Transitional change of colony stimulating factor requirements for erythroid progenitors.

The course of the differentiation and proliferation of the human erythroid burst-forming units (BFU-E) to colony-forming units (CFU-E) was directly investigated using a combination of highly purified BFU-E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU-E with a purity of 45-79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU-E required not only rEP but also rIL-3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 +/- 20-fold above the starting cells, while erythroid progenitors increased 156 +/- 74-fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rEP combined with or without rIL-3 showed the following: 1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. 2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rIL-3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU-E. These studies provide a quantitative assessment of the loss of IL-3 dependency by BFU-E and indicate that the size of the generated erythroid colonies and their IL-3 requirement correlate with the erythroid differentiated state.     

Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors

The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.     

Canine BFU-e progenitors: adaptation of a reproducible assay and anatomical distribution.

In vitro cloning assays are used increasingly in investigative hematotoxicology and in screening candidate compounds for their hematotoxic potential. To expand these applications, a practical cloning assay for erythroid burst-forming units (BFU-e) that uses a microplasma clot (MPC) system was adapted to the dog, a species used extensively in experimental hematology and drug development. This system offers the advantage over the methylcellulose and soft agar culture systems of allowing specimen fixation and, therefore, morphological and cytochemical evaluation. The distribution of BFU-e among various anatomic sites was assessed using the MPC cloning system, which was modified to optimize the BFU-e growth. BFU-e growth required only erythropoietin (Epo) in the culture medium and there was no need for an exogenous source of burst-promoting activity (BPA). The cloning efficiency was linearly proportional to the plating concentrations of Epo and marrow mononuclear cells (MMC) over a range of 0 to 3 U Epo and 1 x 10(5) to 3 x 10(5) MMC per ml of culture, respectively. Increases in concentrations of Epo and MMC beyond these levels were not associated with linear growth. The addition of transferrin and spleen-conditioned medium containing a mixture of growth factors (including BPA) reduced BFU-e growth. The relative concentration of BFU-e was comparable among samples collected from the iliac crest, femur, and humerus. Serial cultures performed on individual dogs were highly reproducible and there was little variation in BFU-e activity among dogs of comparable age. It was concluded that the MPC system is a practical and reproducible cloning system for early (BFU-e), as well as late erythroid colony-forming units (CFU-e) in the dog. The concentration of BFU-e appears comparable throughout the active marrow; therefore, various anatomic sites can be used interchangeably for serial quantitative analysis of this progenitor.     

Effect of L-phenylalanine methyl ester on the colony formation of hematopoietic progenitor cells from human bone marrow

We have previously shown that L-phenylalanine methyl ester (PME) is capable of removing monocytes and enhancing the growth of hematopoietic colonies from human peripheral blood (PB) mononuclear cells (MNC). In the present study, we further compared the effect of PME on the colony formation of bone marrow (BM) and PB. Low density (less than or equal to 1.077 g/ml) MNC were obtained by Ficoll-diatrizoate density gradient centrifugation. Granulocyte/macrophage colony-forming units (CFU-gm) and erythroid burst-forming units (BFU-e) were cultured in agarose with conditioned media (CM) and/or interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage-CSF (GM-CSF). Treatment of BM MNC with 5 mM PME for 15 min at room temperature yielded a nucleated cell recovery of 44.8 +/- 5.0% (mean +/- SE; N = 8). CFU-gm were enriched 2.7-fold (range 2.0 to 4.8). Using CM or CM supplemented with G-CSF or GM-CSF has minimal effect on the enrichment. Leukocyte differentials revealed that 94.3 +/- 3.05% of the monocytes, as well as 91.2 +/- 1.60% of the cells in the neutrophilic maturation series were removed by PME. Incubation for 40 min in PME abolished CFU-gm formation. BFU-e were not enriched by the PME treatment. In contrast, 40 min incubation of PB MNC produced higher enrichment of CFU-gm than that obtained from 15 min of treatment, although lower cell recovery was obtained with the longer treatment time. In conclusion, we have demonstrated that phagocytic cells can be removed from BM or PB MNC by PME treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors

To facilitate the direct study of the molecular events that control the development of human burst-forming units-erythroid (BFU-E), we have developed a method to purify BFU-E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase-treated and IgG-coated sheep erythrocytes, positive panning with anti-My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU-E were purified from 0.04% to 56.6%, a 1,400-fold purification with a 13% yield. More than 90% of purified BFU-E were recombinant interleukin-3 (rIL-3) dependent, which survived for 48 h with rIL-3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rIL-3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony-forming units-erythroid (CFU-E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I-rEP was incubated for 5 h at 37 degrees C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24-72 h, and the presence of erythropoietin (EP) receptors was investigated using autoradiography. Specific binding of 125I-rEP was detected in 19 +/- 7% of the initial day 1 BFU-E. The percentage of 125I-rEP-binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU-E matured. 125I-rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU-E have a much lower number of EP receptors than CFU-E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.     

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.     

The generation of colony-forming cells (CFC) and the expansion of hematopoiesis in cultures of human cord blood cells is dependent on the presence of stem cell factor (SCF)

We have analyzed the effect of stem cell factor (SCF), alone or in combination with other growth factors, on the generation of colony-forming cells (CFC) and on the expansion of hematopoiesisin vitro from light density, soybean agglutinin^–, CD34⁺ cord blood cells under serum-deprived conditions. The growth factors were either added only once at the onset of the culture or added every few days when the cultures were demidepopulated and refed with fresh medium. No growth factor, alone, generated CFC or expanded hematopoiesis under these conditions. However, SCF, in combination with interleukin 3 (IL-3) or with late-acting factors (granulocyte colony-stimulating factor (G-CSF) or erythropoietin (Epo)), generated large numbers of mature cells as well as CFC. The number of CFC generated depended on the refeeding procedure adopted. In cultures never refed, the CFC numbers increased from > 160 CFC/culture at day 0 to > 3000 CFC at day 10. The CFC numbers stayed above the input levels for 25 days before declining. Almost no CFC were detectable after one month. In contrast, in cultures regularly refed, CFC were detectable for at least 40 days. The lineages of the mature cells and the types of CFC generated varied with the different growth factors. In the presence of SCF plus IL-3, erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (GM-CFC) were generated and erythroid as well as myelomonocytic precursors were present among the differentiated cells. In contrast, in the presence of SCF and G-CSF or Epo, the progenitor cells as well as the differentiated cells were dictated by the late-acting growth factor (i.e. mostly G-CFC and myeloid cells in the presence of SCF and G-CSF vs. BFU-E, erythroid colony-forming cells (CFU-E) and erythroblasts in the presence of SCF and Epo). Thus, marked expansion of erythropoiesis and granulopoiesis can be achievedin vitro by as few as two factors — SCF acting as the early factor along with the appropriate late-acting factor.Paper presented in part at the World Congress on Cell Cultures, Washington D.C., 21–24 June 1992.     

CSF-responsive bone marrow cells in liquid culture: characterization and screening applications

In a previous study, colony-stimulating factor (CSF) activity assayed in colony culture correlated closely with 3HTdR uptake by human marrow cells depleted of adherent cells. To use this assay for screening media for CSF and immunotoxins for marrow toxicity, cells growing in liquid culture were compared to conventional granulocyte/macrophage (CFU-gm) colony assays. CSF dose-response relationships for liquid and colony-forming assays were nearly identical. 3HTdR uptake by nonadherent marrow cells was CSF dose-related, and there was a linear relationship between number of cells cultured and 3HTdR uptake. Ricin cytotoxicity curves for liquid cultures and CFU-gm were identical on day 7 but showed some disparity with day 14 cultures. Results with all cultures showed 3HTdR uptake to be most closely correlated with CFU-gm colony, rather than cluster, growth. Myeloid cell differentiation in liquid culture was similar to colony cultures, producing mixtures of granulocytes, macrophages and eosinophils. By combining cell and differential counts, production of various myeloid cells could be quantitated. Cytotoxicity of anti-Ia for CFU-gm and liquid culture cells was compared and the majority of both cell populations expressed Ia-like antigens. Simultaneous staining for surface antigens and DNA content was used to characterize proliferating marrow cells, and the vast majority of cells expressed myeloid markers. Transferrin receptors were displayed by cells in S/G2/M and appeared after CSF stimulation on G0/G1 cells. We conclude liquid cultures can be used to screen conditioned media for human CSF and to screen for cytotoxicity to normal myeloid precursor cells. Behavior of CSF-responsive cells in liquid culture appears most closely related to that of CFU-gm colony-forming cells, and characterization of CSF-stimulated cells allows quantitative as well as qualitative estimates of myeloid cell production.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Enrichment culture and isolation of slow-growing bacteria

 Enrichment containing large numbers of slow-growing bacteria was developed by repeated batch culture under high biomass concentrations (more than 10 000 mg biomass/l). The characteristics of slow-growing bacterial populations were elucidated by application of colony-forming-curve (CFC) analysis. The CFC were obtained by counting the number of visible colonies on agar plates at successive intervals. The enrichment consisted of several groups with different colony-forming rates and the slow-growing bacteria appeared on cell extract/agar plates after 7 days of incubation. It was found that large numbers of slow-growing bacteria survived under starvation conditions. One of the slow colony-forming bacteria, strain TI-X7, was tentatively identified as being of the genus Micrococcus. The enrichment contained a large amount of Micrococcus-like tetrad cells. The dialysate fractions in excess cell extract, permeable through dialysis tubing, were extremely effective for growth of strain TI-X7. Received: 15 December 1995/Accepted: 20 February 1996     

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: comparison of unconjugated antibody with an immunotoxin containing recombinant ricin A chain

We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.     

Growth kinetics of progenitor cell-enriched hematopoietic cell populations in long-term liquid cultures under continuous removal of mature cells

BACKGROUND: During long-term culture of primitive hematopoietic cells large numbers of mature cells are generated that, on the one hand, consume nutrients and cytokines present in the medium and, on the other hand, may produce or elicit the production of soluble factors that limit the growth of primitive cells. Thus it is possible that under standard culture conditions hematopoietic stem and progenitor cells are unable to display their true proliferation and expansion potentials. METHODS: Hematopoietic cell populations, enriched for CD34+ cells, were obtained from both umbilical cord blood (UCB) and mobilized peripheral blood (MPB), and cultured in cytokine-supplemented liquid culture, under continuous removal of mature cells by means of weekly re-selection of primitive, lineage-negative (Lin-) cells. Proliferation and expansion capacities of such cells were determined weekly for a 42-day culture period. RESULTS: As expected, based on our previous studies in standard liquid cultures, throughout the culture period there was a continuous decrease in the proportion of progenitor cells; however, after every re-selection on days 7, 14 and 21, there was a significant enrichment for both CD34+ cells and colony-forming cells (CFC). As a result of such an enrichment, the cumulative increase in the numbers of total cells and CFC in cultures with two, three or four selections was significantly higher than the increments observed in standard cultures, in which only a single selection was performed on day 0. Cultures of UCB cells showed consistently higher levels of both total cells and CFC than cultures of MPB cells. DISCUSSION: Taken together, these results indicate that continuous removal of mature cells from liquid cultures of primitive progenitors results in higher increments in the levels of both total cells and CFC.     

Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture

Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P>0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P>0.75 to P>0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.Abbreviations ANOVA analysis of variance - CHO Chinese hamster ovary cells - CFA colony-forming ability - dhfr gene for dihydrofolate reductase - DHAP dihydroxyacetone phosphate - FBS fetal bovine serum - G-3-P glyceraldehyde-3-phosphate - GloI glyoxalase I - GloII glyoxalase II - GSH reduced glutathione - HPLC high-performance liquid chromatography - IMDM Iscove's modified Dulbecco's medium - MTX methotrexate - 2-MQ 2-methylquinoxaline - 5-MQ 5-methylquinoxaline - MEM minimal essential medium - P_i inorganic phosphate - PCA perchloric acid - o-PD o-phenylenediamine     

Expression of tumor-associated surface membrane antigens on marrow CFU-s, CFC-gm, BFU-e, and brain CFU-s

Antiserum raised against a mouse mast cell line (FMP1) reacts with 90% to 100% of spleen colony-forming units (CFU-s), granulocyte/macrophage colony-forming cells (CFC-gm), erythroid burst-forming units (BFU-e), and 15% of nucleated marrow cells, using a complement-dependent cytotoxicity assay. We demonstrated that bone marrow, spleen, or thymus cells are able to absorb this activity from the antiserum. Although mouse brain cells have low reactivity with anti-FMP1 serum, the cytolysis level was reduced to background when antiserum was absorbed with brain cells. In addition, colony formation by marrow CFU-s, CFC-gm, and BFU-e was no longer prevented when the cells were incubated with brain-absorbed anti-FMP1 serum and complement. These findings suggest the presence of brain-associated antigens on CFU-s, CFC-gm, and BFU-e. To test whether a CFU-s accessory cell population in marrow is affected by treatment with anti-FMP1 serum and complement, antibody-treated marrow cells were mixed with large numbers of thymocytes and injected into recipient mice. Colony formation was not altered, indicating that the antiserum reacted directly with antigens on CFU-s and not on CFU-s accessory cells.     

Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Differential proliferative effects of transforming growth factor-beta on human hematopoietic progenitor cells

Transforming growth factor-beta (TGF beta) regulates cell growth and differentiation in numerous cell systems, including several hematopoietic lineages. We used in vitro cultures of highly enriched hematopoietic progenitor cells stimulated by natural and recombinant growth factors to investigate the biologic effects of TGF beta 1 and TGF beta 2 on erythroid (CFU-E and burst-forming unit (BFU)-E), granulocyte-macrophage (CFU-GM) and multilineage (i.e., granulocyte, erythroid, macrophage, and megakaryocyte; CFU-GEMM) colony-forming cells. In the absence of exogenous CSF, neither TGF beta 1 nor TGF beta 2 supported progenitor cell growth. In the presence of recombinant or natural CSF, picomolar concentrations of TGF beta 1 inhibited growth of CFU-E, BFU-E, and CFU-GEMM and enhanced growth of day 7 CFU-GM. Inhibition of CFU-E and BFU-E by human and porcine TGF beta 1 was similar, ranging from 17 to 73% over a concentration range of 0.05 to 1.0 ng/ml, and was largely independent of the type of burst-promoting activity used (rIL-3 vs cell line 5637-conditioned medium). Inhibition of CFU-GEMM ranged from 79 to 98% over a concentration range of 0.25 to 1.0 ng/ml. The inhibitory effect of TGF beta 1 was progressively lost when its addition was delayed for 40 to 120 h, suggesting a mode of action during early cell divisions. In contrast, growth of CFU-GM stimulated by plateau concentrations of human rG-CSF, rGM-CSF, and rIL-3 was enhanced up to 154 +/- 22% by human TGF beta 1. Porcine platelet-derived TGF beta 2 was essentially without effect on the progenitor populations examined. These results support the hypothesis that TGF beta may play role in the regulation of hematopoietic progenitor cell proliferation by differentially affecting individual lineages and is apparently capable of doing so in the relative absence of marrow accessory cells.     

Correspondence between the development of hemopoietic tissue and the time of colony formation by colony-forming cells

The development of a haemopoietic tissue and the time when colony-forming cells in it formed detectable colonies were studied with in vivo spleen colony-forming units (CFUs) and in vitro high-proliferation-potential colony-forming cells (HPP CFC). Cells that form colonies first are developmentally more mature than those doing so later. Marrow containing mature spleen colony-forming cells formed fewer cells in the femora of recipients than that which contained early colony-forming cells. The growth curve of developmentally early high-proliferation potential-colony-forming cells was steeper than that of later cells. The time period before colony-formation occurs is a property of the colony-forming cell and is not due to regulatory mechanisms in the animal or to regulatory cells in the haemopoietic stroma.     

Comparative studies on the characteristics of proliferation and differentiation of spleen colony-forming cells

Using a single spleen colony transplantation technique and sex chromosome typing as a natural cytogenetic marker, most spleen colony-forming cells (CFC) in adult bone marrow or fetal livers of inbred LACA or C57 mice re-established hemopoiesis in lethally irradiated mice when the spleen colonies were sampled at 13 days after transplantation. However, most of the spleen colony-forming cells in the peripheral blood of normal mice possess little potential for proliferation and are less efficient in the re-establishment of hemopoiesis in lethally irradiated mice. The CFC population is heterogeneous in the mice. From the subsequent retransplantation of colonies from colony-forming cells in the peripheral blood, the simple assessment of spleen colony-forming units (CFU-s) content, based on the number of splenic colonies, does not reliably represent the content of hemopoietic stem cells.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.