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Diagnostic and prognostic significance of peripheral blood cultural characteristics in adult leukemia

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Abstract

A simple liquid culture technique has been used to study peripheral blood from patients with acute myelogenous leukaemia. Evidence is presented that cells from morphologically identical types of leukaemia have differing capacity for "differentiation" from free floating blast cells into plastic-adherent phagocytic, trypsin-resistant macrophage-like cells with Fc and C3 receptors. Preliminary analysis suggests that patients whose cells have the greatest capacity for "differentiation" have a better chance of achieving complete remission.

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Stem cell based therapies have been reported in protecting cerebral infarction induced neuronal dysfunction and death. However, most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here, we used human embryonic stem cell derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen-glucose deprivation (OGD) for 1 hour. For treatment group, different exosomes were derived from neuron, embryonic stem cell, neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell, and added to culture medium 30 min after OGD (100 μg/ml). Western blotting was performed 12 hours after OGD, while cell counting and electrophysiological recording were performed 48 hours after OGD. We found that these exosomes attenuated OGD-induced neuronal death, mTOR, pro-inflammatory and apoptotic signaling pathway changes, as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD-induced neuronal death and dysfunction in human embryonic stem cell derived neurons, potentially through their modulation on mTOR, pro-inflammatory and apoptotic signaling pathways.
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Spontaneous mitoses in the blood of 67 patients with acute leukemia were enumerated and their identity determined by cytogenetic methods. Most patients were children with acute lymphoblastic leukemia. Simultaneous 16- to 20-hour cultures of blood leukocytes (Bu) and of bone marrow (BM) cells were performed without phytohemagglutinin (PHA). Blood leukocytes were also cultured with PHA for 72 hours (BPHA). Mitoses in Bu cultures were counted, and karyotypic analysis performed on cells from the three culture types. In 21 control subjects, Bu cultures usually yielded no mitoses. Relapse and remission patients both displayed significantly more Bu mitoses than the controls. The karyotypes of Bu, BPHA, and BM mitoses in remission patients were normal. Fifty percent of relapse patients displayed cytogenetically abnormal leukemia cell lines; the percentage of their abnormal karyotypes was significantly higher in BM cells than in Bu or BPHA cells. The majority of the mitotic cells in Bu cultures from both relapse and remission patients appear to be of a nonleukemic origin. The number of mitoses could not be correlated with type of leukemia, hematologic parameters, or prognosis.
Peripheral blood leukocytes from 31 out of 48 patients with acute myelogenous leukemia grew in short-term liquid culture. Two distinct types of growth occurred. The first type was dimorphic with supernatant free-floating ("non-sticker") cells and, in addition, a plastic/glass adherent, trypsin resistant, phagocytic population ("stickers"). The second type which occurred less frequently than the first consisted almost entirely of free-floating "non-sticker" cells. Although patients with this second type of growth pattern almost invariably had AML, 44% of AML's produced monocytoid "sticker" cells in culture. Cells from the majority of patients with ALL did not grow in culture.
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The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to encourage communication of the very best cancer research from laboratories and clinics in all countries. The breadth of its coverage, its editorial independence and it consistent high standards, have made BJC one of the world's premier general cancer journals. Its increasing popularity is reflected by a steadily rising impact factor.
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Peripheral blood cells from three patients with acute leukemic have been studied using a suspension culture method previously described.1 Cytogenetic studies in two of the patients permitted the identification of the proliferating cells in the cultures as being derived from a leukemic population. Cell separation studies using velocity sedimentation supported the concept that growth of the leukemic cells in culture is dependent on an interaction between two populations of leukemic cells.
A simple in vitro technique is described for the growth of colonies from single cell suspensions of mouse bone marrow. The system involves the plating of marrow cells in agar on feeder layers of other cells, those from 8-day-old mouse kidney and 17th day mouse embryo being shown to be the most efficient types of feeder layers.
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Peripheral blood cells from three pa- populations. Aneuploid cells present in tients with leukemia in relapse in- direct marrow preparations were also creased in numbers in short-term sus- prevalent in the cultures after 10 days. pension cultures. This increase was The apparent specificity of the in- dependent on the presence of either crease for Ieukemic populations may feeder populations containing a high provide a new approach to the study percentage of blast cells or condi- of leukemic blast cells. honed medium derived from such CELLS FROM PERIPHERAL BLOOD or bone marrow of patients with acute leukemia have been shown to proliferate in suspension culture only after prolonged periods of poor growth'; using viscid media of agar or methyl cellulose, colony formation has been obtained in some patients with acute leukemia, but not in all.28 We have studied three patients with leukemia whose peripheral blood cells increased in number in short-term suspension cultures. In two of these patients, chromosome markers were present in direct marrow preparations and these were also found after 10 days in culture, indicating that leukemic cells were proliferating. The presence of a feeder layer containing leukemic blast cells or conditioned medium derived from such cells was a growth requirement in this culture system.
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Article
THE culture of normal haemopoietic cells was accomplished1 by obtaining colonies of granulocytic and monocytic cells from mouse bone marrow grown in a semisolid agar system. The method was modified for human bone marrow and subsequent studies showed that leukaemia cells from the peripheral blood and marrow of patients with acute myelogenous leukaemia (AML) produced small colonies or clusters1,2. Using this technique, serial sampling and morphological or biochemical studies of the growing cells was difficult. A liquid culture technique was then developed to study the growth of granulocytic or monocytic cells from normal bone marrow3. This in vitro diffusion chamber technique had the advantage of easy sampling but involved the use of large culture vessels. The method has subsequently been used to cultivate AML cells4.
Article
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Article
The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to encourage communication of the very best cancer research from laboratories and clinics in all countries. The breadth of its coverage, its editorial independence and it consistent high standards, have made BJC one of the world's premier general cancer journals. Its increasing popularity is reflected by a steadily rising impact factor.
Article
A system for the cultivation of normal and malignant human bone marrow cells in liquid medium is described. The apparatus used is an in vitro diffusion chamber in which cells grow in suspension and upon a dialysis membrane. Proliferation and maturation of normal granulocytes and macrophages were sustained in culture for several weeks in the absence of exogenous stimulating substances. Cell replication was documented by a rise in viable counts, 3H-thymidine incorporation, and labeled mitoses. The entire maturational sequence of the granulocyte and macrophage series was identified in culture, and the cells were characterized by light and electron microscopy, cytochemical properties, phagocytic ability, and the presence of surface receptors for IgG. Nucleated red cell precursors were observed in some cultures for up to 11 days. Malignant cells from patients with various hematologic neoplasms were also successfully cultured. With this system human myeloma cells were maintained in culture for up to 3 wk on primary explant, and continued to synthesize immunoglobulin. The in vitro diffusion chamber technique permits the cultivation of normal human bone marrow cells in liquid medium and provides a convenient means for studying normal and neoplastic hematopoietic cell differentiation and function in short-term culture.
Article
. Liquid suspension cultures of mouse bone marrow cells at high and low density were prepared in supplemented Eagle's medium containing 10% of a partially purified extract of mouse embryos and pregnant mouse uterus (PMU). In the low cell density cultures the number of cells decreased for 2 days; by 4 days the agar colony-forming cells (agar CFC) had risen ten-fold and the spleen colony-forming units (spleen CFU) had fallen to one tenth; between 4 and 8 days the total cell count showed a four-fold increase and the final cell number exceeded the number of the original culture. The cells produced were mainly macrophages. If PMU was not included in the culture the agar CFC disappeared after 4 days and there was no cell multiplication. In the high cell density cultures a similar pattern was observed; in the presence of PMU the agar CFC showed an increase in number, the spleen CFU decreased and an increase in cell number occurred between 4 and 8 days. However, the cells produced were predominantly granulocytes. In the absence of PMU from this cell culture, agar CFC were maintained for 6 days and the cell population remained predominantly granulocytic. These methods of growing cell enable cell recovery from the cultures to be made at any stage and provide an opportunity to study the kinetics and functional capacity of the cells produced.
Article
Human spleen-conditioned medium can induce the formation in vitro of large granulocyte colonies from normal human bone marrow cells. The granulocyte colonies contained cells in various stages of differentiation, from myeloblasts to mature neutrophile granulocytes. Human spleen-conditioned medium also induced colony formation with rodent bone-marrow cells, whereas rodent spleen-conditioned medium induced colony formation with rodent bone marrow but not with human cells. This in vitro system has been used to determine the potentialities for cell differentiation in bone-marrow and peripheral blood cells from patients with a block in granulocyte differentiation in vivo. The cloning efficiency, colony size, and number of mature granulocytes in bone-marrow colonies from patients with congential neutropenia, whose bone marrow contained only 1% mature granulocytes, were not less than in people whose bone marrow had the normal level of about 40% mature granulocytes. The cloning efficiency of peripheral blood cells from patients with acute myeloid leukemia was 350 times higher, with 10 times larger colonies, than the cloning efficiency of peripheral blood cells from normal people. The cytochemical properties and number of mature granulocytes in colonies from the leukemic patients were the same as in colonies from non-leukemic people. The results indicate that a block in cell differentiation in vivo, in these cases with neutropenia and acute myeloid leukemia, was overcome in vitro, in the presence of an inducer in the conditioned medium. In patients with chronic myeloid leukemia, colony formation was induced only in some of the cases. This indicates that there are blast cells with different potentialities for the development of colonies in different patients.
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In vitro regulation of granulocytic proliferation and differentiation was analyzed by study of marrow granulocytic colony-forming capacity (CFC) and peripheral white-cell provision of colony-stimulating activity (CSA) in patients with acute myeloid leukemia (AML) and preleukemia. Patients with AML in relapse had abnormal marrow CFC, producing either no or excessive numbers of abortive colonies. Furthermore, their leukocytes lacked ability to provide CSA. During complete remission, both these indexes were normal. Sequential studies indicated that marrow CFC correlates with, and probably precedes, detectable changes in clinical and morphologic status. Preleukemic patients showed disordered CFC and CSA, with values similar to those in AML in relapse. AML marrow appears to consist of coexisting normal and leukemic cell clones, and preleukemic marrow to contain either a potentially leukemic clone with greater capacity for differentiation in vivo or a leukemic clone that is held in abeyance.
Article
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Article
Methods and results of culturing human blood and bone marrow cells on glass substrates are reported.A pattern of culture divisible into three successive but overlapping phases was observed. Cells of the first phase, recognisable as normal blood cells, are more suitably studied by other techniques.The large granular cells of the second phase and the fibroblast-like cells of the third phase are described and their origin discussed. Glass substrate cultures of bone marrow cells are a convenient and reliable source of human fibroblast-like cells for morphological, physiological and pharmocological experiments. They have no value as an aid to diagnosis.
Article
Scrum agar cultures of blood and bone marrow cells have been made and the living cells studied by phase contrast microscopy. The general pattern of culture and the appearances and behaviour of some of the cells is described. The blood cells survive for 2 or 3 days. Some multiplication of myelocytes takes place but this is more than offset by cell death. The cells do not usually mature and the cultures provide no information of diagnostic value. The method is suitable for the qualitative studies of the effects of nutrients and drugs upon the cells and results of some of these studies are presented. Attempts to develop the method ouantitativelv have so far failed.
Forming Ability of of AMLCells.Cryo-Expl Acta haemat
  • London
  • Eyre
  • Spottiswoode
London: Eyre and Spottiswoode. Proc. natn. Acad. J. cell. in Tissue Culture. of Human Leukaemic Blood, 38, 500. Forming Ability of of AMLCells.Cryo-Expl Acta haemat.,
Growth of Human Bone Marrow in Liquid Culture Granulopoiesis Leukaemia and Pre-leukaemia
  • Haemat Br
  • F R Balkwill
  • R T D Golde
  • D W Cline
Haemat., Br. r410 F. R. BALKWILL AND R. T. D. OLIVER GOLDE, D. W. & CLINE, M. J. (1973) Growth of Human Bone Marrow in Liquid Culture. 41, 45. GREENBERG, P. L., NICHOLS, W. C. & SHRIER, S. B. (1971) Granulopoiesis Leukaemia and Pre-leukaemia. Med., 284, 1225
Culture de Moelle Osseuse et de Rate C. r
  • A Carrel
  • Ml T Burrows
CARREL, A. & BURROWS, Ml. T. (1911) Culture de Moelle Osseuse et de Rate C. r. Soc. Biol., 69, 299
In vitro Growth of National and Leukaemic Bore Marrow
  • C H Brown
  • P B Carbonne
BROWN, C. H. & CARBONNE, P. B. (1971) In vitro Growth of National and Leukaemic Bore Marrow. J. natn. Cancer Inst., 46, 989
HL-A Antigen Frequency in Patients with AML
  • R T D Oliver
  • A M Williams
  • P Klouda
  • S Lawler
OLIVER, R. T. D., WILLIAMS, A. M., KLOUDA, P. & LAWLER, S. (1976) HL-A Antigen Frequency in Patients with AML. Lancet (in press).
Management of Adult Acute Myelogenous Leukaemia Macrophage Content of Tumours in Relation to Metastatic Spread and Host Immune Reaction The Origin and Role of Blood Borne Monocytes in Rats with a Transplantable Acute Myelogenous Leukaemia
  • C Crowther
  • D Powles
  • R L Bateman
  • C J T Beard
  • M E J Gauci
  • C L Wrigley
  • P F M Malpas
  • Hamilton Fairley
  • G Sir Ronald Bodley Scott Eccles
  • S Alexander
C. r. Soc. Biol., 69, 299. CROWTHER, D., POWLES, R. L., BATEMAN, C. J. T., BEARD, M. E. J., GAUCI, C. L., WRIGLEY, P. F. M., MALPAS, J. S., HAMILTON FAIRLEY, G. & SIR RONALD BODLEY SCOTT (1973) Management of Adult Acute Myelogenous Leukaemia. Br. med J., i, 131. ECCLES, S. & ALEXANDER, P. (1975) Macrophage Content of Tumours in Relation to Metastatic Spread and Host Immune Reaction. Nature, Lond., 250, 667. GAUCI, C. L., WRATHMELL, A. & ALEXANDER, P. (1975) The Origin and Role of Blood Borne Monocytes in Rats with a Transplantable Acute Myelogenous Leukaemia. Cancer Letters, 1, 33. GLICK, A. D. & HORN, R. G. (1974) Identification of Promonocytes and Monocytoid Precursors in Acute Leukaemia of Adults: Ultrastructural Cytochemical Observations. Br. J. Haemat., 26, 395.
Growth of Human Bone Marrow in Liquid Culture. Blood, 41, 45 Granulopoiesis in Acute Myeloid Leukaemia and Pre-leukaemia. New Engl Salt Fractiona-tion of Immunoglobulins
  • F R Balkwill
  • R T D Oliver Golde
  • D W Cline
  • M J Greenberg
  • P L Nichols
  • W C Shrier
  • S B Heide
  • K Schwick
  • H G D M Ed
  • Weir
F. R. BALKWILL AND R. T. D. OLIVER GOLDE, D. W. & CLINE, M. J. (1973) Growth of Human Bone Marrow in Liquid Culture. Blood, 41, 45. GREENBERG, P. L., NICHOLS, W. C. & SHRIER, S. B. (1971) Granulopoiesis in Acute Myeloid Leukaemia and Pre-leukaemia. New Engl. J. Med., 284, 1225. HEIDE, K. & SCHWICK, H. G. (1973) Salt Fractiona-tion of Immunoglobulins. In Handbook of Experimental Immunology, 1. Ed. D. M. Weir.
The Relation Between Morphology and Other Features of Acute Myeloid Leukaemia and Their Prognostic Significance
  • M R C Working Party Report
M.R.C. WORKING PARTY REPORT (1975) The Relation Between Morphology and Other Features of Acute Myeloid Leukaemia and Their Prognostic Significance. Br. J. Haemat., 31, Suppl. 165.
The Growth of Embryonic Chick Tissues in Artificial Media Different Blocks in the Differentiation of Myeloid Leukaemic Cells
  • M R Lewis
  • W H Lewis
  • J Lotem
  • L Sachs
LEWIS, M. R. & LEWIS, W. H. (1911) The Growth of Embryonic Chick Tissues in Artificial Media, Agar and Bouillon. Bull. Johns Hopkins Hosp., 22, 126. LOTEM, J. & SACHS, L. (1974) Different Blocks in the Differentiation of Myeloid Leukaemic Cells. Proc. natn. Acad. Sci. U.S.A., 71, 3507. MARBROOK, J. (1967) Primary Immune Response in Cultures of Spleen Cells. Lancet, ii, 1279. METCALF, D. (1973) Human Leukaemia: Recent Tissue Culture Studies on the Nature of Myeloid Leukaemia. Br. J. Cancer, 27, 191.
Colony Formation by Normal and Leukaemic Human Bone Marrow Cells in Culture
  • Oxford 6 Iscove
  • N N Senn
  • J S Till
  • J E Mcculloch
Oxford: Blackwell Scientific Publications. Ch. 6. ISCOVE, N. N., SENN, J. S., TILL, J. E. & MCCULLOCH, E. A. (1971) Colony Formation by Normal and Leukaemic Human Bone Marrow Cells in Culture. Blood, 37, 1.
Cryopreservation of AML Cells
  • A M Ficat
  • F Oliver
WILLIAMS, A. M., FICAT, F. & OLIVER, R. T. D. (1976) Cryopreservation of AML Cells. Cryobiology (in press).
Salt Fractionation of Immunoglobulins Colony Formation by Normal and Leukaemic Human Bone Marrow Cells in Culture
  • K Schwick
  • H G D M Ed
  • Weir
  • N N Oxford
  • J S Senn
  • J E Till
  • E A Mcculloch
HEIDE, K. & SCHWICK, H. G. (1973) Salt Fractionation of Immunoglobulins. In Handbook of Experimental Immunology, 1. Ed. D. M. Weir. Oxford: Blackwell Scientific Publications. Ch. 6. ISCOVE, N. N., SENN, J. S., TILL, J. E. & MCCULLOCH, E. A. (1971) Colony Formation by Normal and Leukaemic Human Bone Marrow Cells in Culture. Blood, 37, 1.
The Growth of Mouse Bone Marrow Cells In vitro. Aust In vitro Growth of National and Leukaemic Bore Marrow
  • T R Metcalf
BRADLEY, T. R. & METCALF, D. (1966) The Growth of Mouse Bone Marrow Cells In vitro. Aust. J. exp. Biol. med. Sci., 44, 287. BROWN, C. H. & CARBONNE, P. B. (1971) In vitro Growth of National and Leukaemic Bore Marrow. J. natn. Cancer Inst., 46, 989.