Long-term Marrow Cultures Research Articles

Long-term bone marrow culture as a stem cell source for transplantation

Stem cells in bone marrow capable of inducing spleen colonies in lethally irradiated mice can be maintained in liquid culture for up to 100 days if an appropriate marrow-derived feeder layer is provided. Marrow from some mouse strains cannot be maintained in such cultures. Neither duration of culture nor in vitro cultivation of marrow on allogeneic feeder layer in any way modulates the occurrence of graft-versus-host disease. Cocultivation of marrow with allogeneic feeder layers does not induce tolerance to feeder-layer-type skin grafts in lethally irradiated syngenic recipients. Though an excellent source of stem cells, long-term marrow cultures require further modification before successful allogeneic marrow transplantation without graft-versus-host reaction can be carried out.

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia.

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.

Formation of osteoclast-like multinucleated cells is human long-term marrow cultures in controlled by osteotropic hormones

Formation of osteoclast-like multinucleated cells is human long-term marrow cultures in controlled by osteotropic hormones

Differentiation pathways of pluripotent hemopoietic stem cells in long-term mouse bone marrow culture

Differentiation pathways of CFU-S were examined in mouse bone marrow long-term culture system. E/G ratios of non-adherent CFU-S in this system increased from 2 to 7–9 by 2–3 weeks of culture and then fluctuated during the culture period. On the other hand, E/G ratios of adherent CFU-S were lower than that of non-adherent CFU-S, and always remained under 2. Some of the supernatants collected from culture flasks at weekly intervals increased E/G ratios of normal bone marrow CFU-S after 18–20 h of incubation. These results suggest that the differentiation of CFU-S is controlled by humoral factors, secreted from yet unknown cells in bone marrow, in this system.

CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures

The CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long- term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.

CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures

AbstractThe CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long- term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.

Surface Phenotypes of Human Hemopoietic Progenitor Cells Defined by Monoclonal Antibodies

AbstractA panel of ten monoclonal antibodies which react with antigens present on the surface of myeloid leukemic cells was used to investigate the distribution of these antigens on normal hemopoietic stem cells and progenitor cells at various stages of maturity. A population of immature cells, possibly stem cells, that are capable of regenerating CFU–GM in long-term marrow cultures reacts with four antibodies recognizing antigens abundantly expressed in leukemic cells, but does not react with antibodies against la-like molecules or against carbohydrate determinants specific for myeloid cells. Progenitor cells that form mixed colonies in semisolid medium (CFU–GEMM), early erythroid (BFU–E) and early myelomonocytic (type 1 CFU–GM) progenitors retain the antigens present on the hypothetical stem cell population and begin to express la-like antigens. As they differentiate, myeloid and erythroid progenitors undergo a series of quantitative and qualitative shifts in surface phenotype. They begin to express stage-related, lineage-specific antigens and cease expressing antigens common to early cells of different lineages. The identification of antigens present on very immature normal progenitor cells should be valuable in future studies aimed at the detailed characterization of this relatively little-known hemopoietic cell population.

Surface phenotypes of human hemopoietic progenitor cells defined by monoclonal antibodies

A panel of ten monoclonal antibodies which react with antigens present on the surface of myeloid leukemic cells was used to investigate the distribution of these antigens on normal hemopoietic stem cells and progenitor cells at various stages of maturity. A population of immature cells, possibly stem cells, that are capable of regenerating CFU-GM in long-term marrow cultures reacts with four antibodies recognizing antigens abundantly expressed in leukemic cells, but does not react with antibodies against Ia-like molecules or against carbohydrate determinants specific for myeloid cells. Progenitor cells that form mixed colonies in semisolid medium (CFU-GEMM), early erythroid (BFU-E) and early myelomonocytic (type 1 CFU-GM) progenitors retain the antigens present on the hypothetical stem cell population and begin to express Ia-like antigens. As they differentiate, myeloid and erythroid progenitors undergo a series of quantitative and qualitative shifts in surface phenotype. They begin to express stage- related, lineage-specific antigens and cease expressing antigens common to early cells of different lineages. The identification of antigens present on very immature normal progenitor cells should be valuable in future studies aimed at the detailed characterization of this relatively little-known hemopoietic cell population.

Inhibitors of Cholera Toxin-Induced Adenosine Diphosphate Ribosylation of Membrane-Associated Proteins Block Stem Cell Differentiation

Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p-methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)-responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium. These data indicate that these inhibitors of mono-ADP ribosylation can block the commitment and/or differentiation of stem cells and infers that ADP ribosylation may be of some importance in the hematopoietic process.

Depletion of stromal cell elements in human marrow grafts separated by soybean agglutinin

We report studies demonstrating the presence on human marrow stromal cells of binding sites for the soybean lectin (SBA). Marrow cells were separated by agglutination with SBA into an agglutinated cell (SBA+) fraction containing most mature hemic cells including T lymphocytes, and an unagglutinated cell (SBA-) fraction containing the hematopoietic stem cells. The vast majority of fibroblast colony-forming units (CFU- F) (97.2% +/- 1.1%) were in the SBA+ fraction. Mixing experiments using SBA+ and SBA- cells excluded the possibility that these results were caused by an unequal distribution of accessory cells having a regulatory effect on CFU-F growth. In the heterogeneous adherent layers of long-term marrow cultures derived from SBA+ and SBA- cells, endothelial cells, and adipocytes were found almost exclusively in cultures of SBA+ marrow cells. Immunofluorescence studies with fluorescein-labeled SBA revealed the staining of cultured marrow fibroblasts. This staining was inhibited by D-galactose, which is the sugar that specifically binds to SBA. Staining of marrow-derived heterogeneous adherent layers with fluorescein-labeled SBA demonstrated that cultured endothelial cells (identified by rhodamine-labeled antibodies against factor VIII-associated protein) and early adipocytes also had surface glycoproteins binding SBA, although its density on the latter cells was much less important. Thus, our data indicate that human marrow grafts processed with soybean lectin to remove T cells are also depleted of the cellular components of the marrow stroma.

Depletion of Stromal Cell Elements in Human Marrow Grafts Separated by Soybean Agglutinin

We report studies demonstrating the presence on human marrow stromal cells of binding sites for the soybean lectin (SBA). Marrow cells were separated by agglutination with SBA into an agglutinated cell (SBA+) fraction containing most mature hemic cells including T lymphocytes, and an unagglutinated cell (SBA-) fraction containing the hematopoietic stem cells. The vast majority of fibroblast colony-forming units (CFU-F) (97.2% +/- 1.1%) were in the SBA+ fraction. Mixing experiments using SBA+ and SBA- cells excluded the possibility that these results were caused by an unequal distribution of accessory cells having a regulatory effect on CFU-F growth. In the heterogeneous adherent layers of long-term marrow cultures derived from SBA+ and SBA- cells, endothelial cells, and adipocytes were found almost exclusively in cultures of SBA+ marrow cells. Immunofluorescence studies with fluorescein-labeled SBA revealed the staining of cultured marrow fibroblasts. This staining was inhibited by D-galactose, which is the sugar that specifically binds to SBA. Staining of marrow-derived heterogeneous adherent layers with fluorescein-labeled SBA demonstrated that cultured endothelial cells (identified by rhodamine-labeled antibodies against factor VIII-associated protein) and early adipocytes also had surface glycoproteins binding SBA, although its density on the latter cells was much less important. Thus, our data indicate that human marrow grafts processed with soybean lectin to remove T cells are also depleted of the cellular components of the marrow stroma.

915 SPONTANEOUS IN VITRO NEUTROPHIL DIFFERENTIATION IN SEVERE CONGENITAL NEUTROPENIA: EVIDENCE FOR A NORMAL MARROW MICROENVIRONMENT

Marrow cells from 5 patients with severe congenital neutropenia (SCN) associated with maturation arrest at the promyeloeyte stage were found to differentiate spontaneously into segmented neutrophils in long-term marrow culture (LTC). Previous studies have indicated that the granulocyte/macrophage progenitor cells (CFU-GM) in SCN are normal in number and in responsiveness to exogenous colony stimulating activity. This latter observation suggests that part of the defect in SCN may be extrinsic to the granulocytic lineage. To test this hypothesis, we have used autologous LTC to examine the ability of the patient's microenvironment to support granulopoiesis. By week 2 of culture, all 5 patients demonstrated terminal neutrophil differentiation with 2.9±3.6 × 106 (mean ± S.D.) band and segmented neutrophils/flask (12-45% of total cells), a value comparable to that seen in cultures from a normal control (2.0 ±0.6 × 106/flask). Differentiation to mature neutrophils persisted through 6 weeks of culture in all but 1 patient. In that patient the addition of 13-cis-retinoio acid (RA) (10−6M) to culture at week 3 produced a resurgence of neutrophil differentiation at week 6. We conclude that CFU-GM from patients with SCN are able to proliferate and differentiate on an autologous marrow adherent layer, suggesting the development of a normal in vitro microenvironment. Furthermore, the spontaneous differentiation which occurred in these cultures implies the presence of an in vivo suppressor of granulopoiesis. Finally, stimulation of differentiation by RA suggests a possible therapeutic role for this agent.

Effects of in vitro purging with 4-hydroperoxycyclophosphamide on the hematopoietic and microenvironmental elements of human bone marrow

We describe the effects of 4-hydroperoxycyclophosphamide (4-HC) on the hematopoietic and stromal elements of human bone marrow. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-Mix), erythroid (BFU-E), granulomonocytic (CFU-GM), and marrow fibroblast (CFU-F) colony-forming cells and studied in the long-term marrow culture (LTMC) system. The inhibition of colony formation by 4-HC was dose and cell- concentration dependent. The cell most sensitive to 4-HC was CFU-Mix (ID50 31 mumol/L) followed by BFU-E (ID50 41 mumol/L), CFU-GM (ID50 89 mumol/L), and CFU-F (ID50 235 mumol/L). In LTMC, a dose-related inhibition of CFU-GM production was noted. Marrows treated with 300 mumol/L 4-HC were completely depleted of CFU-GM but were able to generate these progenitors in LTMC. Marrow stromal progenitors giving rise to stromal layers in LTMC, although less sensitive to 4-HC cytotoxicity, were damaged by 4-HC also in a dose-related manner. Marrows treated with 4-HC up to 300 mumol/L, gave rise to stromal layers composed of fibroblasts, endothelial cells, adipocytes, and macrophages. Cocultivation experiments with freshly isolated autologous hematopoietic cells showed that stromal layers derived from 4-HC- treated marrows were capable of sustaining the long-term production of CFU-GM as well as controls. In conclusion: (1) Hematopoietic progenitors cells, CFU-Mix, BFU-E, and CFU-GM, are highly sensitive to 4-HC, whereas marrow stromal progenitor cells are relatively resistant. (2) Marrows treated with 300 mumol/L 4-HC that are depleted of CFU-Mix, BFU-E, and CFU-GM can generate CFU-GM in LTMC, suggesting that most primitive hematopoietic stem cells (not represented by CFU-Mix) are spared by 4-HC up to this dose. (3) Consequently, the above colony assays are not suitable tools for predicting pluripotent stem cell survival after 4-HC treatment in vitro.

Effects of In Vitro Purging With 4-Hydroperoxycyclophosphamide on the Hematopoietic and Microenvironmental Elements of Human Bone Marrow

We describe the effects of 4-hydroperoxycyclophosphamide (4-HC) on the hematopoietic and stromal elements of human bone marrow. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-Mix), erythroid (BFU-E), granulomonocytic (CFU-GM), and marrow fibroblast (CFU-F) colony-forming cells and studied in the long-term marrow culture (LTMC) system. The inhibition of colony formation by 4-HC was dose and cell-concentration dependent. The cell most sensitive to 4-HC was CFU-Mix (ID50 31 mumol/L) followed by BFU-E (ID50 41 mumol/L), CFU-GM (ID50 89 mumol/L), and CFU-F (ID50 235 mumol/L). In LTMC, a dose-related inhibition of CFU-GM production was noted. Marrows treated with 300 mumol/L 4-HC were completely depleted of CFU-GM but were able to generate these progenitors in LTMC. Marrow stromal progenitors giving rise to stromal layers in LTMC, although less sensitive to 4-HC cytotoxicity, were damaged by 4-HC also in a dose-related manner. Marrows treated with 4-HC up to 300 mumol/L, gave rise to stromal layers composed of fibroblasts, endothelial cells, adipocytes, and macrophages. Cocultivation experiments with freshly isolated autologous hematopoietic cells showed that stromal layers derived from 4-HC-treated marrows were capable of sustaining the long-term production of CFU-GM as well as controls. (1) Hematopoietic progenitors cells, CFU-Mix, BFU-E, and CFU-GM, are highly sensitive to 4-HC, whereas marrow stromal progenitor cells are relatively resistant. (2) Marrows treated with 300 mumol/L 4-HC that are depleted of CFU-Mix, BFU-E, and CFU-GM can generate CFU-GM in LTMC, suggesting that most primitive hematopoietic stem cells (not represented by CFU-Mix) are spared by 4-HC up to this dose. (3) Consequently, the above colony assays are not suitable tools for predicting pluripotent stem cell survival after 4-HC treatment in vitro.

Long-term marrow culture of cells from patients with acute myelogenous leukemia. Selection in favor of normal phenotypes in some but not all cases.

Long-term cultures were initiated with leukemic marrow aspirate cells from each of 13 newly diagnosed acute myelogenous leukemia (AML) patients. Initial assessment of the clonogenic potential of the marrow suggested that normal hemopoietic progenitors were reduced in most cases and progenitors of abnormal colonies and clusters were present in 10 cases. Subsequent assays of both nonadherent and adherent fractions of long-term cultures revealed two patterns of progenitor cell behavior. The most common pattern (nine cases) featured the detection after 1-4 wk of near normal numbers of typical erythroid, granulopoietic, and mixed colony-forming progenitor cells. Progenitors of abnormal (blast) colonies and clusters initially demonstrable in eight of these nine cases were, in these cases, not sustained in long-term culture and could not be found after 4 wk. Conversion to cytogenetic normalcy in long-term culture was confirmed in two experiments in this group. The second pattern (four cases) was characterized by the failure of progenitors capable of normal differentiation to become detectable in long-term cultures, and the concomitant maintenance of blast progenitors in the two cases in this group where such cells were initially demonstrable. Although progenitors capable of producing abnormal (blast) colonies or clusters in methylcellulose were not detected in either of the other two experiments, the maintenance for 6 wk of a hypercellular nonadherent blast population in one of these suggested the persisting activity of an "adherent layer-dependent" leukemic progenitor cell. Taken together, these findings indicate a strong correlation between the presence of leukemic blasts and their progenitors and a decreased level of normal hemopoiesis. In addition, the failure of leukemic cells to be maintained in long-term marrow cultures from some (but not all) AML patients suggests new applications of this methodology for studies of early stages of leukemic cell development.

Generation of natural killer cell lines from murine long-term bone marrow cultures.

Functionally active natural killer (NK) cells with the ability to lyse 51Cr-labeled YAC-1 lymphoma target cells are no longer detectable by 1 wk of culture in cultured marrow cells harvested from Dexter-type long-term marrow cultures (LMC). Interferon, which enhances NK cell-mediated target cell lysis, fails to induce NK activity from LMC cells even at high effector to target cell ratios. However, such LMC cells, when placed in secondary cultures in the presence of Con A-splenic leukocyte-conditioned medium (spleen-CM) generated a population of cells with NK activity within 1 wk. Kinetic studies showed that the generation of NK activity was not due simply to proliferation of a few surviving NK cells, but suggested derivation from NK precursors through clonal expansion and functional maturation. This NK activity was further shown to be associated with a subpopulation of cells bearing surface Thy-1, Ly-5, and NK-1 as well as asialo-GM1 antigens but lacking Ly-1 antigen. The expression of Ly-2 antigen, however, was variable. Electron microscopy studies of isolated asialo-GM1-positive cells showed a uniform lymphoblastoid morphology with large cytoplasmic to nuclear ratios and prominent electron dense cytoplasmic granules characteristic of large granular lymphocytes. In support of the NK nature of such cultured cells was the ability of anti-asialo-GM1 and complement to abrogate, and of interferon to augment, target cell lysis. Isolated cell lines also showed target selectivity similar to NK cells. The implications of the studies on further analysis of the nature of NK precursors is discussed.

Behavior of human leukemic progenitor populations in long-term marrow culture.

An analysis of mechanisms that underlie abnormal hemopoiesis requires methods that allow the cell populations involved to be characterized and quantitated under conditions that can be defined, and ideally manipulated. Although such methodologies are not yet fully developed, the past 2 decades have seen major advances in several areas relevant to this need. Of major importance has been the establishment of semisolid culture systems capable of supporting the short-term clonal growth and differentiation of individual primitive human hemopoietic progenitor cells of various types. These include pluripotent progenitors [1] as well as different classes of lineage-restricted progenitors [2–4]. Also noteworthy has been the successful exploitation of various genetic markers to examine the cell of origin of the abnormal clone in rare patients who are constitutional mosaics and in whom a clonal hemopoietic neoplasm [5, 6] has subsequently developed.KeywordsChronic Myeloid LeukaemiaPolycythemia VeraAdherent LayerHemopoietic ProgenitorHemopoietic Progenitor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Studies on the self-renewal ability of CFU-S which have been serially transferred in long-term culture or in vivo

The progressive decline in the repopulating ability of bone marrow serially-transferred through a succession of recipients is well documented. A similar series of transfers onto successive long-term culture adherent layers has been carried out using as ‘donor’ cells both adherent layer cells and cells from the culture supernatant. For as long as the in vitro serial transfer regime can be maintained the decline in self-renewal ability (‘quality’) of the CFU-S parallels the similar decline observed in vivo and occurs irrespective of the quality of CFU-S transferred. In both the in vivo and in vitro transfer regimes there is little or no loss of quality of CFU-S as a result of one ‘transfer’ although there may be a reduction in the total numbers of CFU-S in the mouse. However, a second and third transfer in vivo or in vitro leads to a rapid decline in the quality (self-renewal ability) of the CFU-S. Furthermore, despite the fact that the cells are transferred in vitro to a new adherent layer there is no recovery of the quality lost in the second transfer of the CFU-S. This fact implies that self-renewal potential of the CFU-S is a property intrinsic to the cell. The data presented here appear to exclude mitotic history and proliferative stress as factors determining the loss of self-renewal in CFU-S. They also fail to implicate stromal involvement in the decline. It may be that the dilution of an accessory cell or simply the disaggregation of the marrow may be major factors. The work presented indicates that the loss of self-renewal and repopulating ability of haemopoietic stem cells as a result of marrow transplantation may be studied using the long-term marrow culture and yield results relevant to in vivo marrow transplantation.

Responsiveness of the in vitro hematopoietic microenvironment to platelet-derived growth factor

The adherent or stromal cells in human long-term marrow cultures, a possible in vitro counterpart of the in vivo microenvironment, were investigated for responsiveness to platelet-derived growth factor (PDGF). Many stromal cells from cultures derived from normal donors as well as from patients with chronic myelogenous leukemia, bore receptors to PDGF and were stimulated to incorporate [ 3H]-thymidine by highly purified PDGF and to a lesser extent by epidermal growth factor (EGF). These data suggest that PDGF and perhaps EGF may be involved in the regulation of the in vitro microenvironment and, therefore, of normal and possibly neoplastic hematopoiesis.

Nonclonal Hemopoietic Progenitor Cells Detected in Long-Term Marrow Cultures From a Turner Syndrome Mosaic With Chronic Myeloid Leukemia

AbstractWe have investigated the clonality of Ph1-negative hemopoietic progenitor cells appearing in long-term marrow cultures established with cells from a mosaic Turner syndrome patient (46,XX/45,X) with Ph1-positive chronic myeloid leukemia (CML). The Ph1-positive clone had been shown previously to have arisen from a cell of the 45,X lineage. At the time of the present study, the patient was five years postdiagnosis and had been off chemotherapy for two months following a year of treatment for lymphoid blast crisis. All analyzed unstimulated marrow metaphases and each of 23 individually analyzed erythroid and granulocyte colonies produced in assays of the starting marrow were 45,X,Ph1. Pooled granulocyte colonies from the same assays yielded four metaphases that were 45,X,Ph1 and one that was 46,XX. Very few hemopoietic progenitors were detected in long-term cultures at any time; however, all of four individually analyzed large granulocyte colonies and a pooled granulocyte colony preparation obtained from assays of 4- to 6-week-old adherent layers yielded exclusively 46,XX metaphases. These results provide evidence that nonclonal progenitors can persist in patients with CML, even after the onset and treatment of blast crisis, and that the long-term marrow culture system provides a sensitive method for detecting such cells.