Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

Changes in cell kinetics associated with differentiation of a human promyelocytic cell line (HL60)

Abstract. Terminal cell differentiation results in an irreversible arrest in the G₁ phase of the cell cycle and loss of the capacity for cell renewal. In the murine erythroleukaemia cell line (MELC), commitment to erythroid differentiation was found also to be preceded by an early, transient, phase of inhibition of growth due to prolongation of the G₁ phase. We determined the effect of differentiation-inducing agents on the growth kinetics of a human promyelocytic cell line (HL60) which undergoes differentiation into mature granulocyte. At concentrations of inducers optimal for cell differentiation, an early, transient stimulation of cell multiplication was found. DNA synthesis was enhanced in HL60 cells as early as 5 hr after exposure to inducer. Nevertheless, HL60 cell maturation eventually also resulted in a loss of the multiplication ability. The duration of exposure to inducer required for irreversible loss of the potential for self-renewal was determined by the fall in the cloning efficiency of induced cells; the results indicate that it preceded the switch-off of the replication mechanism; the majority of the cells lost their ability to form large colonies at the time of peak DNA synthesis and were able to complete an additional two to three cell cycles at a rate similar to uninduced cells. These changes occurred before HL60 cells became committed and might play a pivotal role in the process of cell differentiation.     

The continued requirement for inducer for the development of macrophage and granulocyte colonies

Rat bone marrow cells were seeded as mass cultures or for cloning together with inducer required for colony formation, and at various times after seeding, the cells were re-seeded for cloning either with or without inducer. The results indicate that the development of both macrophage (M) and granulocyte (G) colonies requires a continued supply of the inducer. No M or G colonies were produced when the inducer was replaced by erythropoietin.     

Regulation of self-renewal of human T lymphocyte colony-forming units (TL-CFUs)

Formation of human T lymphocyte colonies in semisolid medium from T lymphocyte colony-forming units (TL-CFUs) under stimulation of phytohemagglutinin (PHA) has been reported by several authors. These TL-CFUs were present in unsensitized lymphocyte populations. We report here that such TL-CFUs are capable of renewing themselves. This was observed when colony cells from primary T cell colonies that developed in the presence of PHA were replated in methylcellulose medium containing irradiated autologous leukocytes and PHA. We have also been able to demonstrate serial transfer of TL-CFU for up to six passages. At each passage, colony-forming frequency was determined from the proportional relationship between the number of new colonies obtained and the number of colony cells plated. Examination of the number of new colonies derived from each individual T cell colony ("burst size of TL-CFU") showed that most colonies contained very few new TL-CFU and only a very small number of colonies contained many new TL-CFU. The distribution of burst sizes could be well fitted to a gamma distribution, in agreement with prediction from a stochastic model. We have identified an activity that enhanced the mean TL-CFU burst size three to ten times. This work provides the first evidence in vitro that self-renewal of human T lymphocyte progenitor cells can be stimulated by specific regulatory proteins.     

Receptors for C3 and IgG on macrophage, neutrophil and eosinophil colony cells grown in vitro.

Macrophage, neutrophil, and eosinophil colony cells from bone marrow culture in semisolid agar medium were studied for membrane C3 and IgG receptors. The capacity of these cells to bind either erythrocytes-19S antibody-complement (EAC) or erythrocyte-7S antibody (EA7S) complexes was measured using the rosette method. Whereas macrophage and neutrophil colony cells showed receptors for both C3 and IgG, eosinophil colony cells appear to bear only IgG receptors. Studies correlating colony age and the presence of receptors showed that 60 to 70% of the cells from 3-day-old macrophage colonies were reactive for EAC and EA7S contrasting with 80 to 90% of the cells from 6- to 12-day-old colonies. Neutrophils behaved somewhat differently: EAC and EA7S reactive cells were seen in colonies after 4 or 5 days in culture and comprised only 50 to 60% of the colony population. Eosinophilic colonies showed 50 to 60% EA7S reactive cells after 6 to 7 days in culture, but no EAC reactive cells were found among these colonies at any time. The characteristics and properties of the receptors detected on colony cells were similar to those on macrophages and neutrophils from normal peritoneal fluid or bone marrow. Most macrophage colony cells were actively phagocytic whereas neutrophils and eosinophilic colony cells failed to show phagocytosis under the same conditions.     

Changes in self-renewal potential of human leukemic cells (K562): a bidirectional stochastic process

Daughter cells arising from a single cell division in the leukemic cell line K562 have equivalent self-renewal potential with respect to their ability to form clones in semisolid medium. However, individual cells isolated from these clones in sequence have vastly different abilities in their self-renewal potentials. Thus, cells originating from a clone with any particular self-renewal potential exhibit the full range of self-renewal potentials--from highly renewing to none renewing, cells. These results show that self-renewal potential in the K562 cell line is a random, reversible and partially noninherited characteristic. It is suggested that the stochastic variability of the intraclonal self-renewal potential of K562 progeny cells either reflects the initial expression of a differentiation program or the expression of the predeterministic portion of the normal myelopoietic differentiation pathway.     

Differentiation of Mouse Bone Marrow Precursor Cells into Neutrophil Granulocytes by an Activity Separation from WEHI-3 Cell-Conditioned Medium

Colonies comprised exclusively of neutrophil granulocytes have been obtained by growing mouse bone marrow cells in nutrient semisolid agar cultures. A stimulator of predominantly granulocyte colony formation was present in the breakthrough fraction of preparations of colony-stimulating activity separated on DEAE-Sephadex A. The source of colony-stimulating activity was concentrated conditioned medium of a murine myelomonocytic cell line (WEHI-3), which unfractionated stimulated the growth of colonies of granulocytes, macrophages, megakaryocytes, as well as mixed colony types. After stepwise column chromatography of the conditioned medium, the breakthrough fraction was shown to stimulate predominantly granulocyte colony formation, and the fraction eluted with 1 M NaCl was found to induce primarily macrophage colony growth. Colony morphology was independent of the concentration of eluate used. The morphology of colonies varied with increasing concentrations of the breakthrough fraction. At low concentrations, granulocyte colony formation was almost exclusively observed. With increasing concentrations of this fraction, an increasing proportion of the colonies were found to contain macrophages. The effect of concentration of this activity was in marked contrast to previous findings where the incidence of granulocyte colony formation was inversely related to the concentration of colony-stimulating activity. This differential responsiveness of cell to stimulus has previously been interpreted as low concentrations of a growth and differentiation factor being required for macrophage production and high concentrations of the same factor required for granulocyte formation. Separation of these activities by DEAE Sephadex chromatography, and alteration of the dose-response curve, such that granulocyte colony formation varies directly with the amount of stimulator, indicates that the differentiation of these two cell blood lineages may be controlled by separate entities.     

Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages

Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-μm-diameter colonies give rise to mesoderm, while small 200-μm colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages.     

Remission of acute myeloid leukemia: studies of in vitro colony formation by bone marrow cells

Human bone marrow contains cells which form leukocyte colonies in semisolid culture media. Each leukocyte colony arises from a single colony-forming cell which is thought to be a unipotential stem cell, and which is subject to regulation in vitro by colony-stimulating factor. In acute myelogenous leukemia variable abnormalities in colony formation by marrow cells occur. Usually colony formation either fails to occur or the colonies that are formed are small and contain fewer than 50 cells. Similar abnormalities have been described in bone marrow dysfunction preceding overt leukemia. Usually remission of leukemia is accompanied by improved cloning by marrow cells. In this study three patients are reported in whom remission was associated with impaired cloning, and one of these patients has remained in continuous remission for a further 18 months. These observations suggest that remission status is not necessarily associated with repopulation of the bone marrow by normal hematopoietic cells.     

Conditions affecting clonal growth of lymphoma cells in a semisolid matrix

Summary This study demonstrated the importance of the methods used in determining the lymphoma cell colony stimulating activity of factors derived from lymphoma cells. The in vitro colony formation in a semisolid matrix of the AKR mouse lymphoma cell line, SL 12, and three cloned derivatives, SL 12.1, SL 12.3, and SL 12.4, was studied. We show that the use of soft agar or methylcellulose as a semisolid matrix results in colony formation by the lymphoma cells only in the presence of serum. The addition of conditioned medium (CM) from lymphoma cells growing in serum-free medium does not stimulate colony growth. However, when purified agarose is used, colonies grow in a dose-dependent manner in the absence of serum and in the presence of CM. These results indicate that the type of semisolid matrix used can influence results in studies of this nature. Purified agarose provides the best environment when colony formation by lymphoma cells is used to measure the presence of growth factors in test-conditioned media. This research was supported by the Department of Energy, contract DE-AM03-76-SF00012 and National Institutes of Health grant CA12386. Dr. Bessho was a Visiting Scientist from the National Institute of Radiological Sciences, Chiba, Japan.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

Analysis of the growth kinetics of murine erythroleukaemia cells following commitment to terminal differentiation

Abstract Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.     

Analysis of the growth kinetics of murine erythroleukaemia cells following commitment to terminal differentiation

Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.     

Haemopoietic spleen colony growth: a versatile, parsimonious, predictive model

Abstract. Substantial support has been obtained for the stochastic model for stem cell differentiation first proposed by Till, McCulloch & Siminovitch (1964), over 20 years ago. By adding a cell maturation pathway, it is possible to predict (by computer simulation) the total number of cells and consequently the time at which individual colonies appear and disappear.
Only a few uncontroversial assumptions are required to predict that cells, uniform with respect to self-renewal, are capable of producing the high proportions of late disappearing and late appearing colonies observed experimentally in the spleens of irradiated mice that have been injected with normal haemopoietic cells. It is shown that differences in stem cell self-renewal only slightly influence the time of appearance of colonies; whereas changes in the kinetics of the maturing cells, by changing the size of colonies, has a marked effect on the time of appearance and disappearance of colonies and on the average doubling-time of colony-forming cells per colony (but not the doubling-time of individual colonies).
These results (1) seriously question the prevailing view that spleen colonies scored at 8 days measure a separate population (without the capacity for self-renewal), from those scored at 12 days; (2) argue against the existence of multiple sub-populations of stem cells with differing self-renewal and toxicity to cytotoxic agents; (3) help to identify those experiments for which it is obligatory to postulate heterogeneity, and (4) are consistent with self-renewal being regulated by a feedback control of stem cell differentiation, to which only proliferating stem cells can respond and where the stimulus for differentiation decreases at a time when the bone marrow is known to be depleted.     

Further studies on the differentiation of a cell line of myeloid leukemia

A limited time of contact with a conditioned medium from embryo cells induced phagocytotic activity in a cell line of myeloid leukemia followed by the loss of colony forming and leukemogenic capacity. After two days in a high concentration of the conditioned medium, the colonies showed morphological changes which indicated the differentiation of this line of cells. The differentiation-stimulating factor present in the conditioned medium was relatively thermolabile, while the growth-stimulating factor was highly thermostable. Both factors could pass through a dialysis membrane.     

Cloning of Drosophila cells: effect of vitamins and yeast extract components.

Yeast extract, a component of Drosophila cell culture media, is shown to contain substances of high, intermediate, and low molecular weight, that are, respectively, essential, inhibitory, and stimulatory for colony formation in semisolid agar medium. Furthermore, it is shown that high concentrations of pyridoxal greatly increase the cloning efficiency of Drosophilia cells. A cloning method with line Kc is described which routinely gives cloning efficiencies in excess of 20%.     

Colony formation in agar by murine plasmacytoma cells: potentiation by hemopoietic cells and serum

Clonal growth in semisolid agar medium was obtained using cells from 19 of 25 transplanted murine plasmacytomas when the medium was supplemented by whole mouse blood or washed red cells. With different tumors cloning efficiency ranged from 0.01% to 21.6%. With two exceptions, mouse blood did not potentiate colony formation in agar by cells from transplantable myelomonocytic, myeloid, and lymphoid leukemias, reticulum cell sarcomas and fibrosarcomas. The clonal growth of some plasmacytomas was also potentiated by syngeneic thymic, spleen or bone marrow cells. Plasmacytoma colony growth was not stimulated by normal mouse serum but serum from mice injected with endotoxin or polymerised flagellin stimulated colony growth by some plasmacytomas. The active serum factor was not the colony stimulating factor (CSF) and its appearance after antigenic stimulation was not T cell-dependent. Preimmunised mice failed tq respond to antigenic stimulation. Whole body irradiation did not induce a rise in the capacity of serum to stimulate colony formation by plasmacytoma cells.