Pluripotent Haemopoietic Stem Cell Entry Into Dna Synthesis In Vitro After Stimulation By Ara-C Treatment In Vivo

Abstract. Non-cycling pluripotent bone marrow stem cells (CFU-S), taken 3 hrs after injection of 20 mg of Ara-C, have been shown to enter DNA synthesis at 1 to 3 hr after being cultured in α-medium. This phenomenon was observed when bone marrow was incubated as a plug, but not when incubated as a cell suspension in the present experimental conditions.
These results suggest that a medullary structure is necessary in order to observe this effect and/or that accessory cells are destroyed during the process of single cell preparation.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody-positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2 vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2 vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody-positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

Rationale

Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.

Objective

We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.

Methods and Results

Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.

Conclusions

These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.     

The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.     

Biological time and the effects of hydroxyurea on DNA synthetic activity of bone marrow and tumor cells in mice bearing the Ehrlich ascites carcinoma

The objective of this experiment was to attempt to induce, with hydroxyurea (HU), significant quantitative differences in the level of DNA-synthetic activity (DNA-SA) between a neoplastic cell population (the Ehrlich ascites carcinoma or EAC) and bone marrow in the same animal. Mice bearing a 5-day-old EAC were standardized to and kept on an LD 12:12 cycle (light 0600-1800 hr). They were treated with 500 mg/kg HU at 0500 hr (23 hr after lights on, or HALO) or at 1700 hr (11 HALO). DNA-SA was determined by liquid scintillation counting of 3H-thymidine incorporation into chemically isolated DNA. DNA-SA in bone marrow and EAC cells was monitored over the next 60 hr with subgroups of ten mice each killed every 3 hr beginning 3 hr after treatment with HU. The circadian system of the host influenced the response of the bone marrow to HU; i.e., the response to HU administered at 0500 hr was different both qualitatively and quantitatively from that for HU given at 1700 hr. Comparisons of DNA-SA in bone marrow and EAC from the same animal revealed time points after treatment with HU when DNA-SA in the EAC was high, but DNA-SA in bone marrow was low. These differences in the level of DNA-SA between a tumor cell population and bone marrow should be of therapeutic value; i.e., executor doses of anti-DNA-SA drugs such as cytosine arabinoside could be given at that point in time after treatment with HU when DNA-SA in the tumor was high, but DNA-SA in the bone marrow was low.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation I. Regeneration and Quantification of Thymocyte Progenitor Cells In the Bone Marrow

Growth kinetics of the donor-type thymus cell population after transplantation of bone marrow into irradiated syngeneic recipient mice is biphasic. During the first rapid phase of regeneration, lasting until day 19 after transplantation, the rate of development of the donor cells is independent of the number of bone marrow cells inoculated. the second slow phase is observed only when low numbers of bone marrow cells (2.5 × 10⁴) are transplanted. the decrease in the rate of development is attributed to an efflux of donor cells from the thymus because, at the same time, the first immunologically competent cells are found in spleen. After bone marrow transplantation the regeneration of thymocyte progenitor cells in the marrow is delayed when compared to regeneration of CFUs. Therefore, regenerating marrow has a greatly reduced capacity to restore the thymus cell population. One week after transplantation of 3 × 10⁶ cells, 1% of normal capacity of bone marrow is found. It is concluded that the regenerating thymus cells population after bone marrow transplantation is composed of the direct progeny of precursor cells in the inoculum.     

Interleukin 3 enhanced interleukin 2-dependent maturation of NK progenitor cells in bone marrow from mice with severe combined immunodeficiency

Human recombinant interleukin 2 (IL 2) and highly purified murine interleukin 3 (IL 3) were tested for their ability to generate NK activity in bone marrow cells from mice with severe combined immunodeficiency. IL 2 alone could dose dependently induce NK activity in marrow cells as determined by cytotoxicity against YAC-1 target cells. It was demonstrated that IL 3 had dual effects on the generation of NK activity in this culture system. The addition of IL 3 resulted in inhibition of NK cell activity seen at high concentrations of IL 2. In contrast, when IL 3 was added together with low concentrations of IL 2, the generation of NK cells as judged by cytotoxicity assay as well as the appearance of cells with NK phenotypes was markedly augmented. In some experiments, mice were treated with 5-fluorouracil (5-FU) to eliminate relatively differentiated NK precursors from bone marrow cells. It was noted that the residual immature marrow cells from 5-FU-treated mice showed little NK activity even after the culture with high concentrations of IL 2. Importantly, IL 3 could induce the generation of NK activity from 5-FU-treated marrow cells in the presence of IL 2. Kinetic studies indicated that NK activity was appreciably generated from 5-FU-treated marrow cells when preincubated with IL 3 at least for 12 hr and subsequently cultured with IL 2. The cells bearing IL 2 receptors appeared in 5-FU-treated marrow cells, even though cultured only with IL 3, which implied that IL 3 could support the development of very primitive NK cells from IL 2-unresponsive to IL 2-responsive states. These results suggested that IL 3 might play a crucial role for the IL 2-induced generation of NK cells in bone marrow through promoting the expression of IL 2R on NK progenitor cells.     

Regulation of apoptosis in interleukin-3-dependent hemopoietic cells by interleukin-3 and calcium ionophores

An immortalized interleukin-3 (IL-3)-dependent progenitor cell line, BAF-3, undergoes programmed cell death (apoptosis) when deprived of IL-3. This program is characterized by an early degradation of DNA into oligonucleosome-length fragments that precedes by several hours the loss of cell viability. In the absence of IL-3, DNA fragmentation and cell death can be prevented by the calcium ionophores A23187 (1 microM) and ionomycin (0.5 microM). This addition of calcium ionophore maintains cell viability while reversibly arresting the cell cycle. Apoptosis by growth factor deprivation is also a mechanism of cell elimination in bone marrow cells removed from the stromal micro-environment, as DNA fragmentation and cell death was shown to take place in primary cultures of IL-3-responsive bone marrow cells after IL-3 removal.     

Regulation of natural killer progenitors. Studies with a novel immunomodulator with distinct effects at the precursor level

NK cells originate from progenitors in the bone marrow and maturate independently of other lymphoid organs. NK cell maturation represents an important site for regulation of the level of NK activity and constitutes a potentially interesting target for therapeutic intervention. The effect of the immunomodulator Linomide (carboxamide-3-quinoline) on the regeneration of NK cells was studied in vivo after depletion of mature NK cells. Linomide significantly, although to various extent, accelerated the maturation of NK cells after specific depletion with antibodies to asialomonoganglioside, treatment with cyclophosphamide or lethal irradiation and syngeneic bone marrow grafting. Examination of the target cell spectrum lysed by spleen effector cells during Linomide treatment as well as studies of phenotype, clearly indicated that the effector cells studied were NK cells. Treatment of mice for 4 days with Linomide increased the frequency of bone marrow NK cell progenitors from 1/11,900 to 1/6,000 as judged by limiting dilution analysis. Direct addition of Linomide in vitro had no effect on cultures of mature NK cells from spleen, but had an additive effect to IL-2 on the generation of NK cells when added to bone marrow cultures. Our study indicates that different mechanisms exist for the regulation of progenitor and mature NK cells, and that the immunomodulator Linomide represent a potentially important tool for investigating the mechanisms governing NK cell maturation.     

Formation of capillary networks from bone marrow cultured in collagen gel

The growth of capillaries from mouse bone marrow was studied in collagen gel. When the culture contained sufficient bone marrow cells (more than 1 X 10(6) cells) and cell clusters, short capillaries with lumina appeared about one to two weeks after inoculation, following the proliferation of fibroblastic cells and hemopoietic cells. Four weeks after inoculation, these capillaries formed a network among hemopoietic cells and adipocytes. Electron microscopic observations revealed that these capillaries had thin walls and poorly developed basement membranes, similar to the sinusoids of bone marrow. These capillaries did not appear when the amount of inoculated bone marrow was reduced or dispersed to the point that the marrow cell clusters disappeared. The quantity of the inoculum and the clustering of cells, therefore, seems to play important roles in the appearance of the capillaries.     

Time course of the differentiation of granulated metrial gland cells in chimeric mice

Granulated metrial gland (GMG) cell differentiation was examined in deciduomata in lethally irradiated mice which had been reconstituted with rat bone marrow. The time at which the bone marrow reconstitution was carried out was varied in relation to the time of initiating the decidual reaction. GMG cells were examined at various times after bone marrow transplantation to determine whether they had the morphology which characterised them as being derived from host or donor stem cells. Differentiation of donor type GMG cells was seen within the first week after transplantation and occurred even when the bone marrow was transplanted two days after initiating the decidual response.     

Content of ATP and glucoso-6-phosphate in the dogs' bone marrow cells after cryopreservation

The content of ATP and glucoso-6-phosphate in the dogs' bone marrow cells before and after cryopreservation in liquid nitrogen have been studied. It has been found that ATP and glucoso-6-phospate levels in the dog bone marrow cells after cryopreservation in liquid nitrogen without cryoprotectors was very low. Solutions of DMSO and PEO-400 were used as cryoprotectors. The DMSO was more effective than PEO-400, because no metabolic changes were observed in incubated and freezed-thawed cells there.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

The effect of natural killer cells on the development of syngeneic hematopoietic progenitors

To determine whether natural killer (NK) cells are involved in the regulation of hematopoiesis, well-characterized, cell sorter-purified NK cells were incubated with syngeneic bone marrow, and the effect of this interaction on the development of various hematopoietic progenitors was assessed. NK cells were obtained from the peritoneal exudates of CBA/J mice after i.p. infection with live Listeria monocytogenes (LM). These NK cells were nylon wool-nonadherent and were purified by using M1/70, a rat anti-murine macrophage monoclonal antibody, and a fluorescence-activated cell sorter (FACS). Syngeneic bone marrow was incubated overnight with these M1/70-purified NK cells. The cells were then assayed in vitro to determine the effect on the colony formation of the following hematopoietic progenitor cells: the myeloid progenitor that produces mixed granulocyte/macrophage colonies (CFU-G/M), the myeloid progenitor that is committed to macrophage differentiation (CFU-M), and the early erythroid progenitor that is known as the burst-forming unit-erythroid (BFU-E). The marrow cells, after incubation with NK cells, were also injected into lethally irradiated syngeneic recipients to assay for the splenic colony formation capacity of the trilineage myeloid stem cell (CFU-S). Although the formation of BFU-E-, CFU-G/M-, and CFU-M-derived colonies was not adversely affected by the exposure of syngeneic bone marrow to purified NK cells, there was a dramatic decrease in the number of CFU-S-derived colonies. Incubation with NK-depleted cells did not result in an inhibition of colony formation by the CFU-S. Mixing experiments showed that the M1/70-labeled NK cells exerted their effect directly on the CFU-S and not on any accessory cells. The effect of the NK cells on colony formation by the CFU-S could be blocked competitively and selectively by the addition, before incubation, of a classic murine NK tumor target, Yac-1. Another tumor line (WTS) that is poorly recognized by NK cells was less effective in blocking the inhibitory effect of NK cells on CFU-S. The demonstration that purified NK cells can selectively inhibit the development of the tripotential CFU-S may point to the importance of NK cells in the regulation of hematopoiesis, in the development of some types of marrow dysfunction, and in the failure of engraftment of transplanted bone marrow.     

The relative importance of the bone marrow and spleen in the production and dissemination of B lymphocytes.

The relative importance of the bone marrow and spleen in the production of B lymphocytes was investigated in guinea pigs by the combined use of [³H]TdR radio-autography and fluorescent microscopy after the staining of B cells by FITC-F(ab′)₂-goat-anti-guinea pig Ig. Large and small lymphoid cells possess sIg in the marrow and spleen but B cell turnover in the marrow exceeds that in the spleen. That newly generated bone marrow B cells are not derived from an extramyeloid bursa equivalent was demonstrated by the absence of [³H]TdR labeled B cells in tibial marrow 72 hr after [³H]TdR was administered systemically, while the circulation to the hind limbs was occluded. Pulse and chase studies with [³H]TdR showed that large marrow B cells are derived from sIg-negative, proliferating precursors resident in the bone marrow and not from the enlargement of activated small B lymphocytes. The acquisition of [³H]TdR by splenic B cells lagged behind that observed in the marrow. Three days after topical labeling of tibial and femoral bone marrow with [³H]TdR, a substantial proportion of splenic B cells were replaced by cells that had seeded there from the labeled marrow. The studies unequivocally identify the bone marrow as the organ of primary importance in B cell generation and indicate that in the guinea pig rapidly renewed B lymphocytes of the spleen are replaced by lymphocytes recently generated in bone marrow. The rate of replacement of B lymphocytes in the lymph node by cells newly generated in the bone marrow takes place at a slower tempo than in the spleen.     

Germinal center B cells and antibody production in the bone marrow

In secondary antibody (Ab) responses, Ag processing and presentation occur in secondary lymphoid organs but most serum Ab is produced by cells in the bone marrow. Plasma cells in the bone marrow are derived from B cells activated by Ag in secondary lymphoid organs. We hypothesized that germinal center (GC) B cells, which acquire Ag from follicular dendritic cells in draining lymph nodes during the first few days of the secondary response, migrate to the bone marrow to terminally differentiate and produce specific Ab. To test this we looked for GC B cells in the thoracic duct lymph and in peripheral blood after secondary challenge using the peanut agglutininhi phenotype and blast cell morphology as markers for GC B cells. In addition, GC B cells were injected i.v. into naive recipients to determine if they would home to the bone marrow. Finally, to determine if the bone marrow environment supports maturation and Ab production by GC B cells, we cocultured GC B cells with bone marrow cells or bone marrow supernatants. The results indicate that blast cells bearing the GC B cell phenotype were present in both the thoracic duct and the peripheral blood 3 days after antigenic challenge. Day 3 peripheral blood cells secreted specific Ab, whereas cells isolated on day 0, 8, or 11 did not. Furthermore, in adoptive transfer experiments, only the day 3 GC B cells produced specific Ab and migrated to the bone marrow of naive mice. Finally, either bone marrow cells or factor(s) produced by bone marrow cells markedly enhanced Ab production by day 3 GC B cells. These data support the hypothesis that during the first few days after secondary challenge GC B cells seed the bone marrow and differentiate into plasma cells which produce the large quantities of Ab typical of secondary responses.     

Selective retention of T cell precursors in bone marrow. Cell populations after filtration through anti-immunoglobulin columns.

Lethally irradiated mice transplanted with normal syngeneic bone marrow have restored their immunocompetence 2–4 weeks after transplantation. During regeneration T cells seem to develop more slowly than B cells. We have studied the impact of anti-Ig column fractionation of bone marrow cells on their subsequent development into T cells. The results showed that in mice grafted with anti-Ig column-passed bone marrow cells (a) the number of θ-positive cells which develop was very much reduced, and (b) the development of GVH reactive cells and helper cells was much delayed when compared with mice grafted with normal unpassed bone marrow cells. In contrast, the development of B cell function and of the hematopoietic system was always normal. It was concluded that one possible explanation for such findings was the existence of immunoglobulin on T cell precursors.