, and a two-fold increase in myeloid cells expressing the hematopoietic cell surface lineage antigens Mac-1 and Gr-1. However, the hematopoietic deficiencies of op/op mice are not permanent. An age-related progressive remodeling of the bone marrow cavity results in the correction of bone marrow parameters by 22 weeks of age. This correction in marrow hematopoietic activity is accompanied by a resolution of the splenomegaly, a progressive decrease in splenic hematopoietic activity at both the primitive and committed progenitor cell levels, and a correction of the lymphoid:myeloid cell ratio. Negative immunomagnetic selection of splenic hematopoietic progenitor cells from op/op and control littermate mice, followed by analysis of their expansion in liquid culture, demonstrated that primitive hematopoietic progenitor cells of high proliferative potential continued to reside in the spleen of old op/op mice. The response of these mice to a 5-fluorouracil (5-FU) cytotoxic challenge suggested that this pool of primitive progenitor cells acted as a hematopoietic reserve capable of rapidly responding to hematopoietic perturbation.

Classes of primitive murine megakaryocytic progenitor cells.

We describe high proliferative potential colony-forming cells-megakaryocyte (HPP-CFU-Mk) from mouse bone marrow preparations using known cytokine combinations. These primitive precursors, which generate at least 80 megakaryocytes per colony, were detected from bone marrow populations enriched for primitive cells, either following treatment with 5-fluorouracil (5-FU) or after enrichment from normal bone marrow using immunological procedures. HPP-CFU-Mk were most reproducibly grown in the presence of interleukin-1 (IL-1) plus IL-3 plus IL-6, and mostly grew as a single large aggregate, rarely forming multiple foci. Cell separation studies showed that the HPP-CFU-Mk have membrane properties that characterized these cells as being intermediate between high proliferative myeloid primitive progenitors (high proliferative colony-forming cells [HPP-CFC]) and committed megakaryocyte progenitors (CFU-Mk). The data show that HPP-CFU-Mk can be designated as primitive cells on the basis of their being spared in vivo after 5-FU treatment, their proliferative potential to produce megakaryocytes, and their cofractionation with a proportion of primitive myeloid progenitor cells.

Temporal thrombocytopenia after engraftment with defined stem cells with long-term marrow reconstituting activity.

Delayed platelet recovery and altered marrow megakaryocytopoiesis was observed in highly irradiated mice following transplantation with 1000 lineage-negative, stem cell antigen-positive (Lin-Sca-1+) cells. Thirty days after transplantation, the mice were thrombocytopenic. Normal platelet levels were reestablished by 90 days after transplantation. Platelet levels were established from the bone marrow with altered megakaryocytopoiesis, however. Megakaryocyte progenitor numbers were found at reduced levels in the reconstituted marrow at all time points assessed. Bone marrow function was also different in that marrow reserve was also diminished. While the transplantation regime did give long-term reconstitution of blood cells, the quality of the marrow reserve was significantly impaired as revealed by response to further hematopoietic challenge. The data indicate that mice transplanted with a population of highly defined stem cells have perturbed marrow function, one characteristic being an altered process of megakaryocytopoiesis.

An improved negative immunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow.

An improved negative immunomagnetic selection strategy has been devised for the enrichment of primitive hemopoietic cells using the high proliferative potential colony-forming cell (HPP-CFC) assay as an index of stem cell purification. Immunomagnetic selection was carried out using goat anti-rat conjugated M-450 Dynabeads and a cocktail of rat monoclonal antibodies directed against lineage antigens expressed on B-lymphocytes (B220), neutrophils and activated macrophages (7/4), differentiating erythroid cells (YW 25.12.7), and T-lymphocyte subsets (Lyt-2 and L3T4). This negative selection strategy results in the highly reproducible enrichment of HPP-CFC with negligible loss of HPP-CFC at the immunomagnetic selection step. A 30-fold enrichment of HPP-CFC stimulated by interleukin 3 (IL-3) plus colony-stimulating factor (CSF-1), or interleukin 1 alpha (IL-1 alpha) plus IL-3 plus CSF-1, is obtained with simultaneous resolution of HPP-CFC from progenitor cells of low proliferative potential responsive to CSF-1 alone (LPP-CFC). Flow cytometric analysis of these lineage-negative cells reveals that they almost exclusively exhibit the light-scattering characteristics of blast cells and the morphology of a candidate hemopoietic stem cell. Positive fluorescence-activated cell sorting of immunomagnetically pre-enriched normal bone marrow cells using wheat germ agglutinin yields cell preparations with a cloning efficiency of up to 45% and a HPP-CFC content of 20%.

Multiparameter analysis of transplantable hemopoietic stem cells. II. Stem cells of long-term bone marrow-reconstituted recipients.

Marrow obtained from mice (referred to as [X + BM] mice) 3 months after gamma-irradiation (9 Gy) and bone marrow inoculation (0.1 femur equivalents) showed a reduced capacity to reconstitute hemopoiesis of irradiated mice and an increased sensitivity to 5-fluorouracil. Sorting of marrow from (X + BM) mice on the basis of low angle and 90 degrees scatter, and low rhodamine 123 fluorescence, showed that the set of cells that in normal mice is enriched for cells efficient at hemopoietic reconstitution manifested the greatest reduction in hemopoietic reconstituting ability. In spite of this reduction this fraction contained as many 13-day spleen colony-forming units (CFU-S13) and high proliferative potential colony-forming cells (HPP-CFC) as the equivalent fraction from normal littermate mice. This could be explained by postulating that neither CFU-S13 nor HPP-CFC are responsible for hemopoietic reconstitution, but that this is dependent on an earlier, pre-CFU-S13 cell. Alternatively only a subset of either CFU-S13 or HPP-CFC is responsible for long-term hemopoietic reconstitution after lethal irradiation. It would appear that at present there is no adequate method of predicting the hemopoietic reconstituting ability of a given marrow, other than to test it by injection into lethally irradiated hosts.

Characterization of early leukemic cells in spleen and bone marrow of Friend virus infected mice.

Nonidentity of leukemic cell populations of DBA/w mice (TCFU and CRVEI) early after Friend Visus infection has been demonstrated for spleen by different growth kinetics of these cell populations during leukemogenesis and for spleen and bone marrow of DBA/w mice by the velocity sedimentation technique. In addition the velocity sedimentation data indicate a heterogeneity of the TCFU population. Stem cell analysis of TCFU and CFUS derived spleen colonies showed that TCFU colonies contain significantly more CFUEI than the surrounding tissue. The erythroid nature of TCFU colonies is confirmed by the almost complete absence of CFUC from these colonies as compared to CFUS derived spleen colonies.

Multiparameter analysis of transplantable hemopoietic stem cells: I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence.

A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells that preferentially home to the marrow of lethally irradiated mice to be enriched and separated from the majority of spleen colony-forming cells that are assayed 13 days after transplantation (CFU-S13). First, bone marrow cells are centrifuged in a discontinuous bovine serum albumin gradient. Low-density cells (1.060-1.068 g/cm3) are harvested and labeled with the supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled cells are analyzed using a fluorescence-activated cell sorter, and cells are sorted on the basis of relative Rh123 fluorescence within a predetermined forward versus 90 degrees red light scatter window that has been optimized for the recovery and enrichment of cells with marrow repopulating ability (MRA). Cells with MRA were characterized by relatively low Rh123 fluorescence and could be separated from a fraction that fluoresced more intensely and contained the majority of CFU-S13 but low MRA. The ratios of low-fluorescent:high-fluorescent MRA and CFU-S13 were 20:1 and 0.5:1, respectively, and the resultant ratio of MRA:CFU-S13 was 556:1 in the low-fluorescent fraction, and 48:1 in the high-fluorescent fraction. Cells with platelet repopulating ability cofractionate with MRA whereas cells with erythroid repopulating ability remain associated with CFU-S13. High-proliferative-potential macrophage colony-forming cells that form colonies in agar in the presence of the combined stimulus of pregnant mouse uterus extract plus human-spleen-conditioned medium were present in both fractions. Between 20% and 30% of transplantable hemopoietic stem cells has been recovered in approximately 1% of total nucleated cells from the starting preparation, and enrichments of 25- to 30-fold have been achieved permitting hemopoietic reconstitution of lethally irradiated host mice with as few as 400 donor cells.

Characterization and enrichment of macrophage progenitor cells from normal and 5-fluorouracil treated mouse bone marrow by unit gravity sedimentation.

Unit gravity sedimentation has been used to characterize and enrich cells from normal, post-5-fluorouracil (FU) and post 5-fluorouracil plus endotoxin (FUEt) regenerating mouse bone marrow with respect to two classes of macrophage progenitor cells. The two classes of progenitor cells assayed were (1) those responsive to the combined stimulus of pregnant mouse uterus extract (PMUE) plus human spleen conditioned medium (HUSPCM), and (2) those responsive to PMUE alone. In contrast to the bimodal nucleated cell distribution of normal marrow, 7 day post-FU marrow exhibited a unimodal nucleated cell profile. In marrow from 7 day post-FUEt treated mice, in ;which marrow regeneration was accelerated, there was a reemergence of a second nucleated cell peak. In addition, in 7 day post-FU and 7 day post-FUEt marrow there was a shift in the modal sedimentation velocities of both PMUE responsive and PMUE + HUSPCM responsive populations to higher values. The combined effect of these changes resulted in a marked increase in plating efficiencies of the peak enrichment fractions, reaching 12.5% in 7 day post-FUEt marrow. However, the highest yield of progenitor cells responsive to the combined stimulus of PMUE + HUSPCM compared to those responsive to PMUE alone were obtained in 7 day post-FU marrow.

The concentration and resolution of primitive hemopoietic cells from normal mouse bone marrow by negative selection using monoclonal antibodies and Dynabead monodisperse magnetic microspheres.

High proliferative potential colony-forming cells (HPP-CFC) detected in clonal agar culture in the presence of the combined stimulus of colony-stimulating factor 1 (CSF-1) + interleukin 3 (IL-3) + interleukin 1 alpha (IL-1 alpha) are closely related to developmentally early progenitor cells capable of reconstituting the hemopoietic system of lethally irradiated mice following transplantation. Flow cytometric analysis and sorting of normal, unperturbed bone marrow has shown that HPP-CFC are B220- and 7/4-, whereas the committed progenitors of the macrophage lineage responsive to CSF-1 alone (CSFCSF-1) are B220- and 7/4+. Negative immunomagnetic selection using an anti-7/4, anti-B220 antibody cocktail and second-antibody-coupled Dynabead microspheres to replace flow cytometry results in the highly reproducible and specific enrichment of HPP-CFC, and simultaneous resolution of HPP-CFC from CFCCSF-1. The tenfold enrichment of HPP-CFC compared with unfractionated bone marrow cell suspensions was comparable to that obtained by fluorescence-activated cell sorting. Enrichment was achieved with negligible loss of HPP-CFC at the immunomagnetic bead selection step, and 65% of HPP-CFC were recovered. The method is rapid, highly reproducible, and efficient, and has wide application to the separation of rare hemopoietic cells from normal bone marrow.

Developmental hematopoiesis from prenatal to young-adult life in the mouse model.

Five measurements of hematopoietic function were made in the mouse from midfetal life to young adulthood. These included two in vivo (day-8 colony-forming unit-spleen [CFU-S8] and day-12 CFU-S [CFU-S12]) and two in vitro clonal measurements of hematopoietic stem and progenitor cells (high proliferative potential colony-forming cell [HPP-CFC] and CFC of low proliferative potential [LPP-CFC]) as well as an in vitro clonal measurement of colony-forming unit-fibroblast (CFU-F). The appearance, increase, subsequent decrease, and later emergence and increase of each of these parameters in the fetal-liver, newborn, growing-infant, and young-adult bone marrow were correlated and found to be in parallel. Exceptions to this included the earlier appearance in the fetal liver of CFU-F and the relatively differentiated hematopoietic LPP-CFC. The pattern of emergence of these progenitor cell subpopulations in the fetal liver may be related, in part to the timing of the hematopoietic microenvironment development and the relative frequencies of progenitor cell types in the circulation. This developmental study in the mouse model describes additional correlations between in vivo and in vitro colony-forming stem cells and fibroblastic stromal colony-forming cells, and it suggests the dependence of hematopoietic stem cells upon the stromal microenvironment for the necessary conditions for hematopoietic stem cell lodgment, growth, and maturation.

Modified thrombopoietic response to 5-FU in mice following transplantation of Lin-Sca-1+ bone marrow cells.

An experimental murine model of bone marrow transplantation (BMT) has been used to study the mechanisms of platelet production following transplantation. A defined primitive population of hematopoietic bone marrow cells (1000 Lin-Sca-1+) was isolated and transplanted into lethally irradiated (13 Gy) syngeneic recipient mice. Platelet counts, but neither red nor white blood cell (WBC) counts, were low 30 days after transplantation. By 90 days, platelet levels had normalized in transplanted mice, but this occurred from a reduced megakaryocyte progenitor (CFU-Mk) pool, implying that altered bone marrow control was involved in platelet production. To assess the capacity of the bone marrow of these compensated mice to sustain platelet production, the rate and degree of recovery were examined following administration of 150 mg/kg of 5-fluorouracil (5-FU) 90 days after transplantation. Transplanted mice showed a delay, both in platelet recovery and rebound thrombocytosis, after 5-FU administration when compared to normal littermates treated with 5-FU. The regeneration and expansion of bone marrow CFU-Mk and mature megakaryocytes was retarded in the transplanted mice and explained the altered platelet kinetics. The onset of increased platelet and mature megakaryocyte size, however, was not different between the two groups, indicating that the transplanted mice responded normally to the mechanisms controlling megakaryocyte development and platelet formation. The data suggest that following BMT a limitation in the proliferative capacity of primitive hematopoietic cells results in a smaller pool of megakaryocyte precursors. Compensatory adjustment within the megakaryocyte lineage, nevertheless, results in normalization of megakaryocyte and platelet number. The ability of transplanted mice to sustain platelet production when challenged with increased platelet demand is not limited by megakaryocytic maturation but by a restriction in proliferation or differentiation from the stem cell pool.

The relationship between different high proliferative potential colony-forming cells in mouse bone marrow.

Previous work has shown that part of the hierarchical structure of the hematopoietic system can be described by HPP-CFC-1 (primitive high proliferative potential colony-forming cells responding to colony-stimulating factor-1 [CSF-1] + interleukin-3 [IL-3] + IL-1), HPP-CFC-2 (more mature HPP-CFC responding to CSF-1 + IL-3), and mature HPP-CFC responding to the single factors, CSF-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-3. In this study, we have attempted to relate the murine HPP-CFC, stimulated by various combinations of growth factors (GFs)--CSF-1, GM-CSF, IL-3, IL-6, IL-1, stem cell factor (SCF), and transforming growth factor-beta (TGF-beta)--and by CSF-1, GM-CSF, and IL-3 on their own, to these known progenitors. Studies involving regeneration of the bone marrow after 5-fluorouracil (5-FU) treatment, generation of progenitors in liquid cultures in response to different GF combinations, and the HPP-CFC content of lineage-negative rhodamine-sorted bone marrow (BM) fractions have indicated that: 1. the combinations CSF-1 + IL-3 + IL-1 + SCF and CSF-1 + IL-3 + IL-1 + IL-6, and possibly CSF-1 + GM-CSF + IL-3 + IL-1, stimulate pre-HPP-CFC-1; 2. the combinations CSF-1 + IL-1 + GM-CSF, CSF-1 + IL-1 + IL-6, CSF-1 + IL-1 + SCF, CSF-1 + IL-3 + SCF, CSF-1 + IL-6 + SCF, and IL-3 + SCF, appear to overlap with the CSF-1 + IL-3 + IL-1 combination to stimulate the more mature cells of the HPP-CFC-1 compartment; 3. the combinations CSF-1 + GM-CSF, CSF-1 + IL-1, CSF-1 + IL-6, and CSF-1 + SCF may stimulate the more mature cells of the HPP-CFC-2 population, while the single factors CSF-1, GM-CSF, and IL-3, as suggested in other reports, may stimulate HPP-CFC that are more mature than the HPP-CFC-2; 4. the combinations IL-3 + IL-6 and SCF + IL-6 appear to stimulate HPP-CFC that overlap with the HPP-CFC-1 population, while those responding to the combination GM-CSF + TGF-beta overlap with the HPP-CFC-2 population within the hematopoietic hierarchy; and 5. CSF-1 and GM-CSF appear to be interchangeable in the combinations studied.

The resolution, enrichment, and organization of normal bone marrow high proliferative potential colony-forming cell subsets on the basis of rhodamine-123 fluorescence.

Cell sorting on the basis of rhodamine-123 (Rh123) fluorescence has been used in conjunction with negative immunomagnetic selection to analyze the high proliferative potential colony-forming cell (HPP-CFC) compartment of normal murine bone marrow and to resolve and enrich HPP-CFC subpopulations responsive to different combinations of the hemopoietic growth factors interleukin 1 alpha (IL-1 alpha), interleukin-3 (IL-3), and colony-stimulating factor 1 (CSF-1). HPP-CFC with a specific requirement for IL-1 alpha plus IL-3 plus CSF-1 in order to proliferate were resolved and enriched on the basis of their low Rh123 retention (Rh-dull), whereas HPP-CFC that grew in the presence of IL-3 plus CSF-1, IL-3 alone, or CSF-1 alone were Rh-bright. Further addition of IL-1 alpha to IL-3 plus CSF-1 stimulated few additional HPP-CFC in the Rh-bright fraction. Our data confirm the value of Rh123 as a probe for the dissection and analysis of the primitive hemopoietic stem cell (PHSC) compartment. These data also show that the Rh123 staining characteristics of IL-1 alpha plus IL-3 plus CSF-1-responsive HPP-CFC are consistent with the hypothesis that these HPP-CFC are closely related to PHSC with long-term reconstituting capacity in vivo and that they are among the most primitive progenitors yet detected in clonal agar culture.