Enrichment for GM-CFU from human bone marrow using Sambucus nigra agglutinin: potential application to bone marrow transplantation

A new lectin, purified from black elder-berries, Sambucus nigra L. (SNA I, hereafter called SNA), was used for fractionation of normal human marrow cells. The stem cell enrichment capability of SNA was investigated by comparing colony formation (GM-CFU) in unagglutinated cell fractions (SNA-) following agglutination with SNA with that with soybean agglutinin (SBA-). GM-CFU recovery in SNA- was equal or superior to that in the SBA- fraction. A modified procedure was developed to combine stem cell enrichment and depletion of E-rosette-forming T-lymphocytes. Bone marrow cells were exposed in one step to SNA and to untreated sheep red blood cells (SRBC). Nonagglutinated, nonrosetting cells were collected after Ficoll-Hypaque-gradient separation. The procedure, a modification of Reisner's multistep procedure involving the agglutination of SBA+ cells; separation of the unagglutinated SBA- cells from the top of a 5% bovine-serum-albumin gradient; the formation of E-rosettes with SRBC; and the separation of rosettes over a Ficoll-Hypaque gradient, is faster, simpler, and as effective for T-cell depletion and stem cell enrichment.     

Enrichment of myeloid progenitor cells from normal human bone marrow using an immune-rosette technique

In this study we have developed methods for purification of myeloid progenitor cells (CFU-Cs) from normal human bone marrow cells. Bone marrow aspirates were obtained from volunteers, and mononuclear cells (MNCs) were separated by Ficoll-Hypaque gradient centrifugation. T- and B-lymphocytes, monocytes, mature granulocytes, and erythroid precursors were eliminated by an immune-rosette technique using a panel of murine monoclonal antibodies and immunoglobulin (Ig)-coated sheep red blood cells (SRBCs). MNCs were treated with OKT3, B1, M3, Mo5, and EP1 monoclonal antibodies, which are reactive with T cells, B cells, monocytes, granulocytes, and erythroid precursors, respectively. Antibody-treated MNCs were incubated with SRBCs that had been coated with goat antirabbit IgG F(ab')2 and rabbit antimouse Ig for immune rosetting. Rosetted cells were then separated from nonrosetted cells in Ficoll-Hypaque. Nonrosetted cells were, in the second step, treated with an OKIa1 monoclonal antibody and again separated into an Ia+ and Ia- cell fraction by the same manner; 39% +/- 19.2% (mean +/- 1 SD, range 16.3%-75.4%) of CFU-Cs (colonies plus clusters) were recovered in the OKT3-, B1-, M3-, Mo5-, EP1- cell fraction, and the number of CFU-Cs grown in semisolid agar was 149.6 +/- 73.0 (64.0-309.0)/10(4) plated cells in this purified fraction, representing an enrichment of 14.2 +/- 6.4 (6.0-27.3)-fold when compared with unseparated marrow cell fractions. CFU-Cs were enriched 17.7 +/- 8.6 (6.1-28.3)-fold in the Ia+ cell fraction. These purified myeloid precursors would be of value for in-depth studies of the interactions between hematopoietic progenitor cells and regulatory factors that influence their proliferation and differentiation and also of drug metabolism and determinants of cytotoxicity.     

Enrichment of erythroblasts from human bone marrow using complement-mediated lysis: measurement of ferritin

S ummary . Sequential lysis of human bone marrow cells with a monoclonal antibody directed against myeloid cells (TG1) and a rabbit antiserum raised against peripheral blood mononuclear cells gave preparations in which 78–97% of the nucleated cells were erythroid, with a 24–77% recovery. Viability was high, morphology was good and the cells were able to divide and differentiate in culture. No metabolic experiments were carried out but the ferritin content of the erythroblasts was measured in four experiments and found to be about 200–2000 times higher than that found in normal erythrocytes. The H/S ratio was high in both erythroblasts and erythrocytes. Fractionation on the basis of density of two erythroblast preparations, one from a patient with sideroblastic anaemia and one from a patient with megaloblastic anaemia, showed that the most immature erythroblasts contained the highest content of ferritin and that this fell with maturation. The H/S ratio stayed the same or fell with maturation. It was concluded that this method would be valuable for the study of the role of erythroblast ferritin in normal and pathological situations.     

Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow

Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.     

Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)- treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5- FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this "lineage-negative" (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.     

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.     

Enrichment of hematopoietic progenitor cells from human bone marrow on percoll density gradients

Summary Hematopoietic progenitor cells were fractionated on continuous and discontinuous Percoll density gradients. It could be shown, that Percoll provides a suitable gradient medium for analytical as well as preparative density gradient procedures without major methodological problems. Advantages compared to albumin and Ficoll-Metrizoate are discussed.     

Enrichment of progenitor cells from human marrow

Suspensions enriched for myeloid and erythroid colony-forming cells were prepared from human marrow by simultaneous treatment of low-density cells with the monoclonal antibodies Campath-1 and 80H.3, followed by immune-rosetting; lymphocytes and monocytes were successfully removed by this method. The final suspension contained 22% of blasts and 70% primitive myeloid-monocytic cells. GM-CFCs, counted after 14 days of culture, were enriched 21-fold. Of all cells present in the final suspension, 10.8% were day 8 GM cluster-forming cells, 2.3% were day 8 GM colony-forming cells, 2.4% were day 7 CFU-E, 0.9% were day 14 GM cluster-forming cells, 1.1% were day 14 GM colony-forming cells, and 0.6% were day 14 BFU-E. After enrichment, BFU-E became markedly more dependent on the addition of 5637-conditioned medium as a source of growth factors, suggesting that lymphocytes and/or monocytes support erythroid progenitor growth in cultures of unfractionated marrow. By removing these cells, we obtained a sensitive assay for burst-promoting activities in conditioned media. This procedure can be used to study the roles of marrow lymphocytes and monocytes in hemopoiesis as well as providing a basis for the purification of normal and aberrant progenitors.     

Survival of CFU-C in recipient blood after bone marrow transplantation

In six patients undergoing allogeneic bone marrow transplantation granulocyte colony and cluster forming units were detectable in the blood up to 24 hours after intravenous marrow infusion. The mean surviving fraction 30 minutes after the end of the infusion was 0.3 for colonies and 0.42 for total aggregates. CFU-C declined logarithmically with time, but a small secondary rise in blood CFU-C occurred 2-6 hours after the end of the marrow infusion. Three patients were plasma-exchanged immediately before the marrow graft for major donor-recipient ABO incompatability. The proportion of total groups remaining in the blood at 30 minutes was significantly higher in the plasma-pheresed patients (P = < 0.05), and the secondary rise in CFU-C was less marked, but no correlation of the blood CFU-C decay curve with the subsequent fate of the graft was observed.     

Peanut agglutinin shows specificity for bone marrow plasma cells

A panel of lectins was used to study surface carbohydrate expression on myeloma cells with the aim of finding a possible agent for in vitro bone marrow purging. Peanut agglutinin (PNA, galactose beta 1,3 N-acetylgalactosamine-binding) bound to all plasma cells in 33/34 bone marrow samples from myeloma patients and to all plasma cells in 11 bone marrow from patients with monoclonal gammopathy of undetermined significance and 10 normal bone marrow samples. Bone marrow and peripheral blood monocytes reacted weakly with PNA except in one case of acute monoblastic leukaemia and two of chronic myelomonocytic leukaemia in which monocytes were strongly positive. The only case of plasma cell leukaemia studied was PNA negative. All other bone marrow mononuclear cells were negative for PNA but became positive after sialidase treatment. Peanut agglutinin may have potential as an agent to be used in myeloma for in vitro marrow purging prior to autologous transplantation in combination with high dose chemotherapy.     

Differential agglutination by soybean agglutinin of human leukemia and neuroblastoma cell lines: potential application to autologous bone marrow transplantation.

Normal human bone marrow cells were mixed with radioactively labeled tumor cells from different leukemia and neuroblastoma cell lines, and the cell mixtures were separated by differential agglutination with soybean agglutinin. It is shown that the cell fraction unagglutinated by soybean agglutinin, which was previously found to be capable of reconstituting the hematopoietic system of lethally irradiated recipients, can be purged of tumor cells with varying efficiency depending on the tumor cell expression of soybean agglutinin receptors as detected by flow cytofluorimetry with fluoresceinated soybean agglutinin.     

Enrichment of human marrow lymphocytes with monoclonal antibodies to murine antigens.

Two monoclonal antibodies, prepared against a murine B lymphoma and characterized as binding to a cell surface antigen represented primarily on cells of the B lineage, were found to bind to human hemopoietic cells. These antibodies recognize similar populations of cells in mice and humans. Antibodies from clones 177.17 and 83.4 bound to 6% of human bone marrow nucleated cells. This included all cells with detectable cell surface Ig (sIg+) and those that lack sIg but have detectable cytoplasmic mu (sIg-, c mu+), considered to be the immediate precursors of B lymphocytes (pre-B cells). In addition, these antibodies bound to a subpopulation of T cells and a proportion of null lymphocytes in marrow, spleen, and peripheral blood. An unexpected finding was that established pre-B cell lines were not recognized by these antibodies and possible reasons for this are considered. By using antibody-coated polystyrene plates for cell depletion and recovery, highly enriched preparations of c mu+, sIg- cells have been obtained. These antibodies and enrichment procedures should prove valuable in establishing the minimal requirements for maturation of these putative precursors in vitro, for comparative studies of immunodeficiency/autoimmune diseases in man and experimental animal models, and for monitoring the outcome of therapeutic marrow transplantation.     

Enrichment of natural suppressor activity in a wheat germ agglutinin positive hematopoietic progenitor-enriched fraction of monkey bone marrow

Natural suppressor (NS) activity, the capacity of unprimed cells to suppress immunologic responses, is present in mouse, rabbit, and human bone marrow (BM). In this study we characterize NS activity in bone marrow cells of the rhesus monkey. Greatest NS activity was found in low-density cells (1.0600 to 1.0655 g/mL) obtained by density centrifugation on a discontinuous Percoll gradient. NS activity was further enriched when cells were separated by affinity for wheat germ agglutinin (WGA). Cells with high affinity to WGA demonstrated potent NS activity, whereas cells with low affinity to WGA had no NS activity. A significant relationship between NS activity and hematopoietic activity was demonstrated using in vitro assays of colony formation (CFU-GM and CFU-MIX). NS activity was not affected by treatment with monoclonal antibodies (MoAbs) to human Fc gamma receptors (Leu, 11a,b,c) or treatment with MoAbs to monkey natural killer cells. These findings extend our prior observations by showing that cells with NS activity, which apparently have WGA receptors, are present not only in murine BM but also in monkey bone marrow, and suggest that such cells may be involved in immunoregulation by primitive cells of BM.     

T cell depletion from human bone marrow using magnetic beads

We have studied a recently described method to purge human marrow of T cells using magnetic beads conjugated to various anti-T cell monoclonal antibodies. Using anti-CD2 and anti-T cell receptor/CD3 monoclonal antibodies, we show that up to 2.76 log T cell purge can be achieved while recovery of hemopoietic activity is 82%. As a control, our clinically established T cell depletion protocol, namely soybean agglutination combined with E-rosetting, provided 3.39 log T cell purge and 59% recovery of hemopoietic activity. The impact of these results on T cell depletion strategies in HLA-nonidentical bone marrow transplantation is discussed.     

Canine cyclic haematopoiesis: bone marrow adherent cell influence of CFU-C formation

Canine cyclic haematopoiesis (CH) appears to be a multipotential stem cell defect, possibly due to an intrinsic marrow defect. The in vitro adherent marrow cells of the cyclic haematopoietic (CH) dog were cultured as underlayers beneath normal dog nonadherent marrow cells. The marrow granulocyte-committed colony forming units (CFU-C) of the normal dog nonadherent cells were cyclically influenced by the CH adherent cell underlayers. The CH adherent cells collected on the ninth or tenth day following the onset of neutropenia did not stimulate CFU-C formation while those collected on the sixth day stimulated as many as 108 colonies. The CH adherent cells collected on other cycle days supported increased CFU-C formation with the exception of cycle day 3 which inconsistently stimulated growth. These data show that the CH marrow in vitro adherent cells alternately stimulate and inhibit in vitro granulopoiesis of normal dog marrows.     

Enrichment of interleukin-2-responsive natural killer progenitors in human bone marrow

Natural killer (NK) cells can be cultured in interleukin-2 (IL-2)- containing medium from selected human bone marrow (BM) cells obtained after the elimination of mature T and NK cells. To isolate and characterize IL-2-responsive NK progenitors in the selected BM cells, we investigated the expression of IL-2 receptors (IL-2R) on these cells. Neither CD25 (IL-2R alpha) nor IL-2R beta antigen was observed on the selected BM cells before culture. However, CD25+ cells without detectable levels of IL-2R beta antigen appeared 24 hours after culture in IL-2-containing medium. Cells were sorted from each fraction of the selected BM cells 24 hours after culture after staining with anti-CD33, anti-CD34, and anti-CD25 monoclonal antibodies. The generation of NK cells (CD3- CD56+ cells) and NK activity were observed only from the CD33-/CD34-/CD25+ cell fraction after culture in IL-2-containing medium. The frequency of IL-2-responsive NK progenitors relative to the fraction was 1/231 (95% confidence range, 1/156 to 1/289), which corresponded to the frequency relative to the total number of selected BM cells when the frequency relative to the CD33-/CD34-/CD25+ cell- fraction was converted according to the percentage of these cells in the total number of selected BM cells. These results indicated that IL- 2-responsive NK progenitors were enriched in the CD33-/CD34-/CD25+ cell fraction.     

Growth kinetics of CFU-GEMM, BFU-E, and CFU-C in diffusion-chamber cultures of normal human bone marrow cells

The proliferation and differentiation of human pluripotent hemopoietic precursor cells CFU-GEMM as well as of the committed precursors BFU-E and CFU-C within conventional diffusion chambers (DC) was studied. Low-density human bone marrow cells from eight normal donors were cultured in DC implanted into 750-rad-preirradiated mice. Cells harvested from DC after 1-14 days were assayed for the content of CFU-GEMM, BFU-E, and CFU-C. The mean number of progenitor cells inoculated per DC on day 0 was 59 (range 12-144) for CFU-GEMM, 367 (range 165-878) for BFU-E, and 632 (range 122-1168) for CFU-C. After an initial decrease in the number of all progenitors, an increase of CFU-GEMM was observed in five out of eight experiments and in the number of BFU-E and CFU-C in seven and five experiments, respectively. The assessment of the percentage of precursor cells sensitive to ara-C demonstrated a rise in the proportion of cells in S phase after implantation into DC consistent with reentry into active proliferation. From the observed behavior of the CFU-GEMM it is concluded that the earliest detectable human precursor cells do proliferate within DC.     

Characterization of fibroblastic stromal cells from murine bone marrow

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.     

Separation of antibody helper and antibody suppressor human T cells by using soybean agglutinin.

Soybean agglutinin (SBA) binds specifically to mouse B cells and has been used in the past to separate mouse B and T spleen cells by differential agglutination of the B cells. In the present study it was found that a major T-cell subpopulation of human peripheral blood mononuclear cells is agglutinated by SBA along with the B cells and monocytes. Tests of such cell surface markers as Fc receptors for IgG and IgM, as well as functional assays of antibody production by B cells, revealed that the SBA-agglutinated cell fraction contains the antibody helper T cells whereas the unagglutinated fraction is enriched with antibody suppressor T cells. Similar observations were made in tests of the proliferative response to mumps antigen. A recently prepared monoclonal antibody, anti-Leu 2a, which recognized the same thymus-dependent antigen previously defined by a heterologous anti-human T cell serum (alpha TH2), was found to define by indirect immunofluorescence a subpopulation of SBA- cells of intermediate staining intensity which was not detectable in the SBA+ population.