Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.     

Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.     

Growth of hemopoietic cells after incubation with VP 16-213

We examined the effect of various concentrations of VP 16-213 (25-125 microM/l, 2-h incubation on normal and complete remission bone marrow from patients with acute leukaemia and on leukaemic blasts. The maximal tolerated dose of the drug for normal bone marrow GM-CFC was between 75 and 100 microM/l whereas that for complete remission bone marrow was distinctly lower. More early stem cells measured by aid of LTBMC were more resistant in normal, but not in every remission bone marrow. We have to examine if these LTBMC results are influenced by a damaged microenvironment by using 2 stage LTBMC. Spontaneous leukaemic cells showed a different, sometimes lower sensitivity to VP 16-213 doses maximally tolerated by normal hemopoietic cells so that the VP 16-213 incubation must not be effective for every leukaemia.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

Production and differentiation of NK lineage cells in long-term bone marrow cultures in the absence of exogenous growth factors.

Neither lytic NK cells nor IL-2-responsive NK precursors were produced in myeloid (Dexter) long-term bone marrow cultures (LTBMC). However, when myeloid LTBMC were switched to lymphoid (Whitlock-Witte) conditions and reseeded ("recharged") with fresh bone marrow cells (BMC), nonadherent cells with NK lytic activity and NK 1.1+ phenotype were produced within 1-2 weeks without the addition of exogenous IL-2 to the cultures. NK- and T cell-depleted BMC proliferated extensively in switched cultures and in 2 weeks generated cells that lysed the NK target YAC-1 but not the LAK target P815. The presence of NK precursors in the cultures was confirmed by reculturing nonadherent cells harvested from recharged LTBMC in fresh medium containing 50 U rIL-2/ml. High levels of NK lytic activity were generated. Sequential expression of NK 1.1 and IL-2 responsiveness followed by lytic activity was demonstrated by harvesting cells early after recharge, prior to the appearance of lytic cells. Elimination of NK 1.1+ cells depleted the ability to respond to IL-2 in secondary culture. Our studies demonstrate that myeloid-to-lymphoid switched LTBMC support the proliferation and differentiation of NK lineage cells from their NK 1.1-, nonlytic progenitors in the absence of an exogenous source of growth factors.     

Human granulocyte colony stimulating factor: effects on human long-term bone marrow cultures

Human granulocyte colony stimulating factor (G-CSF) was previously shown to support the survival and proliferation of early myeloid progenitors (pre-CFU) that are capable of generating more mature CFU-GM progenitor cells. To evaluate the scope of action of G-CSF in the hierarchy of hematopoietic stem cells, we studied the effects of recombinant G-CSF (rhG-CSF) on long-term cultures of normal human bone marrow cells (LTBMC). We found that rhG-CSF predominantly influenced initial cell proliferation and expansion of CFU-GM progenitor cells in LTBMC before establishment of a confluent adherent layer. In rhG-CSF-treated LTBMC, the stromal cell layer was associated with a higher proliferative capacity and progenitor cell content as compared to control cultures. This effect was pronounced early after layer confluence and was gradually lost with culture time. rhG-CSF did not alter the duration of the productive phase of LTBMC, suggesting that it may not be active on the hematopoietic stem cells responsible for LTBMC propagation. Alternatively, stromal cells may exert tight regulatory control over progenitor cells, even in the presence of rhG-CSF.     

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.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Haemopoietic Inductive Capacity of Irradiated Stromal Cell Layers In Human Micro Long-Term Bone Marrow Cultures

Abstract. In a micro long-term bone marrow culture (LTBMC) system the effects of irradiation on confluent stromal cell layers were studied. In order to individually analyse the number of granulocyte-macrophage colony-forming cells (GM-CFC) per LTBMC a miniaturized human GM-CFC assay was established. the normalized GM-CFC numbers in the micro-assay compared well with data by the conventional GM-CFC assay. Pre-formed stromal cell layers were irradiated with doses up to 20 Gy and subsequently recharged with allogeneic bone marrow cells (BMC). Immediately before recharge the BMC were stromal cell-depleted by nylon wool filtration. When stromal cell-depleted BMC were inoculated on empty culture dishes, in vitro haemopoiesis rapidly declined. Sustained GM-CFC production, however, was seen when these cells were used as a second inoculum. It is concluded that irradiation doses of up to 20 Gy do not cause alteration of the haemopoietic inductive capacity of confluent stromal cell layers.     

Haemopoietic inductive capacity of irradiated stromal cell layers in human micro long-term bone marrow cultures

In a micro long-term bone marrow culture (LTBMC) system the effects of irradiation on confluent stromal cell layers were studied. In order to individually analyse the number of granulocyte-macrophage colony-forming cells (GM-CFC) per LTBMC a miniaturized human GM-CFC assay was established. The normalized GM-CFC numbers in the micro-assay compared well with data by the conventional GM-CFC assay. Pre-formed stromal cell layers were irradiated with doses up to 20 Gy and subsequently recharged with allogeneic bone marrow cells (BMC). Immediately before recharge the BMC were stromal cell-depleted by nylon wool filtration. When stromal cell-depleted BMC were inoculated on empty culture dishes, in vitro haemopoiesis rapidly declined. Sustained GM-CFC production, however, was seen when these cells were used as a second inoculum. It is concluded that irradiation doses of up to 20 Gy do not cause alteration of the haemopoietic inductive capacity of confluent stromal cell layers.     

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

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.     

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.     

A comparative morphometric study on the ultrastructure of adherent cells in long-term bone marrow culture from normal and congenitally anemic mice

The relationship between structure and function of bone marrow stromal tissue in adherent layers of long-term bone marrow cultures (LTBMCs) from normal and congenital anemic mice (C57BL, Sl/Sld, Sl+/Sl+, W/Wv, and W+/W+) was investigated. Many previously reported features were confirmed. However, in LTBMC from all strains of mice examined, isolated cilia with the axonemal structure of a 9 + O pattern with obvious dynein arms were observed in the blanket cells. The frequency of cilia was approximately 2%-5% of total number of profiles of blanket cells examined. Crystalloid inclusions (CI) were observed in cultured macrophages similar to those reported in vivo in all strains of murine LTBMC. The CI could be classified into four types according to their structure in the same way as in vivo (type A to type D), with a predominance of type A in the cultures. Viral particles were also apparent in adherent cells of all strains (except W/Wv and W+/W+), which were compatible with a type C retrovirus. Gap junctions occurred regularly between the adherent cells of LTBMC, particularly between blanket cells and preadipocytes. The most frequent appearance of gap junctions was found in Sl/Sld cultures. The phenomena of normal and abnormal hematopoiesis appear to be accurately reproduced in culture, thus retaining the same relationship between function and structure as occurs in vivo. The surface of isolated cilia of blanket cells, CI of macrophages, viral particles among adherent cells, and gap junctions between blanket cells and preadipocytes is discussed.     

Response kinetics of radiation-induced micronucleated reticulocytes in human bone marrow culture

The frequency of micronucleated reticulocytes (MN-RETs) in the bone marrow or peripheral blood is a sensitive indicator of cytogenetic damage. While the kinetics of MN-RET induction in rodent models following irradiation has been investigated and reported, information about MN-RET induction of human bone marrow after radiation exposure is sparse. In this report, we describe a human long-term bone marrow culture (LTBMC), established in three-dimensional (3D) bioreactors, which sustains long-term erythropoiesis. Using this system, we measured the kinetics of human bone marrow red blood cell (RBC) and reticulocyte (RET) production, as well as the kinetics of human MN-RET induction following radiation exposure up to 6Gy. Human bone marrow established in the 3D bioreactor demonstrated an average percentage of RBCs among total viable cells peaking at 21% on day 21. The average percentage of RETs among total viable cells reached a maximum of 11% on day 14, and remained above 5% by day 28, suggesting that terminal erythroid differentiation was still active. Time- and dose-dependent induction of MN-RET by gamma radiation was observed in the human 3D LTBMC, with peak values occurring at approximately 3 days following 1Gy irradiation. A trend towards delayed peak to 3-5 days post-radiation was observed with radiation doses ≥2Gy. Our data reveal valuable information on the kinetics of radiation-induced MN-RET of human bone marrow cultured in the 3D bioreactor, a synthetic bioculture system, and suggest that this model may serve as a promising tool for studying MN-RET formation in human bone marrow, thereby providing opportunities to study bone marrow genotoxicity testing, mitigating agent effects, and other conditions that are not ordinarily feasible to experimental manipulation in vivo.     

T-cell depletion and haematological side effects of two cytotoxic monoclonal antibodies.

We investigated two cytotoxic monoclonal antibodies of BL-series (BL-IIIB4 and BL-IIG2) according to T-lymphocyte depletion from bone marrow. Both antibodies work together with human complement similar Campath-1, which was tested parallelly. The extent of T-cell depletion is about 95% for all three antibodies. On the other hand the haemopoietic side effects tested by CFU-GM recovery and LTBMC are for the BL-antibodies not as strong as for Campath-1, especially in view of LTBMC. T-cell regeneration could be shown in long term cultures. Our results indicate a possible suitability of the two investigated antibodies for T-cell depletion of bone marrow.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

Erythropoiesis in murine long-term bone-marrow cell cultures: dependence on erythropoietin and endogenous production of an erythropoietic stimulating activity

Erythropoiesis was obtained in murine long-term bone-marrow cell cultures (LTBMCs) in the presence of erythropoietin (Epo) when the medium was frequently renewed. The level of the erythropoietic differentiation was shown to be a function of the erythropoietin concentration. In response to Epo addition, an activity which stimulates CFU-E proliferation in semisolid cultures of fresh bone marrow cells was detected in the LTBMC supernatants. These results suggest that another factor, whose synthesis may be under Epo control, participates in the stimulation of erythropoiesis in vitro.     

Expression of CD23 by human bone marrow stromal cells.

CD23 is a surface antigen expressed by a variety of human hematopoietic cells and shown to display multiple biological functions. In present work, we assayed CD23 expression by human bone marrow (BM) or by stromal cells derived from this tissue. While freshly isolated BM-cells showed low CD23 expression, a subset of long term BM-culture (LTBMC)-derived stromal cells expressed CD23 mRNA at high levels in their steady state and secreted soluble CD23 in their culture supernatants. To assay the role of CD23 in LTBMC, these cultures were initiated in the presence of neutralizing anti-CD23 mAb. A dramatic decrease in total numbers of hematopoietic cells and CFU-GM recovery was observed in these cultures as compared to controls. These data suggest a role of CD23 expression in stroma cell functions and further confirm the ability of this antigen to regulate human hematopoietic cell development.