scholarly journals Improved Plasma Culture System for Production of Erythrocytic Colonies In Vitro: Quantitative Assay Method for CFU-E

Blood ◽  
1974 ◽  
Vol 44 (4) ◽  
pp. 517-534 ◽  
Author(s):  
David L. McLeod ◽  
Mona M. Shreeve ◽  
Arthur A. Axelrad
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Culture System ◽  
Fetal Calf Serum ◽  
Bone Marrow Cells ◽  
Calf Serum ◽  
Quantitative Assay ◽  
Assay Method ◽  
Wide Range

Abstract An improved plasma culture system is described for the production of erythrocytic colonies by mammalian adult hemopoietic cells in vitro under the influence of erythropoietin. The concentration of fetal calf serum in the medium used for dilution of the cells was critical for erythrocytic colony formation when low numbers of cells were plated. Optimal concentrations were found for plasma, fetal calf serum, bovine serum albumin, and L-asparagine in the culture medium, and the colony-forming efficiency was shown to depend on the concentration of erythropoietin. With erythropoietin at plateau concentration, the number of erythrocytic colonies produced was directly proportional to the number of bone marrow or spleen cells plated, over a wide range of cell concentrations. Colony numbers per culture conformed to a Poisson distribution. Thus, the improved plasma culture system may be used for the quantitative assay of CFU-E. The method is rapid (2 days), reliable, convenient, and inexpensive. Since the improved plasma culture system also supports granulocytic colony formation by bone marrow cells in the presence of conditioned medium (CSA), and the number of granulocytic colonies produced is proportional to the number of cells plated, the same hemopoietic cell suspensions can be simultaneously assayed for CFU-E and CFU-C under virtually identical conditions.

scholarly journals Cell-mediated amegakaryocytic thrombocytopenia associated with systemic lupus erythematosus

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 479-483
Author(s):  
T Nagasawa ◽  
T Sakurai ◽  
H Kashiwagi ◽  
T Abe
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Bone Marrow ◽  
T Cells ◽  
Lupus Erythematosus ◽  
Culture System ◽  
Bone Marrow Cells ◽  
Rare Complication ◽  
Systemic Lupus ◽  
Marrow Cells

We studied a patient with a rare complication of amegakaryocytic thrombocytopenia (AMT) associated with systemic lupus erythematosus (SLE). To investigate the underlying pathogenesis of AMT, the effects of peripheral blood T cells and serum on human megakaryocyte progenitor cells were studied using in vitro coculture techniques. Mononuclear bone marrow cells (2 X 10(5) from normal donors produced 33.6 +/- 8.8 (n = 10) colony-forming unit-megakaryocytes (CFU-M) in our plasma clot system. When 2 X 10(5) of the patient's T cells were added to the culture system, the number of CFU-M decreased to only 3.5 +/- 0.6/2 X 10(5) bone marrow cells. No evidence of inhibitory effects was found by the addition of the patient's serum and complement to the culture system. The T cells stored at -80 degrees C on admission were also capable of suppressing autologous CFU-M after recovery from AMT. These results indicate that in vitro suppression of CFU-M from allogenic and autologous bone marrow cells by this patient's T cells provides an explanation for the pathogenesis of AMT associated with SLE.

scholarly journals Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2859-2870 ◽  
Author(s):  
OJ Borge ◽  
V Ramsfjell ◽  
OP Veiby ◽  
MJ Jr Murphy ◽  
S Lok ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Calf Serum ◽  
Myeloid Cell ◽  
Promoting Effect ◽  
Multipotent Progenitors

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability-promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum-supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca-1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.

scholarly journals Serial in vitro bone marrow fibroblast culture in human leukemia

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 589-592 ◽  
Author(s):  
T Nagao ◽  
K Yamauchi ◽  
M Komatsuda
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Colony Formation ◽  
Calf Serum ◽  
Culture Method ◽  
Fibroblast Culture ◽  
Human Leukemia ◽  
Chronic Myelocytic Leukemia ◽  
Myelocytic Leukemia

Abstract Human fibroblast colony formation from bone marrow was performed in liquid culture. Fetal calf serum was used as a stimulator of the fibroblast colony formation. The colony formation took place not only in normal donors, but also in patients with acute leukemia and chronic myelocytic leukemia. At the diagnosis of the disease, significant colony suppression was observed in most cases of acute leukemia, while the number of colonies increased in half of the cases of chronic myelocytic leukemia. However, there was no correlation between the colony-forming efficiency and the initial number of peripheral platelets or bone marrow megakaryocytes that contained growth-promoting factor. The number of colonies increased after chemotherapy, recovered at the stage of complete remission, and then decreased to low levels at relapse in the patients with acute leukemia; it decreased after treatment with busulfan in the patients with chronic myelocytic leukemia. This fibroblast culture method is useful for counting fibroblast colony-forming cells in the bone marrow of human leukemia.

scholarly journals Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1733-1741 ◽  
Author(s):  
M Kaleko ◽  
JV Garcia ◽  
WR Osborne ◽  
AD Miller
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Dna Sequences ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Helper Virus ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Wide Range

Abstract A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

scholarly journals Parvovirus B19-induced perturbation of human megakaryocytopoiesis in vitro

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 1997-2004 ◽  
Author(s):  
A Srivastava ◽  
E Bruno ◽  
R Briddell ◽  
R Cooper ◽  
C Srivastava ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Replication ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Northern Analysis ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Marrow Cells

Abstract Parvovirus B19 infection leads to transient aplastic crises in individuals with chronic hemolytic anemias or immunodeficiency states. An additional unexplained sequela of B19 infection is thrombocytopenia. Because B19 is known to have a remarkable tropism for human erythropoietic elements, and is not known to replicate in nonerythroid cells, the etiology of this thrombocytopenia is uncertain. We sought to define the pathobiology of B19-associated thrombocytopenia by examining the role of B19 on in vitro megakaryocytopoiesis. B19 infection of normal human bone marrow cells significantly suppressed megakaryocyte (MK) colony formation compared with mock-infected cells. No such inhibition was observed with a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The B19-MK cell interaction was also studied at the molecular level. Whereas low-density bone marrow cells containing erythroid precursor cells supported B19 DNA replication, no viral DNA replication was observed in B19-infected MK-enriched fractions as determined by the presence of viral DNA replicative intermediates on Southern blots. However, analysis of total cytoplasmic RNA isolated from B19-infected MK fractions showed a low-level expression of the B19 genome as detected by quantitative RNA dot blots as well as by Northern analysis. Furthermore, a frame-shift mutation in a recombinant AAV-B19 hybrid genome segment that encodes the viral nonstructural (NS1) protein significantly reduced the observed inhibition of MK colony formation. These studies indicate tissue- tropism of B19 beyond the erythroid progenitor cell, and lend support to the hypothesis that B19 genome expression may be toxic to cell populations that are nonpermissive for viral DNA replication.

Granulocyte-macrophage colony formation in vitro using human non-phagocytic bone marrow cells

1988 ◽  
Vol 188 (6) ◽  
pp. 405-409 ◽  
Author(s):  
N. Ohhara ◽  
S. Okamura ◽  
S. Hayashi ◽  
T. Otsuka ◽  
Y. Niho
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Macrophage Colony ◽  
Marrow Cells

scholarly journals Abnormal in vitro proliferation and differentiation of T colony-forming cells in patients with lymphadenopathy syndrome

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1063-1069 ◽  
Author(s):  
Y Lunardi-Iskandar ◽  
V Georgoulias ◽  
W Rozenbaum ◽  
D Klatzmann ◽  
MC Coll ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymph Node ◽  
Growth Factors ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Interleukin 2 ◽  
Proliferation And Differentiation

Abstract Patients with acquired immunodeficiency syndrome (AIDS) present impaired colony growth and in vitro differentiation capacity of peripheral blood and bone marrow T colony-forming cells (T-CFC). We show that peripheral blood, bone marrow, and lymph node T-CFC from patients with persistent lymphadenopathy syndrome (LAS), a syndrome that can precede AIDS, displayed similar abnormalities. Indeed, peripheral blood T-CFC generated a low number of colonies in seven out of 12 patients, and almost no colonies were obtained from bone marrow cells of all patients. The simultaneous study of T-CFC from peripheral blood and lymph node mononuclear cells seems to provide a reliable indicator for the risk of developing AIDS. The six patients who developed AIDS displayed extremely low numbers of peripheral blood T- CFC (13 +/- 17 colonies per 5 X 10(4) cells), and in two of them, no colonies could be obtained from lymph node T-CFC. The remaining patients who had not developed AIDS displayed a higher number of peripheral blood T-CFC (141 +/- 113 per 5 X 10(4) cells) and lymph node T-CFC, which, in addition, preserved their clonogenic capacity. In some patients, peripheral blood and lymph node, but not bone marrow, T-CFC were capable of generating colonies in the absence of added growth factors or mitogens, whereas in others, colony formation was obtained with purified interleukin 2 (IL 2) alone. Both spontaneous and IL 2- induced colony formation was abrogated by a monoclonal antibody against the IL 2 receptor. Taken together, these findings suggest that at least some T-CFC expressed IL 2 receptors. Colonies generated either in the presence or in the absence of added growth factors were composed of T4+, T6+, and T8+ cells, indicating impaired in vitro T-CFC differentiation. These findings indicate that a dramatic quantitative and qualitative impairment of the proliferation and differentiation of peripheral blood and lymph node T-CFC precedes the clinical evolution from LAS to AIDS.

scholarly journals Modulation of murine erythropoiesis in vitro by syngeneic thymocytes: interactions of enhancing and suppressing subpopulations with fluorescent anti-theta antibody and polyamino acids

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 845-850 ◽  
Author(s):  
F Sieber ◽  
SJ Sharkis
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Surface Density ◽  
Bone Marrow Cells ◽  
Present Evidence ◽  
Density Fraction ◽  
Large Numbers ◽  
Negative Surface ◽  
Negative Surface Charge

Abstract As we have shown previously, cocultures of bone marrow cells with large numbers of syngeneic thymocytes enhance erythroid colony formation in plasma clots, whereas cocultures with low numbers of thymocytes suppress erythroid colony formation. In this article, we present evidence that the enhancing and suppressing functions of thymocytes are most likely mediated by at least two separate subpopulations. When thymocytes were fractionated on the basis of cell surface density of the theta antigen, enhancing cells were limited to the high theta density fraction, whereas suppressing cells were accumulated in the low theta density fraction. When thymocytes were fractionated on the basis of negative surface charge, the enhancing cells were recovered among the more negatively charged cells. A short in vitro incubation with polyamino acids selectively abrogated the suppressor function.

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