Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Histological study of splenic colonies at different times following the transplantation of hematopoietic cells from embryonic liver

Histological analysis was carried out on the hemopoietic spleen colonies within 7 and 11 days after transplantation of the embryonic liver cells. Large superficial colonies were always present and were, predominantly, erythroid and mixed. Small superficial colonies consisted of undifferentiated cells and, unlike large colonies, appeared on the 11th day only. In the spleen thickness erythroid colonies predominated. The possibility of formation of small superficial 11 day colonies at the expense of pre-CFU-S is discussed.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Granulocyte colony-stimulating factor and drugs elevating extracellular adenosine act additively to enhance the hemopoietic spleen colony formation in irradiated mice.

The effects of combined administration of two drugs elevating extracellular adenosine, namely dipyridamole (DP) and adenosine monophosphate (AMP), and granulocyte colony-stimulating factor (G-CSF) on hemopoietic stem cells in vivo were investigated. The experiments were performed on mice using the endogenous spleen colony formation in gamma-irradiated animals as an endpoint. The results have shown that DP and AMP act additively with G-CSF to enhance spleen colony formation and thus the erythroid repopulation of the spleen. These findings indicate that the signaling pathways of G-CSF and drugs elevating extracellular adenosine can interact at the level of primitive hemopoietic stem cells. The enhancement of hemopoiesis-stimulating effects of G-CSF by DP and AMP, which are low-priced and clinically available drugs, could improve the cost-effectiveness of the therapy with G-CSF.     

Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

Stimulant effect of antilymphocytic serum on early stages of hematopoietic recovery in the spleen of radiation chimeras]

Experiments conducted on 236 BALB/c mice were aimed at the study of the effect of the antilymphocytic serum on the hemopoiesis recovery in the spleen of the radiation chimeras, since the antilymphocytic serum was known to increase the number of macroscopic hemopoietic colonies. This increase was found to be due to the intensification of the first hemopoietic recovery stage, i.e. to the acceleration and intensification of the reticular cell activation in the recepient's spleen.     

Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.     

The action of the cells of hemopoietic organs in chick embryos on mouse CFUdc and CLFUdc]

The humoral influence of cells of hemopoietic organs of chicken embryos of different terms on the development of the colony and cluster formation of mononuclears of the bone marrow of mice was studied in joint cultivation in two-compartment cylindrical diffuse microchambers. The process of formation of colonies and clusters is inhibited by cells of the yolk sac on the 2nd-4th day of the development, by cells of the liver on the 8th-12th day, of the spleen on the 13th-18th day and of the bone marrow--on the 15th day. The yolk sac cells were found to have most considerable inhibiting influence on proliferation and differentiation of cells on the 2nd day of the development of chicken embryo. The yolk sac cells on the 6th day stimulate the formation of colonies and clusters. The yolk sac, beginning from the 4th day of the development, and the liver release humoral factors promoting the formation of erythroid colonies. The erythroid colonies are formed but when cultivated on the vascular membrane of the chicken embryo; the erythroid colonies are not formed when cultivated in the abdominal cavity of mice. Local erythropoietinoid factors are not synthetized by the spleen and bone marrow cells. A supposition is put forward that a combination of the local inhibiting and erythropoietic effects promotes the erythroid differentiation of cells.     

Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody,YBM/42

A rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS-II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25-fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU-E). In the most enriched fraction, 20% of the cells are day 2 CFU-E. Day 7 CFC can therefore be well separated from day 2 CFU-E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G-CFC), macrophage (M-CFC), mixed granulocyte/macrophage (GM-CFC), pure erythroid (E), or mixed erythroid (E_mix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM-CFC and M-CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM-CFC and M-CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.     

The need of B-helpers for the T-lymphocyte regulation of the process of hematopoietic stem cell differentiation

We have detected and characterized a subpopulation of immunoregulatory cells, i.e., B-helpers capable to enhance the activity of Td-lymphocytes and controlling differentiation of syngeneic hemopoietic stem cells in mouse spleen and bone marrow. B-helpers found in the spleen and lymphatic nodes are resistant to radiation (at a dose of 6 Gr) but are impaired when irradiated at 9 Gr. Manifestation of the helper activity does not require either DNA or RNA synthesis but depends on protein synthesis and is mediated by soluble transmitter substances. Initial activation of B-helpers by lipopolysaccharide or alloantigens does not affect their helper functions. In the absence of T-lymphocytes B-cells do not affect differentiation of hemopoietic stem cells; interaction of B-helpers with differentiating Td-lymphocytes is not genetically restricted. Using preparative electrophoresis, we could isolate fractions of Td-lymphocytes which require or do not require B-helper cells in order to induce change in differentiation of hemopoietic stem cells from mainly erythroid to preferentially granulocyte pathway.     

Clonal origin of murine hemopoietic colonies with apparent restriction to granuclocyte-macrophage-megakaryocyte (GMM) differentiation

We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte-macrophage-megakaryocyte (GEMM) colonies could be distinguished from granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y-chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day-8 and day-12 colonies and in seven out of ten day-14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU-E and normoblasts in all of the 50 day-8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU-E, CFU-EM, CFU-GEM, and CFU-GEMM. In contrast, the three day-14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte-macrophage-megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.     

A Study of Hemopoiesis on Sublayer of Peritoneal Cells in the Presence of Hemopoietic Cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

Susceptibility to merocyanine 540-mediated photosensitization: a differentiation marker on murine hematopoietic progenitor cells

Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane-bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540-mediated photosensitization. Late erythroid progenitors (CFU-E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU-E), megakaryocyte progenitors (CFU-Meg), day 7-spleen colony forming cells (day 7-CFU-S), granulocyte/macrophage progenitors (CFU-GM), and day 11-spleen colony forming cells (day 11-CFU-S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies ("clusters"). We conclude that sensitivity to MC 540-mediated photosensitization is develop-mentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late-appearing spleen colonies are the progeny of two distinct classes of progenitor cells.     

Influence of major histocompatibility complex H-2LD class I molecules on spleen colony-forming units

To test whether the major histocompatibility complex class I genes are involved in the regulation of hemopoiesis, the stem cell activities of BALB/c-H-2dm2 (Dm2) mice, which are defective in the expression of H-2L antigens, have been compared with those of the wild-type, BALB/c-Kh, in in vivo and in vitro stem cell assays. In spleen colony-forming unit assays, Dm2 as hosts consistently supported a smaller number of colonies than did BALB/c-Kh. However, both Dm2 and BALB/c-Kh supported a comparable number of colonies in in vitro granulocyte-macrophage colony-forming unit and erythroid colony-forming unit assays. These observations together suggest that the mutation in Dm2 has not affected the hemopoietic potential of the stem cells but may probably affect the hemopoietic microenvironment for the development of the stem cells.