The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes

The platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), defined by the CD31 monoclonal antibody (MoAb), was initially described as a cell-cell adhesion molecule mediating both homotypic and heterotypic adhesion. In this report, we show that enriched CD34+ human hematopoietic progenitor cell populations, containing early myeloid, erythroid, and multipotential progenitor cells, are CD31+. Analyses of CD34+ cell lines representing early myeloid, multipotential, and pre- pre-B-lymphoid progenitors indicate that precursors of both myeloid and B-lymphoid cells express PECAM-1 at high levels. Three-color flow- cytometric analyses also show that normal human bone marrow CD31+ CD34+ subsets coexpress myeloid (CD33) or B-lymphoid (CD19, CD10) markers. Except for the monocytic cell line, U937, all CD34- cell lines tested, which represent more mature stages of the myeloid, erythroid, and lymphoid lineages, expressed substantially lower or negligible levels of PECAM-1. Western blotting studies indicated that the CD31 MoAb, JC/70A, detected molecules in the 120- to 140-kD molecular weight range on the monocytic CD34- CD33+ CD31+ cell line, U937; on the CD34+ CD31+ CD33+ CD19- multipotential/lymphomyeloid precursor cell lines, KG1 and KG1B; on the CD34+ CD31+ CD19+ CD10+ CD33- precursor pre-pre-B-cell line, MIK-ALL; and on a CD34(+)-enriched precursor cell population from normal human bone marrow. A single molecular weight species was generally observed with enriched membrane preparations, whereas two PECAM-1 molecules were present in whole-cell lysates of cell lines and the CD34+ bone marrow cell subset. Preliminary studies show that a proportion of the PECAM-1 molecules on the lymphomyeloid/multipotential progenitor cell line, KG1, and on the monocytic cell line, U937, binds to heparin-sepharose. A soluble form of PECAM-1 also binds heparin- sepharose. The high level of expression of PECAM-1 on CD34+ cells suggests that this glycoprotein may function as a heterotypic adhesion molecule, possibly mediating multipotential, myeloid, and early-B- lymphoid precursor cell interactions with stromal cells and extracellular matrix molecules via heparan sulfate proteoglycans. It may also act as a homotypic adhesion molecule by interacting with PECAM- 1 on bone marrow stromal macrophage-like cells and endothelial cells or on endothelial cells during stem/progenitor cell migration. Thus, this molecule has the potential importance of directing both lineage commitment and trafficking of early hematopoietic progenitor cells.     

T-lymphoid differentiation potential measured in vitro is higher in CD34+CD38-/lo hematopoietic stem cells from umbilical cord blood than from bone marrow and is an intrinsic property of the cells

Background

Human bone marrow and umbilical cord blood are sources of allogeneic hematopoietic stem cells for transplantation, which is a life-saving treatment in a variety of diseases but is burdened by delayed T-cell reconstitution. Observational studies evaluating T-cell reconstitution in post-transplant recipients suggest that cord blood hematopoietic stem cells have a more effective capacity for T-cell reconstitution. This study focuses on the comparison of the capacity of cord blood and bone marrow hematopoietic stem cells to generate T cells in vitro.

Design and Methods

Hematopoietic stem cells were cultured in OP9-delta-like-1 and OP9-green fluorescent protein co-cultures to estimate T and myeloid generation capacity, respectively. Phenotypic markers of T-lineage or myeloid differentiation were measured by flow cytometry and used to analyze their kinetics as a function of culture time. Hematopoietic stem cells were labeled with carboxyfluorescein diacetate succinamidyl ester and analyzed after culture to track their phenotypic progression in consecutive generations. Mixed OP9-delta-like-1 co-cultures were done with either carboxyfluorescein diacetate succinamidyl ester-labeled bone marrow and unlabeled cord blood hematopoietic stem cells, or vice versa, to evaluate their mutual influence on T-lineage differentiation. The T-cell potential of hematopoietic stem cells was addressed quantitatively by limiting dilution analysis.

Results

Bulk cultures showed faster and more extensive T-cell differentiation by cord blood hematopoietic stem cells. Furthermore, the T-lymphoid differentiation capacity of cord blood and bone marrow hematopoietic stem cells can be discriminated very early based on the coordinated expression of CD34 and CD7. Mixing experiments with cord blood hematopoietic stem cells and bone marrow hematopoietic stem cells showed that these differences are cell intrinsic. Quantitative clonal analyses demonstrated that CD34⁺CD38^−/lo hematopoietic stem cells from cord blood contained a two-fold higher T-lineage generation capacity than CD34⁺CD38^−/lo bone marrow hematopoietic stem cells, whereas the myeloid differentiation was similar.

Conclusions

Our data shows that cord blood hematopoietic stem cells have higher T-lymphoid differentiation potential than bone marrow hematopoietic stem cells and that this property is cell autonomous.     

Identification of B cells in multilineage hematopoietic colonies derived from cells of patients with lymphocytic lymphoma.

Pluripotent stem cells from human bone marrow can be identified in culture by their ability to form multilineage hematopoietic colonies containing different myeloid lineages and T cells of different phenotypes. The observation of a common progenitor of myeloid and lymphoid cells in normal and disturbed hematopoiesis prompted the question of whether B cells are part of the differentiation program of stem cells. The availability of hybridomas of azaguanine-resistant T-cell lines secreting monoclonal growth factors for B cells and clinical conditions that are considered to originate from malignant B cells might facilitate this investigation. We were able to identify surface immunoglobulin and B-cell-associated antigen-positive cells within such colonies, indicating that B cells are generated from a myelolymphopoietic stem cell. This report describes the presence of B cells in these colonies derived from bone marrow cells of patients with non-Hodgkin lymphoma.     

Expression of intercellular adhesion molecule-1 (CD54) on hematopoietic progenitors.

The distribution of intercellular adhesion molecule-1 (ICAM-1), a ligand for lymphocyte function antigen-1, on hematopoietic tissue was determined using the anti-ICAM-1 monoclonal antibody CL203.4 with flow cytometry and short-term semi-solid hematopoietic progenitor cultures. After timed incubation in media with fetal bovine serum, 29% of erythroid burst-forming units (BFU-E), 24% of erythroid colony-forming units (CFU-E), and 52% of granulocyte-macrophage colony-forming units (CFU-GM) bone marrow progenitors expressed ICAM-1. This finding, which is consistent with the detection of ICAM-1 on acute non-lymphoblastic leukemic blasts, is at variance with recent reports. ICAM-1 was also detected on bone marrow blasts, proerythroblasts, promyelocytes, and cells of monocyte/macrophage lineage, but was not detected on erythroblasts, normoblasts, neutrophilic myelocytes, metamyelocytes, bands, or on most lymphocytes. These results indicate that maturation of cells of the erythroid and myeloid lineage is associated with loss of ICAM-1. The distribution of ICAM-1 on bone marrow progenitors, early precursor cells, and accessory cells in conjunction with the function of this molecule in cell-cell interactions suggests that ICAM-1 may play a role in the cell-cell and cell-stromal interactions that regulate hematopoiesis.     

Purification and partial characterization of a human hematopoietic precursor population.

This study reports the development of an assay, the Pre-colony-forming unit (CFU) assay, which detects human hematopoietic precursors. The Pre-CFU assay is based on the observation that precursors to CFU-granulocyte-macrophage (CFU-GM) that are undetectable in clonogenic assays differentiate into CFU-GM preferentially following treatment in suspension culture with recombinant human interleukin-1 alpha (rhIL-1 alpha) combined with rhIL-3. Using the Pre-CFU assay, hematopoietic precursors were detected in human bone marrow depleted of CFU-GM progenitors and differentiated hematopoietic elements via 4-hydroperoxycyclophosphamide treatment coupled with selection for CD34+ cells (4-HCresistant/CD34+ marrow). Additionally, the Pre-CFU assay detected recovery of hematopoiesis substantially earlier than the CFU-GM assay in primates following myeloablation with 5-fluorouracil. The Pre-CFU assay was used to asses purification of a phenotypically defined hematopoietic precursor population, the lin-CD34+ population. The lin-CD34+ population lacks detectable surface markers for T-cell, B-cell, natural killer cell, and myeloid lineage, possesses the CD34 antigen, is devoid of CFU-GM progenitors, and yields Pre-CFU assay values comparable with 4-HCresistant/CD34+ marrow. Using a combination of phenotypic analysis and Pre-CFU assay analysis, the action of rhIL-1 alpha plus rhIL-3 treatment on lin-CD34+ cells was further characterized. The data indicate that rhIL-1 alpha plus rhIL-3 treatment induces proliferation and differentiation of early hematopoietic precursors into progenitors and terminally differentiated cells, without inducing a significant expansion of the precursor population itself.     

Preparation and characterization of monoclonal antibodies recognizing two distinct differentiation antigens (Pro-Im1, Pro-Im2) on early hematopoietic cells

A series of monoclonal antibodies recognizing myeloid differentiation antigens were prepared by immunizing Balb/c mice with HL-60 cells. Hybrids secreting antibodies reactive with HL-60 cells but unreactive with peripheral blood mononuclear cells were isolated and further cloned. One clone was found to produce an IgG2a antibody recognizing an 85,000-dalton molecular weight surface glycoprotein, and a second clone was found to produce an IgM antibody recognizing a heat-stable determinant present on a glycolipid. We have termed these antigens Pro- Im1 and Pro-Im2, respectively (Pro for using HL-60 promyelocytes as an immunogen and Im for the presence of these antigens on immature cells). alpha Pro-Im1 and alpha Pro-Im2 were used to investigate the surface expression and tissue distribution of these two antigens. Pro-Im1 and Pro-Im2 were found to be brightly expressed on a fraction of fetal liver hematopoietic and bone marrow cells. Both antibodies mediated complement-dependent inhibition of CFU-GM, BFU-E, and CFU-GEMM formation assayed by soft agar colony and burst formation, indicating the expression of these antigens by early hematopoietic precursor cells. This was further confirmed by the induction of HL-60 cells by TPA to differentiate into more mature monocytes and macrophages, accompanied by the loss of both antigens. Pro-Im1 and Pro-Im2 were absent from peripheral blood monocytes, erythrocytes, and platelets, but Pro-Im2 was expressed on granulocytes. Both antigens were absent from thymocytes and peripheral T cells. Cytofluorographic analysis suggested their absence from peripheral blood B cells but that both were expressed on a minority of tissue B cells. Analysis of 150 cases of various myeloid and lymphoid malignancies demonstrated Pro-Im1 and Pro-Im2 expression on myeloblasts and promyelocytes from some acute myelogenous leukemias as well as some B cell malignancies, suggesting that these antigens are shared by early hematopoietic cells and a subset of B cells.     

Effect of bone morphogenetic PROTEIN-5 on hematopoietic stem cells and cytokine production in normal human bone marrow stroma

Normal human bone marrow stroma cells include stem cells for both hematopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and II receptors. As a member of the TGF-beta superfamily, BMP-6 binds to both BMP receptors and is involved in the developmental processes of renal and hepatic systems as well as of human fetal intestine. BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal stem cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), which represent the functional primitive repopulating hematopoietic stem cell in long-term marrow culture. The effect of BMP-6 on marrow stroma production of interleukin-6, interleukin-11 and their common transducing receptor gp130, which is expressed in primitive hematopoietic stem cells and is indispensable for the proliferation and tri-lineage differentiation of these hematopoietic stem cells, was examined. The effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive hematopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88 ± 10 in control cultures in a dose-dependent manner to only 48 ± 3 per 105 cells in 50 ng/ml BMP-6– treated marrow stroma cultures, p < 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 to significantly reduce IL-6 and IL-11 production from human marrow stroma on day 5, p < 0.01. BMP-6 significantly increased soluble gp130 release by 7.4 fold in 50 ng/ml BMP-6-treated marrow stroma cultures. This profound rapid increase in the natural antagonist of human IL-6 cytokine family could abrogate the gp130 signalling required for proliferation and/or tri-lineage differentiation of human primitive hematopoietic stem cells and may lead to hematopoietic stem cell survival. This study revealed that in 50 ng/ml BMP-6-treated marrow stroma cultures, the released soluble VCAM-1 increased by 2-fold. Marked increases in the soluble adhesion molecule could exert a significant antagonist effect on the function of VCAM-1 (ligand for VLA4) and may allow the mobilization of human hematopoietic stem cells. Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilize hematopoietic CD34⁺ cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilized CD34⁺ cells in the peripheral blood than on bone marrow CD34⁺ before mobilization in the same individuals. BMPs are currently being used in clinical trials for bone repair and fracture healing; the present results suggest a possible novel therapeutic role for BMP-6 in mobilizing CD34⁻ cells for transplantation. However, the potential therapeutic use of BMP-6 as a mobilizing agent should only be applied in patients with myeloid leukemia and not other tumors due to the expression of BMP-6 in solid tumors and lack of BMP expression in myeloid leukemia cells.     

Rb is dispensable for self-renewal and multilineage differentiation of adult hematopoietic stem cells

Stem cells have been identified as essential for maintaining multiple organ systems, including the hematopoietic system. The distinct cell fates of self-renewal and differentiation of hematopoietic stem cells (HSCs) depend on cell division. Recently, several negative regulators of the cell cycle, such as the cyclin-dependent kinase inhibitors p21(Cip1), p27(Kip1), and p16(INK4a)/p19(ARF), have been demonstrated to have a role in regulating HSC fate decisions, suggesting that regulation of the G(1)-S phase transition can contribute to HSC self-renewal. Because the retinoblastoma protein, Rb, plays a central role in the regulation of the G(1)-S phase cell cycle, we sought to determine whether it has an intrinsic role in the regulation of HSC fate. Surprisingly, we found that HSC function was essentially normal in the absence of Rb. Rb(Delta/Delta) HSCs contributed normally to both myeloid and lymphoid lineages in both primary and secondary recipients, and no evidence of transformation was observed. Additionally, we observed a mild myeloid expansion and decrease in mature B cells within the Rb(Delta/Delta) bone marrow but a similar contribution to phenotypic HSC populations compared with nondeleted bone marrow. The Rb family members p107 and p130 were not deregulated in cells in which Rb had been deleted, as determined by quantitative RT-PCR on the highly enriched stem and primitive progenitor cell lin(-)c-Kit(+)Sca-1(+) population. These studies demonstrate that Rb is not intrinsically required for self-renewal and multilineage differentiation of adult HSCs.     

Interleukin-1 inhibition of B lymphopoiesis is reversible

Interleukin-1 (IL-1) has multiple effects on the hematopoietic system. The present data demonstrate that IL-1 and/or products induced by it reversibly suppress B-cell differentiation. Upon the addition of 50 U/mL (2.4 ng/mL) of recombinant IL-1 alpha (rIL-1 alpha) to lymphoid long-term bone marrow cultures at their initiation, very few B lymphocytes could be detected, and the majority of cells present were myeloid. This inhibition of B lymphopoiesis did not appear to be due to effects on proliferation of mature B cells because IL-1 did not affect the proliferative response of B cells to form B-cell colonies (CFU-B). The actions of the monokine were further examined by using myeloid and lymphoid long-term bone marrow culture systems. The transfer of myeloid long-term bone marrow cultures to lymphoid conditions usually results in the cessation of myelopoiesis and initiation of B lymphopoiesis. Exposure of early B-cell precursors present under the myeloid conditions to 50 U/mL of RIL-1 did not affect their subsequent differentiation into B cells upon transfer of the cultures to lymphoid conditions. However, myelopoiesis was sustained, and B lymphopoiesis did not initiate if 50 U/mL of rIL-1 was added to myeloid bone marrow cultures at the time of their transfer to the lymphoid conditions and during biweekly feedings thereafter. Upon removal of IL-1, myelopoiesis ceased, and B lymphopoiesis initiated. Thus, the effects of IL-1 on inhibition of B lymphopoiesis are reversible.     

Isolation of a candidate human hematopoietic stem-cell population.

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.     

Lentiviral-mediated RNAi inhibition of Sbds in murine hematopoietic progenitors impairs their hematopoietic potential

Shwachman-Diamond syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, multilineage hematopoietic dysfunction, and metaphyseal chondrodysplasia. Bone marrow dysfunction is present in nearly all patients with SDS, with neutropenia being the most common abnormality. The majority of patients with SDS have mutations in the Shwachman Bodian Diamond syndrome (SBDS) gene. We have developed a strategy to examine the consequences of lentiviral-mediated RNA interference (RNAi) of Sbds on hematopoiesis. Here, we report that both Sbds RNA and protein expression can be efficiently inhibited in primary murine hematopoietic cells using lentiviral-mediated RNAi. Inhibition of Sbds results in a defect in granulocytic differentiation in vitro and impairs myeloid progenitor generation in vivo. In addition, short-term hematopoietic engraftment was impaired, which is due in part to reduced homing of hematopoietic progenitors to the bone marrow. Finally, we show that inhibition of Sbds is associated with a decrease in circulating B lymphocytes, despite evidence of normal B lymphopoiesis. These data provide the first evidence that loss of Sbds is sufficient to induce abnormalities in hematopoiesis.     

Tyrosine phosphorylation of proteins in primary human myeloid leukemic cells stimulated by macrophage colony-stimulating factor: Analysis by disease type and comparison with normal human hematopoietic cells

We investigated tyrosine phosphorylation of proteins in primary human leukemic cells stimulated by macrophage colony-stimulating factor (M-CSF) in 60 patients with acute myeloid leukemia (AML) and 5 patients with chronic myelomonocytic leukemia and compared the findings for leukemic cells with those of normal human monocytes and bone marrow immature hematopoietic cells. M-CSF induced tyrosine phosphorylation of p140-200, p110, p60, p44, and p42 frequently, and that of p95 and p55 less frequently, in primary myeloid leukemic cells, whereas M-CSF-induced phosphorylation of proteins was not detected in the normal human hematopoietic cells tested. Of these phosphoproteins, p140-200 was phosphorylated in all patients who responded to M-CSF and was considered to be almost identical to Fms, a product of the c-fms proto-oncogene. M-CSF-induced tyrosine phosphorylation was observed frequently (89%) in AML of French-American-British class M4 and infrequently in all other subtypes of AML, including M5. In chronic myelomonocytic leukemia, M-CSF-induced protein phosphorylation was prominent in blast crisis but was not detected in the chronic phase. Both bone marrow immature cells and mature monocytes showed low responsiveness to M-CSF. These findings for responsiveness to M-CSF were correlated with the amount of Fms in each type of cell. We also identified tyrosine phosphorylation of Vav, Shc, and extracellular signal-regulated kinase by M-CSF in some cases. These findings clarified an M-CSF-specific pattern of protein tyrosine phosphorylation and the responsiveness to M-CSF of primary human myeloid cells. Particularly, enhanced phosphorylation responses to M-CSF and increased amounts of Fms protein were observed in restricted human hematopoietic cells with a premature myelomonocytic character.     

An 80,000-kd glycoprotein cell surface antigen found only on nonhematopoietic cells in human bone marrow.

The murine IgG2a monoclonal antibody (MoAb) 6-19 binds to the surface of human nonhematopoietic cells but not to hematopoietic cells. As previously described, it can be used with complement to selectively kill nonhematopoietic cells prior to culture of human bone marrow. It is now shown that the 6-19 MoAb recognizes a specific antigenic determinant of apparent molecular mass 80 kd, detected by western blotting on nonhematopoietic tumor cell lines, endothelial cells, and bone marrow stromal cells. Mouse L cells were transfected with human DNA, and the 6-19 monoclonal antibody selected cells that expressed an antigen with similar characteristics. The antigenic determinant is absent in hematopoietic tumor cell lines, peripheral blood cells, and bone marrow hematopoietic cells. Investigation of the biochemical nature of the antigen, using various enzymes, lectin-binding studies, and western blotting suggests that the 6-19 epitope is at least partially carbohydrate and that the protein has N-linked sugars but not O-linked. The affinity (Ka approximately 10(9) M-1) of MoAb 6-19 binding is similar in tumor cells and normal fibroblasts and endothelial cells. The identification of a specific antigenic determinant of 80 kd may help to discriminate between hematopoietic and nonhematopoietic cells in human bone marrow.     

Infection of hematopoietic progenitor cells by human cytomegalovirus.

The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.