Placental Conditioned Medium Research Articles

A characteristic CFU-GM growth pattern in acute promyelocytic leukemia

Bone marrow and/or peripheral blood cells from 12 patients with acute promyelocytic leukemia (APL) were cultured in soft agar or methylcellulose in the presence of 15% human placental conditioned medium as a source of colony stimulating factor. Buffy coat cells, taken from ten patients when APL was diagnosed, produced a growth pattern in soft agar that was characterized by a high incidence of small, relatively uniform clusters of promyelocytes, which, when stained in situ, reacted strongly with myeloperoxidase, Sudan black B and chloroacetate esterase. In six cases, the leukemic origin of the cluster forming cells was demonstrated by the presence of the t(15;17) in these cells. During periods of complete remission the small clusters were replaced by larger and more diverse aggregates which had a normal karyotype. At relapse the small cluster growth pattern returned. The growth pattern of small clusters is more commonly associated with APL than with other types of acute myeloid leukemia.

Fractionation of CSF activities from human placental conditioned medium.

Colony stimulating factors (CSFs) are required for the survival, proliferation and differentiation of hemopoietic progenitor cells in vitro and possibly in vivo. Recently, several human CSFs have been purified and molecularly cloned from human tumor cell lines. These include granulocyte colony stimulating factor (G-CSF), from the bladder carcinoma cell line 5637 [1, 2] and the squamous cell carcinoma cell line CHU-2 [3], and granulocyte-macrophage colony stimulating factor (GM-CSF), from the Mo T-lymphoblast cell line [4, 5] and the monocytic cell line U937 (personal communication, Dr. J. DeLamarter). In addition, human erythroid potentiating activity (EPA) has been purified and molecularly cloned from the Mo T-lymphoblast cell line [6, 7].

Murine eosinophil differentiation factor. An eosinophil-specific colony-stimulating factor with activity for human cells.

A purified murine lymphokine, eosinophil differentiation factor (EDF), was found to be a selective stimulus for the clonal proliferation and differentiation of murine eosinophil progenitor cells, establishing it as the murine eosinophil colony-stimulating factor (Eo-CSF). EDF was also active on human eosinophil progenitors and mature blood eosinophils, but had no effect on neutrophil or macrophage precursor cells, nor on blood neutrophils. In culture of human bone marrow cells, EDF stimulated equal numbers and equal sizes of eosinophil colonies to develop when compared with human placental conditioned medium, a source of human CSFs, suggesting that all responsive progenitor cells were stimulated. Clone transfer experiments and the linear relationship between number of bone marrow cells plated and colonies produced confirmed that the action of EDF was directly on eosinophil progenitor cells. EDF increased the capacity of human blood eosinophils, but not neutrophils, to kill antibody-coated tumor cells and to phagocytose serum-opsonized yeast cells. This functional activation was associated with the enhanced expression of functional antigens (GFA-1, GFA-2, and the receptor for C3bi) on eosinophils. The possession by EDF (Eo-CSF) of all the properties expected of a human eosinophil CSF raises the possibility that a human analog of this molecule exists, and is involved in the regulation of production and function of human eosinophils in vivo.

Myeloid progenitors in acute non-lymphocytic leukemia: Prognostic value in remission

The prognostic value of in vitro cloning of bone marrow and blood cells was tested in 32 patients with acute non-lymphocytic leukemia (ANLL) at various stages of disease. Using placental conditioned medium (PCM) as a stimulus and panoptical staining methods for the analysis of colony morphology, four different growth types could be distinguished at presentation. Poor response to colony-stimulating activity (GM-CSF) in vitro seemed to be associated with poor survival. Probably due to low patient numbers, this association could not be proven statistically. During remission a progressive decrease of morphologically normal colonies was recognized in the bone marrow and peripheral blood. Thus by in vitro cloning methods the quality of remission could be substantiated.

Assessing cultured colonies automatically

The number of colonies formed by macrophage colony-forming cells and high proliferation potential colony-forming cells [10] was assessed by an image processor. The processor counted and sized colonies accurately, reproducibly, rapidly (2 s/dish) and objectively. The processor also measured the amount of light (in grey levels) the colonies transmitted. The optical density of a colony (the sum of its grey levels) was related to its cellularity. Thus the image processor compared both the number of colonies in samples and their cellularity. Samples of marrow containing high proliferation potential colony-forming cells of different proliferative capacity were prepared by injecting fluorouracil into mice and collecting their marrow 2–10 days later (marrow samples called FU2–FU10) [10]. These samples were cultured with one of three sources of synergistic factor titrated over seven dilutions. Colonies contained approx. 5 × 10 4 cells after 11 days culture but the way that FU2–FU10 marrow grew depended on the interval between treating donors with fluorouracil and collecting their marrow. Samples collected 2–4 days after fluorouracil formed more colonies containing more cells with small increases of synergistic factor whereas samples collected after 8–10 days did neither. It was important to culture samples of marrow with the appropriate synergistic factor for the interval after fluorouracil. Factor(s) derived from the 5637 cell line [12] acted optimally on high proliferation potential colony-forming cells in samples collected 2–8 days after fluorouracil, and factor(s) derived from Wehi 3B cells on high proliferation potential colony-forming cells in samples collected 6–10 days after fluorouracil [16]. Factor(s) derived from placental conditioned medium [11] acted well on samples collected between 2 and 10 days. The proliferative capacity of samples of marrow could also be compared by estimating growth curves for high proliferation potential colony-forming cells in samples collected at successive intervals after fluorouracil.

Serum-free culture of primitive murine hemopoietic colony-forming cells.

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.

Human placental conditioned medium reverses apparent commitment to differentiation of human promyelocytic leukemia cells (HL60)

Using a system of sequential daughter cell transfers in semisolid medium, we have analyzed self-renewal and differentiation of human promyelocytic leukemia cells (HL60) in presence of all-trans retinoic acid and human placental conditioned medium (HPCM). We find that retinoic acid induces coordinated losses of self-renewal potential which are followed by phenotypic differentiation. The latter occurs as an all-or-none event and is reversible in the presence of HPCM. Thus, HL60 cells that apparently had terminally differentiated (as estimated by the ability to reduce nitroblue tetrazolium [NBT]) can lose their differentiation marker and reenter the proliferative pool.

Human Placental Conditioned Medium Reverses Apparent Commitment to Differentiation of Human Promyelocytic Leukemia Cells (HL60)

Using a system of sequential daughter cell transfers in semisolid medium, we have analyzed self-renewal and differentiation of human promyelocytic leukemia cells (HL60) in presence of all-trans retinoic acid and human placental conditioned medium (HPCM). We find that retinoic acid induces coordinated losses of self-renewal potential which are followed by phenotypic differentiation. The latter occurs as an all-or-none event and is reversible in the presence of HPCM. Thus, HL60 cells that apparently had terminally differentiated (as estimated by the ability to reduce nitroblue tetrazolium [NBT]) can lose their differentiation marker and reenter the proliferative pool.

A new method for the growth of myeloid progenitor cells on nitrocellulose paper

A new method has been developed by which human myeloid progenitor cells can be grown on nitrocellulose paper. The method uses human placental conditioned medium for colony-stimulating activity in an agar layer while the cells grow on the overlying nitrocellulose paper in compact colonies containing granulocytic and macrophage cells. The importance of this method is discussed in the light of its usefulness in carrying out molecular studies differentiating hematopoietic cells.

The Clonal Proliferation In Vitro of Enriched Populations of Human Promyelocytes and Myelocytes

The proliferative capacity of normal human promyelocytes and myelocytes was demonstrated and characterized on the basis of clonal proliferation in agar. An enriched population of normal human promyelocytes and myelocytes was obtained from bone marrow using the monoclonal antibody WEM G11 and the fluorescence-activated cell sorter (FACS). In cultures stimulated by placental-conditioned medium, these cells generated peak total clone numbers between days 3 and 5 of culture. Clones disappeared rapidly thereafter. These clones were mainly of subcolony size at day 7, although some colonies were generated by this population. The clones were primarily neutrophilic in type. These cells had a plating efficiency of up to 50%, and clonal proliferation was dependent on stimulation by colony-stimulating factor (CSF).

The clonal proliferation in vitro of enriched populations of human promyelocytes and myelocytes

The proliferative capacity of normal human promyelocytes and myelocytes was demonstrated and characterized on the basis of clonal proliferation in agar. An enriched population of normal human promyelocytes and myelocytes was obtained from bone marrow using the monoclonal antibody WEM G11 and the fluorescence-activated cell sorter (FACS). In cultures stimulated by placental-conditioned medium, these cells generated peak total clone numbers between days 3 and 5 of culture. Clones disappeared rapidly thereafter. These clones were mainly of subcolony size at day 7, although some colonies were generated by this population. The clones were primarily neutrophilic in type. These cells had a plating efficiency of up to 50%, and clonal proliferation was dependent on stimulation by colony-stimulating factor (CSF).

Marrow culture studies in adult acute myeloid leukemia at diagnosis and during complete remission.

We used the human placental conditioned medium stimulated single layer agar culture technique to study the in vitro growth of marrow cells from 62 adult patients with acute myeloid leukemia (AML). Bone marrow cells were cultured from 50 patients at the time of initial diagnosis, 19 patients in early remission and 20 patients during their full complete remission. Marrow cultures from untreated patients exhibited heterogeneous growth patterns ranging from complete growth failure to excessive microcluster formation. We classified the growth patterns into 4 groups: (1) Gr I: normal growth, (2) Gr II: no growth, (3) Gr III: decreased growth, (4) Gr IV: excessive growth of microclusters. At presentation, none had Gr I growth; Gr II growth was observed in 23; Gr III in 14 and Gr IV in 13. A predominance of no growth were seen in M1 and M3 subtypes, while Gr IV growth was more commonly observed in M2 or M4 subtype. We were unable to correlate the culture findings with age or white cell count. The present results not only indicated that AML at diagnosis was characterized by abnormal granulopoiesis but also demonstrated that leukemic progenitor cells were heterogeneous with different capacities to express their proliferating potential in vitro. Except few with decreased growth, the growth characteristics generally returned to normal with successful remission induction. Both Gr II and Gr IV growth patterns were not observed either in early remission or during full complete remission.

The proliferative effects of human GM-CSFα and β and murine G-CSF in microwell cultures of fractionated human marrow cells

Blast cell-enriched and promyelocyte-myelocyte-enriched fractions of human marrow were prepared by fluorescence-activated cell sorting using an antineutrophil monoclonal antiserum. Cells from both fractions proliferated in microwell cultures when stimulated by placental or bladder cancer cell conditioned medium containing colony stimulating factor. Semipurified preparations of both GM-CSFα and β from bladder cancer cell conditioned medium were effective proliferative stimuli for both cell populations. Pure murine G-CSF, GM-CSF, M-CSF and Multi-CSF failed to stimulate detectable proliferation in blast cell fractions containing granulocyte-monocyte progenitors. However, G-CSF was an effective proliferative stimulus for human promyelocytes and myelocytes leading to the formation of differentiating granulocytic progeny. G-CSF also stimulated the proliferation of human promyelocytic leukemic cells. Promyelocyte-myelocyte-enriched fractions of human marrow appear to be useful target cells for monitoring the proliferative effects of human-active CSFs in microwell cultures.

Effect of retinoic acid on bone-marrow committed stem cells (CFU-c) from chronic myeloid leukemia patients.

The effect of retinoic acid (all-trans) on the clonal growth of chronic myeloid leukemia marrow was studied. The results indicate that 3 X 10(-5) M, 3 X 10(-6) M, 3 X 10(-7) M and 3 X 10(-8) M of retinoic acid inhibits the colony stimulating activity of placental conditioned medium.

The partial purification of colony-stimulating factors from human placental-conditioned medium

Conference Article| April 01 1984 The partial purification of colony-stimulating factors from human placental-conditioned medium WILLIAM S. GILMORE WILLIAM S. GILMORE 1Department of Biochemistry, School of Life Sciences, Ulster Polytechnic, Shore Road, Newtownabbey, Co. Antrim BT37 0QB, N. Ireland, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1984) 12 (2): 261. https://doi.org/10.1042/bst0120261 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation WILLIAM S. GILMORE; The partial purification of colony-stimulating factors from human placental-conditioned medium. Biochem Soc Trans 1 April 1984; 12 (2): 261. doi: https://doi.org/10.1042/bst0120261 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1984 Biochemical Society1984 Article PDF first page preview Close Modal You do not currently have access to this content.

Enrichment of high proliferation potential colony forming cells from mouse marrow by selecting low-density cells expressing receptors for wheat germ agglutinin

Suspensions enriched for high proliferation potential colony forming cells (HPP-CFC's) were prepared from mouse marrow by selecting low-density cells stained with fluoresceinated wheat germ agglutinin on a fluorescence activated cell sorter. HPP-CFC's were 12-fold more concentrated in the final suspensions and co-enriched with a population of primitive CFU s detected in the spleens of irradiated mice reconstituted 13 days earlier with marrow. This is consistent with previous observations suggesting that these populations are closely related. The degree of enrichment for other haemopoietic progenitors was in the order HPP-CFC > day 8 CFU s > BFU e > GM-CFC > CFU e. “Extra” colonies developed when human placental conditioned medium (HPCM) was added to cultures of enriched suspensions already containing pregnant mouse uterus extract (PMUE). HPP-CFC's probably formed most of these and we discuss why counting these extra colonies may be more reliable than the conventional “size” assay for HPP-CFC's.

Requirements for growth of human erythroid progenitors in nutrient agar.

Human erythroid bursts and neutrophil colonies from fetal liver, term cord blood, and adult bone marrow were grown in nutrient agar, and the colonies were scored using a fixed whole plate method. Human placental conditioned medium was shown to contain burst promoting activity, and low oxygen tension markedly enhanced growth of erythroid bursts. Morphological analysis of colonies showed that eosinophil colonies were more numerous in bone marrow than fetal liver and conversely macrophage and neutrophil-macrophage colonies were more numerous in fetal liver. Erythroid bursts were more numerous and larger in fetal liver. Human fetal liver cells do not produce endogenous colony-stimulating factor.

Preparative purification of human placental colony-stimulating factors.

For preparative purification of colony-stimulating factors (CSF) from human placental conditioned medium, cation exchange chromatography, gel filtration, and isoelectric focusing were combined. The individual fractions were tested using the growth in agar of cryopreserved human bone marrow (dose-response curves). Furthermore, the active preparations were characterized by analytical isoelectric focusing. Two fractions with highly enriched biological activity were isolated. They have the isoelectric points 5.4 and 5.6, respectively. Their specific activity exceeds that of the crude material by a factor of 60. Like crude material, they induce predominantly granulocytic colonies. The biochemical heterogeneity of biological activity in placental supernatants is discussed.

Eosinophil activation by colony-stimulating factor in man: metabolic effects and analysis by flow cytometry

Substantial increases in the killing capacity of human eosinophils after in vitro incubation with human placental conditioned medium (HPCM), a standard source of colony-stimulating factor (CSF), have recently been described. In this article, the interaction between HPCM and purified human eosinophils is analyzed by flow cytometry and by effects on iodination, superoxide production, and protein synthesis. HPCM increased the intensity of natural eosinophil autofluorescence (aFlu) (460 nm) after the absorption of ultraviolet light (360 nm) in a manner that was both time and dose dependent. Measured in arbitrary units, eosinophil aFlu was 72 +/- 7.3 (arithmetic mean +/- SEM) and 121 +/- 3.2 after 18-hr incubations in the absence or presence of HPCM, respectively. The activity in HPCM responsible for these changes cochromatographed on Ultrogel AcA44 columns with CSF and with the less hydrophobic variant of CSF (CSF-alpha) on phenyl Sepharose. Mouse spleen, but not mouse lung, conditioned medium was also active on human eosinophils in this assay. Both CSF-alpha and mouse spleen conditioned medium also contain eosinophil colony-stimulating activity (CSA), whereas inactive CSFs with no effect on mature eosinophils, CSF-beta, and mouse lung conditioned medium also lack eosinophil CSA. CSF-alpha stimulated superoxide production of resting eosinophils (from 0.03 +/- 0.03 to 0.47 +/- 0.08 nmole cytochrome-c reduced/10(5) eosinophils) and of eosinophils incubated with preopsonized zymosan (from 0.15 +/- 0.06 to 0.73 +/- 0.07). It also stimulated iodination by resting eosinophils (from 0.76 +/- 0.16 to 2.60 +/- 0.72 nmoles l/10(7) eosinophils/hr) and of eosinophils incubated with preopsonized zymosan (from 7.52 +/- 2.08 to 29.8 +/- 1.32). In contrast, CSF-beta was inactive in these assays. CSF-alpha also stimulated, between 2- and 15-fold, the new protein synthesis of eosinophils. Thus, substances that stimulate the differentiation of progenitor cells into eosinophils also interact with peripheral mature eosinophils, and the activation of postmitotic cells may be a physiologic role of CSF-like molecules.

Eosinophil Activation by Colony-Stimulating Factor in Man: Metabolic Effects and Analysis by Flow Cytometry

Substantial increases in the killing capacity of human eosinophils after in vitro incubation with human placental conditioned medium (HPCM), a standard source of colony-stimulating factor (CSF), have recently been described. In this article, the interaction between HPCM and purified human eosinophils is analyzed by flow cytometry and by effects on iodination, superoxide production, and protein synthesis. HPCM increased the intensity of natural eosinophil autofluorescence (aFlu) (460 nm) after the absorption of ultraviolet light (360 nm) in a manner that was both time and dose dependent. Measured in arbitrary units, eosinophil aFlu was 72 +/- 7.3 (arithmetic mean +/- SEM) and 121 +/- 3.2 after 18-hr incubations in the absence or presence of HPCM, respectively. The activity in HPCM responsible for these changes cochromatographed on Ultrogel AcA44 columns with CSF and with the less hydrophobic variant of CSF (CSF-alpha) on phenyl Sepharose. Mouse spleen, but not mouse lung, conditioned medium was also active on human eosinophils in this assay. Both CSF-alpha and mouse spleen conditioned medium also contain eosinophil colony-stimulating activity (CSA), whereas inactive CSFs with no effect on mature eosinophils, CSF-beta, and mouse lung conditioned medium also lack eosinophil CSA. CSF-alpha stimulated superoxide production of resting eosinophils (from 0.03 +/- 0.03 to 0.47 +/- 0.08 nmole cytochrome-c reduced/10(5) eosinophils) and of eosinophils incubated with preopsonized zymosan (from 0.15 +/- 0.06 to 0.73 +/- 0.07). It also stimulated iodination by resting eosinophils (from 0.76 +/- 0.16 to 2.60 +/- 0.72 nmoles l/10(7) eosinophils/hr) and of eosinophils incubated with preopsonized zymosan (from 7.52 +/- 2.08 to 29.8 +/- 1.32). In contrast, CSF-beta was inactive in these assays. CSF-alpha also stimulated, between 2- and 15-fold, the new protein synthesis of eosinophils. Thus, substances that stimulate the differentiation of progenitor cells into eosinophils also interact with peripheral mature eosinophils, and the activation of postmitotic cells may be a physiologic role of CSF-like molecules.