Tissue Culture Of Bone Marrow Research Articles

The construction of high efficiency human bone marrow tissue ex vivo

The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.

Inosine analogs as chemotherapeutic agents for African trypanosomes: metabolism in trypanosomes and efficacy in tissue culture.

Certain purine analogs, the pyrazolopyrimidines, are effective chemotherapeutic agents against Leishmania spp. and Trypanosoma cruzi both in vitro and in some clinical models. Heretofore they have not been effective against the African trypanosomes; this suggested that these organisms were not comparable to the other pathogens with respect to their purine metabolism. We have studied the efficacy and metabolism of the pyrazolopyrimidine bases allopurinol and thiopurinol, their respective ribonucleosides, and the C-nucleosides formycin B and 9-deazainosine in Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense. The efficacy of these compounds was dependent on the purine content of the culture medium. The C-nucleosides were the most effective, with 90% effective doses for formycin B and 9-deazainosine of 0.01 and 2 micrograms/ml, respectively. Metabolism was the same in both the bloodstream and culture forms and identical to that reported for Leishmania spp. and T. cruzi. Both agents were phosphorylated to the ribonucleotide and then aminated to produce adenine nucleotide analogs. Growth inhibition studies were performed with three inosine analogs (allopurinol riboside, formycin B, and 9-deazainosine) on trypomastigotes grown in bone marrow tissue culture. Both C-nucleosides eradicated the infection at a concentration of 0.25 micrograms/ml. Unlike formycin B, 9-deazainosine is not known to be aminated by mammalian cells and appears to be relatively nontoxic in three different mammalian tissue culture systems. This nucleoside was very active against all pathogenic leishmaniae and trypanosomes investigated and is worthy of further study.

Tissue culture of bone marrow. V. Effect of 5Beta(H) steroids and cyclic AMP on heme synthesis

In mice 5beta(H) steroids have been shown tostimulate heme synthesis in vivo. The current studies were undertaken to evaluate the effect of these steroids on heme synthesis by use of the "well" method of marrow culture. The 5beta(H) steroid metabolites 3alpha-hydroxy-5beta-pregnane-11, 20-dione, 3beta-hydroxy-5beta-pregnan-20-one, 3 alpha,17alpha-dihydroxy-5beta-androstan-3-one stimulated Fe uptake by human marrow explants cultured in wells. The dibutyrl analog of adenosine 3',5'-monophosphate (db cAMP) also stimulated Fe uptake. The effect of the latter compound was enhanced by theophylline. There was no additive nor synergistic effect between db cAMP and the 5beta(H) steroid metabolites. There was no inhibition of the steroid metabolite effect by either antierythropoietin serum or cyproterone. Uptake of fe was also enhanced by the cations Ca++ and Co++.

Bleomycinの造血器に及ぼす影響に関する研究

In order to observe the influence of Bleomycin on the bone marrow thrombopoiesis, clinical tissue culture of bone marrow from normal guinea pigs was conducted. Bleomycin solutions at various concentration were added directly to the culture media and observations were carried out to see effect of Bleomycin on the function of megakaryocytes of bone marrow.The results obtained show that the thrombopoietic functions of bone marrow were not inhibited in the medium containning 0.0005mg/ml, 0.05mg/ml and 5mg/ml of Blomycin.

Haemoglobin synthesis in human bone marrow culture

A bone marrow tissue culture system has been established and the pattern of whole haemoglobin and individual globin chain synthesis examined over a period of 10 days. Total protein synthesis declines in culture with time, haemoglobin A synthesis decreasing at a faster rate than that of non-haemoglobin proteins. Erythropoietin increased and prolonged haemoglobin A synthesis and to a lesser extent stimulated total protein synthesis. One component of the non-haemoglobin proteins has similar chromatographic properties to the γ-chains of haemoglobin F but is easily distinguished from the latter by its higher molecular weight and peptide composition. Haemoglobin F is not produced under these culture conditions.

キノリン誘導体の骨髄造血機能に及ぼす影響に関する研究

In order to observe the influences of chloroquine on the bone marrow thrombopoies, clinical tissue culture of bone marrow from normal guinea pigs was conducted. Chloroquine diphosphate sulutions at various concentrations were added directly to the culture media and observations were carried out to see effect of chloroquine on the functions of megakaryocytes of bone marrow.As a result, it has been seen that the thrombopoietic functions of bone marrow were hardly inhibited in the medium containning 2γ/dl and 20γ/dl of chloroquine, but were markedly inhibited in the medium containning 200γ/dl and 2000γ/dl of chloroquine.

骨髄体外組織培養による白血病の螢光顕微鏡学的研究

骨髄体外組織培養による白血病の螢光顕微鏡学的研究

骨髄体外組織培養による白血病の螢光顕微鏡学的研究

骨髄体外組織培養による白血病の螢光顕微鏡学的研究

Cytochrome Cの骨髄造血機能に及ぼす影響に関する研究

By means of bone marrow tissue culture method deviced by Hiraki, the author investigated the effects cytochrome C on the thrombopoietic function in bone marrow and obtained the following results.1) Cytochrome C diluted in several concentrations was added to the bone marrow tissue culture of normal guinea pigs, normal persons and patients with various diseases. Average number of megakaryocytes in the growth zone and its thrombopoietic function were examined.As a result, it has been seen that the thrombopoietic functions of normal bone marrow added with an optimal amount of cytochrome C were moderately accelerated both in guinea pigs and human beings as compared with that of control added with saline.2) The cytochrome C and each of three kinds of carcinostatic substances; mitomycin C, trenimon, and carzinophyllin were added to the bone marrow tissue culture of normal persons the effects of these drugs on the thrombopoietic functions in the bone marrow were investigated. On the other hand, the bone marrow of guinea pigs of which bone marrow had been rendered to hypoplastic by repeated administration of the above described carcinostatic substances and then treated with repeated administration of cytochrome C were cultured. The thrombopoietic functions of megakaryocytes in these bone marrow tissue culture treated with cytochrome C were maintained moderately high as compared with the control treated with carcinostatic substances and saline only.From these results it could be concluded that cytochrome C has the accelerating effects on the thrombopoietic function of normal bone marrow as well as the protecting effects on the thrombopoiesis from the myelodegenerative especially thrombopoiesisinhibiting action of several carcinostatic substances.

新ビタミンB群の骨髄組織培養に及ぼす影響

新ビタミンB群の骨髄組織培養に及ぼす影響

新ビタミンB群の骨髄組織培養に及ぼす影響

新ビタミンB群の骨髄組織培養に及ぼす影響

Adenosine triphosphate (ATP)の骨髄造血機能に及ぼす影響に関する研究

In order to observe the influences of ATP on the bone marrow thrombopoiesis, clinical tissue culture of bone marrow from normal guinea pigs, normal persons and patients with hypoplastic anemia was conducted. ATP solutions at various concentrations were added directly to the culture media and observations were carried out to see effect of ATP on the functions of megakaryocytes of bone marrow.As a result, it has been seen that by adding an optimal amount of ATP the functions of megakaryocytes were accelerated.

Adenosine triphosphate (ATP)の骨髄造血機能に及ぼす影響に関する研究

In order to observe the influences of ATP on the bone marrow leukopoiesis, clinical tissue culture of bone marrow from normal rabbits, normal persons and patients with hypoplastic anemia was conducted. ATP solutions at various concentrations were added directly to the culture media and observations were carried out to see effect of ATP on the functions of neutrophils of bone marrow.As a result, it has been demonstrated that by adding an optimal amount of ATP the ontgrowth, cell density and wandering velosity of neutrophils were increased.

MY肉腫移植による実験的類白血病反応に関する研究

Leukemoid-reaction inducing factor was searched in mice with myelogenous type of leukemoid reaction indnced by MY sarcom.The following results were obtained.1) Leukemoid reaction was not induced by subcutaneous injection of the extract of MY sarcom. Tissue culture of bone marrow of mice injected with the extract did not show difference from the controls. Therefore, it was presumable that the extract of MY sarcom did not contain leukemoid-reaction inducing factor.2) Serum of mice with leukemoid reaction induced by MY sarcom was demonstrated to produce hyperplasia of the bone marrow of normal mice. This seemed to indicate that serum of the mice with leukemoid reaction contained leukemoid-reaction inducing factor.

抗白血病剤の骨髄造血機能に及ぼす影響に関する研究

In order to compare the influences of various anti-leukemic agents on the bone marrow thrombopoiesis, the clinical tissue culture of bone marrow from normal guinea pigs and healthy persons was conducted. Various anti-leukemic agents were added to the culture media at the maximum concentration where they would not inhibit the bone marrow growth in culture, and observations were carried out to see the influences of these agents on the function of megakaryocytes of bone marrow. As the results, the author arrived at the follwing conclusions.1. Urethane. Nitromin. Demecolcin, Actinomycin C and Mitomycin C inhibit the thrombopoietic function of bone marrow to a high degree.2. Thio-TEPA, 8-Azaguanine, Carzinophilin, and Chromomycin demonstrate a moderate inhibitory action on the thrombopoietic function of bone marrow.3. 6-MP slightly inhibits the thrombopoietic function of bone marrow.4. Myleran, and the combined use of 6-MP and Prednisolone show no inhibitory effect on the thrombopoietic function of bone marrow.5. On the other hand, Prednisolone alone rather augments the bone marrow thrombopoietic function.

サポニン投与による実験的類白血病反応に関する研究

In this study attempts were made to investigate how one could differentiate the leukemoid reaction induced in C57BL mice by saponin from true leukemia.The results obtained were as follows.1. Observation of the growth zone of bone marrow tissue culture (Hiraki's tissue culture slide No.1; Depth 0.2 mm) of these mice revealed that immature blood cells were seen densely packed in the central portion around the explant, but in the intermediate to the peripheral zones the cells were scattered less dense; in these latter zones were found mainly leukocytes that were more mature and had more vigorous migratory capacity.Namely, the growth pattern in the leukemoid reaction of these mice was normal and distinctly different from that of mouse leukemia. The growth zone of leukemic bone marrow extremely dense in cellularity and its boundary was sharply defined.Neutrophils that appeared in the growth zone proportionally increased in number to the progression of the leukemoid reaction in these mice. The growth area, wandering velocity of neutrophils and cell density in the leukemoid reaction were subnormal than those of normal mice.The cytomorphologic structure of blood cells studied by vital staining was normal in the leukemoid reaction and did not show atypical cells observed in mouse leukemia. Besides, characteristic crystals were seen in each cytoplasm of neutrophils that appeared in the growth area of bone marrow tissue culture from saponin-treated mice.Megakaryocytes that appeared in the growth zone of bone marrow tissue culture (Hiraki's tissue culture slide No.2; Depth 0.6 mm) from saponin-treated mice were decreased in number, but their function was well preserved.2. The growth pattern of spleen tissue culture (Hiraki's tissue culture slide No.1) from saponin-treated mice was similar to that of bone marrow.Megakaryocytes appeared abundantly in the growth zone of spleen tissue culture (Hiraki's tissue culture slide No.2) in proportion to the progression of the leukemoid reaction but were not seen in the normal control.3. In normal bone marrow and spleen tissues cultured in the medium with an addition of saponin saline solution, both the growth area and cell function were suppressed than in the control without an addition of saponin saline solution.4. On the fluorescence microscopic observation of bone marrow tissue culture from saponin-treated mice, the growth area was diffusely red, because the myeloid series contained red fluorescent granules.Among them were scattered yellow and green fluorescence due to the presence of lymphocytes, monocytes, and various degenerated cells. These pictures were the same as the normal control.In myelogenous mouse leukemia, the growth zone had green colour with reddish stipplings and its boundary was well defined. This was because blasts were green and a few mature leukocytes contained granules of a reddish orange colour.Next, the growth zone of spleen from the leukemoid reaction observed under a fluorescence microscope, the zone was diffusely reddish orange in colour and intermingled with scattered yellow green colour.In the normal control, the growth zone exhibited diffusely yellow green colour, in which orange fluorescence was scattered.In myelogenous mouse leukemia, the growth area of spleen showed a similar colour and pattern to those of the bone marrow tissue culture.

核酸の骨髄体外組織培養に及ぼす影響

核酸の骨髄体外組織培養に及ぼす影響

核酸の骨髄体外組織培養に及ぼす影響

核酸の骨髄体外組織培養に及ぼす影響

腎性貧血の成因に関する研究

The hematopoietic function of the bone marrow of renal patients and influences of azotemia on the normal bone marrow were studied by means of clinical bone marrow tissue culture. The results obtained are as follows.1) The marrow myelopoietic function of nephrotic patients was increased, when normal human serum was added to the medium.2) The myelopoietic function in acute glomerulonephritis showed no significant change in regard to the cell density index but the relative growth rate and cellular migratory velocity were slightly decreased, when the bone marrow was cultured in a medium containing normal human serum.3) When the bone marrow from patients with chronic glomerulonephritis was cultured in a medium containing normal human serum, the marrow function became activated in a slightly elevated NPN group while it became dep essed in a highly elevated NPN group.4) The bone marrow from uremic patients cultured in a medium containing normal human serum showed a growth pattern simulating that of hypoplastic anemia. The relative growth rate was half the control value and the migratory velocity of neutrophilic cells and cell density index were markedly decreased.5) The bone marrow from patients with chronic renal insufficiency was cultured, to investigate the megakaryocytic function, in a medium containing normal human serum. There was reduction in both megakaryocyte number and percentage of motile forms of megakaryocytes appearing in the growth zone.6) The normal bone marrow showed a reduced relative growth rate and migratory velocity of neutrophilic cells, when cultured in a medium containing azotemic serum.7) Similarly, the megakaryocytic function became depressed by an addition of azotemic serum to the medium.From the evidence obtained, it appears that azotemia accompanying chronic renal insufficiency exerts a depressing action on both myelopoietic and megakaryocytic functions. It is conceivable that the bone marrow under such an inhibitory action of a long standing will gradually show a transition to a hypoplastic state.

核酸の骨髄体外組織培養に及ぼす影響

By means of clinical culture method, the author added RNA and DNA at various directly to the media in which was cultured the bone marrow of normal rabbit, normal person, and aplastic anemia, with the purpose to see the effect of these nucleic acids on the erythropoiesis, and obtained the foliowing results, 1. The addition of RNA and DNA at optimal concentration to the bone marrow tissue culture of normal rabbit clearly accelerated erythropoiesis.2. The addition of RNA and DNA at optimal concentration revealed a slignt accelerating effect of erythropoiesis of normal human bone marrow in tissue culture.3. In the case of the bone marrow tissue of aplastic anemia patient, likewise the addition of RNA and DNA at opitimal concentration showed a slignt accelrating effect of erythropoiesis.