Analysis of human dysplastic haematopoiesis in long-term bone marrow culture

In this paper we have analysed the behaviour of myelodysplastic marrow in a long-term bone marrow liquid culture system (LTBMC) from eleven patients with myelodysplastic syndromes with regard to cellularity, day-7 and day-14 CFU-GM growth, cluster formation, adherent cells and CFU-F formation. An altered CFU-GM pattern was found in 64% of cases at diagnosis, while normal growth was seen in the remaining cases, all of which were affected by refractory anaemia. The levels of CFU-GM, as well as cellularity, were reduced in myelodysplastic marrows compared to normal controls over the whole duration of LTBMCs. Cases with a normal CFU-GM level at diagnosis also showed pathological behaviour when examined in LTBMC. The duration of dysplastic haematopoiesis was significantly shorter than that of controls. The proliferative ability of CFU-F was reduced in 50% of cases as shown by replating experiments. In conclusion, myelodysplastic marrow shows an abnormal behaviour in LTBMC, even in those cases which present normal CFU-GM growth at diagnosis.     

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.     

The effect of 1,25-dihydroxyvitamin D3 on hematopoiesis in long-term human bone marrow cultures

The modulatory effect of 1,25-dihydroxyvitamin D3 (vit D) on the growth of myeloid progenitors and on the composition of the stromal layer in human bone marrow long-term cultures was studied. Vit D (2 X 10(-8) M) caused an enhancement in myeloid progenitor cell (CFU-C) growth in the nonadherent and adherent layers during the entire 5-week incubation period. The vitamin did not alter the differentiation pattern of CFU-C (monocyte-macrophage progenitors CFU-M, granulocytic progenitors CFU-G, or monocyte-granulocyte progenitors CFU-GM). Vit D caused a marked increase in the percentage of lipid-containing cells in the adherent layer and an increase in the number of cells that specifically bound My4 monoclonal antibody (McAb), that reacted positively to fluoride-sensitive alpha-naphthyl acetate esterase, and that phagocytosed Candida albicans (CA). Concentrated supernatants harvested from control cultures showed significant levels of myeloid colony stimulating factor (CSF) activity. The addition of vit D to cultures for 5 weeks did not alter CSF levels. These results suggest that vit D may play a role in hematopoiesis by acting directly on the progenitor cells or via the stromal cell production of stimulatory factor(s).     

FADD deficiency impairs early hematopoiesis in the bone marrow

Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas, FADD is required for the recruitment and activation of caspase 8. In addition, a death receptor-independent function of FADD is essential for embryogenesis. In previous studies, FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers, as shown by Rag1(-/-) blastocyst complementation assays. However, T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers, whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types, including bone marrow cells, by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoproliferation diseases, unlike Fas-deficient mice. Instead, a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow, a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor-enriched population. Furthermore, FADD-deficient bone marrow cells were defective in their ability to generate lymphoid, myeloid, and erythroid cells. Thus, the results revealed a temporal requirement for FADD. Although dispensable during lymphopoiesis post lineage commitment, FADD plays a critical role in early hematopoietic stages in the bone marrow.     

Clonal nature of fibroblast colonies formed by stromal bone marrow cells in culture

The clonal nature of CFUf-derived fibroblast colonies was tested in mixed cultures of CBA and CBAT6T6 bone marrow cells. Inoculation of marrow cell suspensions into flasks coated with poly-I-lysin has proved that no stromal aggregates were present among cells subjected to explantation. Marrow cell cultures depleted of macrophages and myeloid cells were used for chromosome analysis. The coincidence of karyotypes within a stromal colony was found in mixed cultures, which proves that CFUf-derived fibroblast colonies are cell clones.     

Hybrid resistance to precursor cells of bone marrow stroma]

A focusl of hemopoiesis appearing after the transplantation of a bone marrow fragment of C57BL mice to syngeneic mice (under the kidney capsule) contained more hemopoietic cells than in transplantation to the semisyngeneic (CBA X C57BL) FI recipient. Experiments were conducted with a secondary seeding by intravenous injection of hemopoietic cells of the C57BL transplant genotype into the transplant depopulated by irradiation; it was shown that these differences were caused by lesser dimensions of the hemopoietic microenvironment in the focus in the hybrid organism in comparison with such in the syngeneic system. Thus, the hybrid resistance was expressed not only to the hemopoietic cells, but also to the stromal precursors transferring the hemopoietic microenvironment.     

Induction and modification of erythropoiesis in a suspension culture system derived from long-term bone marrow culture of Syrian hamster

Cells in a subculture system derived from long-term hamster bone marrow culture were induced to differentiate into hemoglobin-producing cells in response to added erythropoietin. Treatment of the subculture with dimethyl sulfoxide or other 'Friend inducers' but not 5-azacytidine effected no remarkable induction, though they enhanced the effect of externally added erythropoietin. 3-Aminobenzamide, a weak inducer of Friend cell differentiation [Morioka, K., K. Tanaka, M. Ishizawa and T. Ono: Dev. Growth Differ. 24, 507-512 (1982)], had a strong inhibitory effect on erythropoiesis in the subculture system. Phorbol esters and glucocorticoids (both are known to be strong inhibitors of Friend cell differentiation) acted as strong inhibitors of the differentiation in our system as well. The present system was shown to provide a useful in vitro model to compare the differentiation of normal erythroid progenitors with the previously established systems of neoplastic cells.     

Preparation of a lymphoblastoid B-cell line from a long-term suspension culture of human bone marrow

Lymphoblastoid cell line was obtained from long-term human bone marrow culture. The cell line was characterized using methods of immunological phenotyping, cytochemistry and cytogenetics. This cell line represents different stages of B-cell differentiation, karyotype 46 XX. The cells of this line were able to support their growth during more than 10 months without exogenous stimulation.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

The regulation of hematopoiesis following bone marrow transplantation

Allogeneic bone marrow transplantation requires that donor stem cells home to the recipient bone marrow, proliferate and differentiate under normal physiologic regulatory mechanisms. Recent observations that T cell depletion of donor bone marrow leads to a greatly increased incidence of graft failure mandate a detailed understanding of the engraftment process. Post-transplant hematopoietic deficiencies appear to be related to several sources: decreased number of stem cells, activation of donor hematopoietic suppressor cells, rejection of donor stem cells by residual recipient lymphocytes and abnormal function of accessory cells that produce hematopoietic growth factors. A better understanding of the relative roles of these factors should lead to a better understanding of engraftment as well as graft failure and its prevention.     

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.     

Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro.

Rat bone marrow stromal cells were cultured in vitro. At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later. The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules. Next, these mineralized nodules were electron-microscopically examined. The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae. Some lysosomes and microfilaments were also visible in the cytoplasms. Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix. The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen. A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules. In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed. That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils. From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process. This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms.     

Self-renewal capacity and clonal succession of haemopoietic stem cells in long-term bone marrow culture

The CFU-s proliferative potential varied greatly during long-term cultivation. Most of the CFU-s in the cultures were represented by cells with low renewal capacity. Pre-CFU-s cells capable of producing multipotential colonies in methylcellulose, which contained CFU-s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. The findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre-CFU-s, but not the CFU-s themselves, are clonogenic progenitors.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part II

A mathematical model of mouse granulopoiesis in long-term bone marrow culture was constructed, based on established in vivo cell kinetic parameters. We applied the model to the cell kinetic experiment presented in Part I. Comparing model-predicted cell kinetics with the experimental data led to iterative testing of several hypotheses. In the final model, the cell kinetics of intact tissue culture flasks were reconstructed, using the experimental data from 10 days of tube culture. Among other things, our analysis suggests that the parameters of normal in vivo granulopoiesis apply to bone marrow culture.     

Role of the thymus in regulating bone marrow hematopoiesis in the stress reaction

The role of thymus in the regulation of erythropoiesis during stress has been studied in experiments on immobilized mice. The development of pronounced leukopenia due to stimulation of erythro- and granulocytopoiesis was demonstrated. Thymectomy, performed a month before immobilization, abolished the phenomenon of erythropoietic cellularity, indicating an important role of thymus in the regulation of myelopoiesis during stress.